https://microbewiki.kenyon.edu/api.php?action=feedcontributions&user=Millerk&feedformat=atommicrobewiki - User contributions [en]2024-03-28T09:53:59ZUser contributionsMediaWiki 1.39.6https://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44383Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T19:19:44Z<p>Millerk: /* Conclusion */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. The dotted line in the figure represents the <i>Lima's</i> course, and shows the first instance of both human and satellite documentation of the same milky sea event. The satellite image combined with human documentation of this event solidifies that it really occurred. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It is an important contributor to the carbon cycle, and produces much of the world's atmospheric oxygen. <i>Pyrocystis fusiformis</i> remains a valued food source for many marine filter-feeding organisms, and constitutes a large proponent of the primary producing marine trophic level. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has also proven to be an excellent organism to use in bioassay toxicity tests.<br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44373Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T19:09:13Z<p>Millerk: /* The "Milky Sea" Phenomenon */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. The dotted line in the figure represents the <i>Lima's</i> course, and shows the first instance of both human and satellite documentation of the same milky sea event. The satellite image combined with human documentation of this event solidifies that it really occurred. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44371Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T19:08:17Z<p>Millerk: /* The "Milky Sea" Phenomenon */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. The dotted line in the figure represents the <i>Lima's</i> course, and shows the first recorded instance of both human and satellite documentation of the same milky sea event. The satellite image combined with human documentation of this event solidifies that it really occurred. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44369Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T19:07:02Z<p>Millerk: /* The "Milky Sea" Phenomenon */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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<br />
[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
<br />
The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. The dotted line in the figure represents the <i>Lima's</i> course, and shows the first recorded instance of both human and satellite documentation of the same milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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<br />
Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44359Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T19:02:12Z<p>Millerk: /* Why does Pyrocystis fusiformis Bioluminesce? */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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<br />
QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
<br />
==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
<br />
[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
<br />
[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
<br />
[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44354Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:59:58Z<p>Millerk: /* Why does Pyrocystis fusiformis Bioluminesce? */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed, because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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<br />
[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
<br />
==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
<br />
[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
<br />
[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44352Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:58:08Z<p>Millerk: /* Why does Pyrocystis fusiformis Bioluminesce? */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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A study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44351Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:57:31Z<p>Millerk: /* Why does Pyrocystis fusiformis Bioluminesce? */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44350Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:56:03Z<p>Millerk: /* Introduction */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44349Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:55:17Z<p>Millerk: /* Introduction */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen. This reaction occurs in the microsources. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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<br />
The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<br />
<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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<br />
Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
<br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44344Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:52:30Z<p>Millerk: /* Introduction */</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem by providing an essential food source. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
<br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<br />
<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
<br />
<br />
Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
<br />
<br />
QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
<br />
<br />
[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
<br />
<br />
Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
<br />
==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
<br />
[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
<br />
[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
<br />
[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
<br />
[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
<br />
[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
<br />
[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44343Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:51:55Z<p>Millerk: /* Introduction */</p>
<hr />
<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeding organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
<br />
<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
<br />
==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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<br />
One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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<br />
Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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<br />
The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
<br />
==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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<br />
The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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<br />
In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<br />
<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
<br />
<br />
[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
<br />
The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
<br />
<br />
QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
<br />
<br />
[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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<br />
Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
<br />
==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
<br />
[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
<br />
[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
<br />
[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
<br />
[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
<br />
[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44341Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T18:51:16Z<p>Millerk: /* Introduction */</p>
<hr />
<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeder organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
<br />
==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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<br />
One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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<br />
The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
<br />
==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
<br />
<br />
The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
<br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
<br />
==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
<br />
<br />
[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
<br />
==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
<br />
==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
<br />
[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
<br />
[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
<br />
[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
<br />
[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
<br />
[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
<br />
[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
<br />
[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
<br />
Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Project_Index_2009&diff=44208BIOL 238 Project Index 20092009-05-01T16:29:34Z<p>Millerk: /* Title and Author */</p>
<hr />
<div>== Title and Author ==<br />
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<br> [[The Effect of Acetylation and Deacetylation in Post-translational Regulation in Bacteria]]<br />
<br> Sarah Cook<br />
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<br> [[Community-Acquired Methicillin-Resistant Staphylococcus Aureus (CA-MRSA)]]<br />
<br> Tom Hardacker<br />
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<br> [[Pharmacokinetics of botulinum neurotoxin serotypes: Implications for Infant Botulism]]<br />
<br> Sarah Hall<br />
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<br> [[Bacteroides Influence on Host Behavior]]<br />
<br>Hannah Regan<br />
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<br> [[Dental Caries Prevention by Camellia sinensis]]<br />
<br> Mary Barker<br />
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<br> J.T. Knight [[(+) Sense RNA Virus: West Nile Virus]]<br />
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<br> [[Application of Wolbachia in Invertebrate Vector Control]]<br />
<br> Chinagozi Ugwu <br />
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<br> [[Microbial Infection of Burn Wounds]]<br />
<br> Erin Pienciak<br />
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<br>[[Evolution of Thermophilic Archaea]].<br />
<br> Julia DeNiro<br />
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<br> [[Snottites]]<br />
<br> Paige Roberts<br />
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<br>[[Chemotrophy Along Seafloor Hydrothermal Vents]]<br />
<br> Pamela Moriarty<br />
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<br> [[Origins of a Homochiral Microbial World]]<br />
<br> Maggie Taylor<br />
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<br> Charlie Halsted<br />
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<br> [[Chlorobium FMO antenna complex characterisation]]<br />
<br> Khalid Eldahan<br />
<br />
<br> [[Prion Propagation]]<br />
<br> Kenny Farabaugh<br />
<br />
<br> [[Dental Plaque Biofilms]]<br />
<br> Anna Frutiger<br />
<br />
<br> [[Hospital-acquired Methicillin Resistant Staphylococcus Aureus (MRSA)]]<br />
<br> Anthony Alexander<br />
<br />
<br> [[Diagnosis and Prevention of Neisseria meningitides Induced Meningitis ]]<br />
<br> Emily Staudenmaier<br />
<br />
<br> [[Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi]]<br />
<br> Kim Miller <br />
<br />
<br> [[Feline Immunodeficiency virus]] <br />
<br> Emily Nutt<br />
<br />
<br> [[Trypanosome Life Cycle]]<br />
<br> Rex Rodriguez<br />
<br />
<br> [[Potential Therapeutics Isolated from Salinispora]]<br />
<br> Christina Kucher<br />
<br />
<br> [[Host Dependency of Mycobacterium leprae]]<br />
<br> Jack Hornick<br />
<br />
<br> [[Adeno-Associated Viruses as Gene Therapy Vectors]]<br />
<br> Chad Kurylo<br />
<br />
<br> [[Glycylcycline Antibiotics]]<br />
<br> Kristina Buschur</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Bioluminescence_in_Pyrocystis_fusiformis_and_Vibrio_harveyi&diff=44206Bioluminescence in Pyrocystis fusiformis and Vibrio harveyi2009-05-01T16:28:42Z<p>Millerk: New page: ==Introduction== [[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Do...</p>
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<div>==Introduction==<br />
[[Image:Dinos.jpg|thumb|400px|right|<i>Pyrocystis fusiformis</i> [http://www.lifesci.ucsb.edu/%7Ebiolum/organism/pictures/dinos.html picture 1] picture taken by Martin Dohm]]<br />
<br> <i>Pyrocystis fusiformis</i> is a marine dinoflagellate. Dinoflagellates are marine unicellular planktonic organisms. A few species are found in freshwater environments, however 90% of dinoflagellate species are marine. These organisms are found throughout the world’s oceans, concentrating at the top euphotic zone of the ocean’s water column [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.].Dinoflagellates can perform photosynthetic metabolism, heterotrophic metabolism, or both. <i>P. fusiformis</i> is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. As photosynthesizing organisms, dinoflagellates produce a substantial amount of the world’s oxygen, and consume a large proportion of the atmosphere’s carbon dioxide. Dinoflagellates can be found in large numbers in the ocean, and as a result can consume a considerable amount of carbon dioxide. Their consumption of carbon dioxide creates a major carbon sink in the carbon cycle [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. This carbon sink is crucial for the function of the global carbon cycle. Dinoflagellates are also important in marine food webs and ecosystems. Dinoflagellates consume other planktonic species, as well as provide a food source for marine filter-feeder organisms such as fish, whale sharks, and baleen whales. Dinoflagellates contribute to the producer trophic level of the marine food web, and help to maintain the diversity of marine organisms seen in the marine ecosystem. Some photosynthetic dinoflagellate species live as endosymbionts in marine invertebrates such as sponges and corals [http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8.].<br />
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<br>''P. fusiformis'' produces bioluminescence on a circadian rhythm, meaning that it photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] <i>P. fusiformis'</i> bioluminescence, or emitted blue-green light, originates from microsources found evenly distributed throughout the cytoplasmic layer surrounding the large central vacuole. A single <i>P. fusiformis</i> cell contains an average of 4,500 microsources. During the day, the microsources migrate from the cell’s periphery to a spherical region distal to the nucleus. During their migration from the periphery, they are replaced by chloroplasts. This results in a lack of daytime bioluminescence. The microsources return to the periphery at night, and produce bioluminescence [http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7.] Microsources are composed of a round mass of vesicles which contain electron-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Their high electron density allows the microsources to produce bioluminescence [http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6.]. <br><br />
[[Image:Latz_p1.jpg|thumb|400px|left| [http://curiousexpeditions.org/wp-content/uploads/2008/03/latz_p1.jpg picture 2] Researcher Mike Latz swirls <i>Pyrocystis fusiformis</i> specimens in an Erlenmeyer flask]]<br />
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==Why does <i>Pyrocystis fusiformis</i> Bioluminesce?==<br />
<br>According to the ‘bulgar alarm theory’, <i>Pyrocystis fusiformis</i> bioluminesces in order to attract attention to its predator. When attacked by a predator, <i>P. fusiformis</i> bioluminesces and illuminates itself as well as its predator. When its predator is illuminated, it greatly increases the chance that the predator itself will be preyed upon. This benefits the <i>P. fusiformis</i> species as a whole, because the dinoflagellates tend to exist in groups. The chance that <i>P. fusiformis’</i> predator will be seen and eaten as a result of bioluminescence is greater than the chance that the entire <i>P. fusiformis</i> colony will be preyed upon, which favors the survival of the <i>P. fusiformis</i> group as well as the luminescent genotype. <br />
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One study by [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Fleisher and Case (1995) 1] supports the ‘buglar alarm theory’. The study shows that two species of squid (<i>Sepia officinalis</i> and <i>Euprymna scolopes</i>) use <i>P. fusiformis</i> bioluminescence to locate and capture nonluminous prey. <i>Holmesimysis sculpta</i> (mysids), <i>Palaemonetes pugio</i> (grass shrimp), and <i>Gambusia affinis</i> (mosquito fish) prey upon <i>P. fusiformis</i>, and were used as squid prey in the study. The squid prey, <i>P. fusiformis</i>, and the squid were placed in dark tanks. The amount of squid prey consumed was measured with and without the presence of <i>P. fusiformis</i>. The interaction was viewed using infrared video cameras. The predation of the three types of squid prey on <i>P. fusiformis</i> caused <i>P. fusiformis</i> to bioluminesce, allowing for easier prey capture by the squid because their prey was illuminated. Mosquito fish were observed to trigger luminescence with each tail stroke, which the squid monitored closely. Grass shrimp appendages also triggered luminescence, and the attention of the squid predators. The average number of mysids, grass shrimp, and mosquito fish consumed by the two species of squid significantly increased with increasing <i>P. fusiformis</i> concentration in the experimental tank.<br />
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[[Image:Figure_4.png|thumb|400px|left| [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E Figure 1.] Average number of mysids consumed by the squid species <i>Sepia officinalis</i> as a function of concentration of luminescent dinoflagellates <i>Pyrocystis fusiformis</i>. Error bars represent standard errors. ]]<br />
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Figure 1 shows that the presence of <i>P. fusiformis</i> in the experimental tanks significantly increased the number of mysid individuals that were consumed by the squid. Higher concentrations of <i>P. fusiformis</i> in the tanks resulted in significantly more mysid individuals consumed because higher concentrations of <i>P. fusiformis</i> led to increased illumination of the mysids. Thus, predation of squid on nonluminescent prey in the dark is positively correlated with the presence of bioluminescent <i>P. fusiformis</i> and supports the ‘buglar alarm theory’.<br />
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The presence of <i>P. fusiformis</i> also allowed the squid to capture nonluminescent prey at a quicker rate. In the study, 11 squid in the presence of luminescent <i>P. fusiformis</i> took less than 10 minutes to capture prey, and all prey was consumed within 20 minutes. In the absence of <i>P. fusiformis</i> only one nonluminescent prey individual was consumed, and this attack occurred after 30 minutes. Thus, the presence of luminescent <i>P. fusiformis</i> greatly increased predation on nonluminescent squid prey. This study shows that cephalopods are able to use the light of dinoflagellates such as <i>P. fusiformis</i> to locate their nonluminescent prey [http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1].<br />
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==The "Milky Sea" Phenomenon==<br />
<br>On many occasions over the centuries, mariners have testified to witnessing unusual nocturnal ocean displays where the surface of the ocean produces an intense glow that seems to extend in all directions for many miles. This phenomenon has been labeled the ‘milky seas’ phenomenon, and little is known about its formation mechanisms, spatial extent, global distribution, and ecological implications. The sparse information that is known about this effect is derived almost entirely from archived ship logs, and this information is subject to error resulting from human perception and interpretation. The information known results from 235 documented cases reported since 1915. Based on this information, milky seas are known to occur only at night, glow continuously over an extensive area, are independent of wind speeds, last anywhere from several hours to several days, and may be associated with oceanographic fronts or biological slicks. More than 70% (171 cases) of documented milky seas were observed in the northwest Indian Ocean, most commonly during the summer southwest monsoon. These observation statistics seem to be biased towards active shipping routes, however reports of milky seas from other heavily used shipping routes are exceedingly infrequent [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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The ‘milky seas’ phenomenon was initially hypothesized to be a result of large aggregations of <i>Pyrocystis fusiformis</i> individuals producing bioluminescence. This hypothesis has been discounted, however, due to the fact that <i>P. fusiformis</i> emits brief, bright flashes in response to mechanical disturbance. It seems unlikely that brief flashes, even if repeated, could produce the ‘milky sea’ effect. It also seems unlikely that a sustained uniform mechanical stimulation exists that would allow <i>P. fusiformis</i> to cause the ‘milky sea’ phenomenon. A more likely hypothesis explaining the ‘milky sea’ phenomenon states that luminous bacteria such as <i>Vibrio harveyi</i> could be the source of emitted light because they can emit a continuous glow that can persist for days under specific conditions. This hypothesis suggests that the ‘milky sea’ effect results from bioluminescent bacteria living in microalgal blooms in the surface waters of the ocean. A study by [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Miller et al. (2005)] supports the second hypothesis, and details the first satellite observations of the ‘milky seas’ phenomenon. <br />
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In the study, a 15,400-km squared area of the northwestern Indian Ocean, roughly the size of Connecticut, was observed to glow over 3 consecutive nights. Measurements from the U.S. Defense Meteorological Satellite Program constellation of satellites were used that detected low-light emissions picked up from the area where the milky sea was observed by a passing British merchant ship, the SS <i>Lima</i> on January 25th, 1995. The ship’s log states,<br />
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<i>“25, January 1995. At 1800 [GMT] (2100 local time) on a clear moonless night while 150 nautical miles east of the Somalian coast, a whitish glow was observed on the horizon, and after 15 minutes of steaming, the ship was completely surrounded by a sea of milky-white color with fairly uniform luminescence. The bioluminescence appeared to cover the entire sea area, from horizon to horizon…and it appeared as though the ship was sailing over a field of snow or gliding over the clouds…thick patches of kelp appeared black against the white water. [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]”</i><br />
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Unprocessed OLS nighttime visible satellite imagery from approximately one-half hour into the <i>Lima’s</i> encounter with the milky sea indicates the presence of a large, bright feature near the <i>Lima’s</i> location, which was observed to persist over the next two nights. Positions of the <i>Lima</i> reported over the course of its encounter coincide closely with the boundaries of the satellite-observed bright feature. This occurrence constitutes the first time a milky sea has been recorded by both human visual and satellite observation. Miller et al. explain that this milky sea most likely resulted from <i>Vibrio harvei</i> living in association with a microalga <i>Phaeocystis</i> bloom, as the <i>Lima</i> observed patches of “kelp” in the milky sea. The area where the milky sea was observed is known to be a preferred habitat for phytoplankton colonies and an area where algae blooms often occur. The researchers also discovered the presence of a cold-core eddy where the milky sea was observed, which most likely contributed to maintaining the conditions necessary to keep the algae bloom in place and allowed the milky sea to occur [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3]. <br />
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[[Image:Figure_1.jpg|thumb|500px|right| [http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P Figure 2.] Study areas (top) corresponding to unfiltered (A-C) and filtered (D-F) satellite imagery on the night of the SS <i>Lima</i> observations. (A and D) Jan. 25, 1995, 1836 GMT. (B and E) Jan. 26, 1995, 1804 GMT. (C and F) Jan. 27, 1995 1725 GMT. Arrowheads in F indicate low signal-to-noise ratio artifacts. Shown in D are the ship track (dashed line) and positions at time of first sighting on the horizon (point a) and exit from the glowing waters (point b), based on details of the ship report. ]]<br />
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The researchers explain that the emitted light of the milky sea was produced by high concentrations of <i>Vibrio harveyi</i> participating in quorum sensing. <i>V. harveyi</i> only bioluminesce in high cell density, and use quorum sensing to determine if there is a high enough concentration of other individuals of its species in order to do so. The <i>lux</i> system mediates <i>V. harveyi’s</i> bioluminescence. In this system, the LuxI protein synthesizes an acyl homoserine lactone autoinducer (AI). AI diffuses into the surrounding enviroment, where it accumulates. When it reaches a high enough concentration, AI diffuses back into the <i>V. harveyi</i> cell and binds to the activator protein LuxR, resulting in increased transcription of the lux operon. The lux operon activates transcription of the luciferase target genes that allow <i>V. harveyi</i> to bioluminesce [http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5]. In this system, the more <i>V. harveyi</i> individuals that are producing AI, the more the individuals will bioluminesce. Figure 2 displays the satellite image of the milky sea event. <br />
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<i>V. harveyi</i> individuals are able to quorum sense and bioluminesce during a milky sea event because they aggregate with algal blooms. The microalga bloom seen in the milky sea event in 1995 allowed the bacteria to attach to a solid substrate, which aided the bacteria’s quorum sensing because it kept the bacteria close together and allowed them to sense the AI concentration. Miller <i>et al.</i> (2005) hypothesize that algal blooms may be a necessary component of milky sea events because they allow the bacteria to maintain high enough levels in order to bioluminesce. Luminous bacteria associate with algal species in the ocean in order to gain nutrients from material produced by the algae. The bacteria grow on the algae in colonies, producing and accumulating AI and ultimately emitting light. When algal blooms occur, the bacteria also bloom due to the increase in nutrients. Milky seas occur as large algal blooms begin to break down and as massive amounts of decomposing lipids and hydrocarbon-rich microbial material accumulate as a surface film, where the growth of a luminous bacterial species is favored. Confined to the film, where nutrients from the decomposing algae accumulate, AI accumulates and luminescene is induced [http://aem.asm.org/cgi/content/full/72/4/2295 4]. <br />
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Milky seas remain a lesser studied phenomenon. The use of satellite imaging will hopefully allow researchers to discover more about milky sea formation mechanisms, global distributions, and ecological implications.<br />
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==Industrial Use for Bioluminescence in <i>Pyrocystis fusiformis</i>==<br />
<br><i>Pyrocystis fusiformis</i> is considered a useful organism industrially because of its use in bioassays. Phytoplankton bioassays are currently used as biological tools to determine the extent of contamination in a given area. Phytoplankton are useful test organisms in bioassays because they are simple and inexpensive organisms in comparison to fish and vertebrate species. In 1989, a bioluminescent dinoflagellate bioassay termed “QwikLite” was developed using <i>P. fusiformis</i>. The bioassay measured the light output from bioluminescent dinoflagellates to assess acute and sublethal toxic effects. QwikLite specifically aided in the testing for toxicity at naval facilities. Most naval facilities are located within harbors and bays and therefore have direct impacts on marine flora and fauna, particularly plankton. The development of the dinoflagellate bioassay allowed for the identification of toxins in the area near the naval base in both the water and sediment within a few days, while other bioassay tests need several weeks to determine the outcome of the tests. <br />
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QwikLite was compared with other conventional toxicity tests using minnows and shrimp to assess storm water outfalls and industrial wastewater treatment plant effluent collected at the U.S. Naval Shipyard, Norfolk Virginia [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. Water samples were collected from various storm water outfalls as well as dry dock outfalls during storm events from September through June 1996. <i>P. fusiformis</i> was placed in solution containing a portion of the collected water samples, and put in a Laboratory Plankton Test Chamber (LPTC). The LPTC measured light output from <i>P. fusiformis</i>, and its readings allowed researchers to determine the toxicity of the sample. <i>P. fusiformis</i> lost its ability to bioluminesce following exposure to heavy metals, organic compounds such as ammonia and polycyclic aromatic hydrocarbons, and sediments bound with metals and organics. The QuikLite bioassay proved to be an effective and sensitive indicator of toxicity in contaminated waters and sediments. <br />
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[[Image:Table_3a.jpg|thumb|500px|right|[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 Table 1.] Norfolk Naval Shipyard storm event monitoring toxicity test results]]<br />
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Table 1 shows the concentration of test material that caused a 50% reduction or inhibition of bioluminescence in the QwikLite bioassay (IC50), and a 50% reduction of test individuals in the other bioassays used (LC50). The first sample in the table displayed toxicity, while the other samples did not. The first sample caused a 50% reduction in bioluminescence when <i>P. fusiformis</i> was treated with 45% of the test material, and caused a 50% reduction in shrimp with 48% of the test material. The other samples did not contain toxins because it took more than the entire sample to reach a 50% reduction in bioluminescence as well as the other bioassay test organisms [http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2]. The data indicates that QuikLite can be used as an bioassay and yield accurate results. <i>Pyrocystis fusiformis</i> can be used in bioassays to provide a fast and sensitive assessment of present environmental conditions.<br />
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==Conclusion==<br />
<br><i>Pyrocystis fusiformis</i> is an interesting marine dinoflagellate that produces bioluminescence. It was thought to be the cause of the “milky sea” phenomenon, but this theory has been discredited. <i>Pyrocystis fusiformis</i> has proven to be an excellent organism to use in bioassay toxicity tests. <br />
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==References==<br />
<br>[http://www.jstor.org/sici?sici=0006-3185%281995%29189%3A3%3C263%3ACPFBDL%3E2.0.CO%3B2-E 1. Fleisher, K.J. and J.F. Case. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biological Bulletin 189: 263-271]<br />
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[http://journals.ohiolink.edu/ejc/article.cgi?issn=0025326x&issue=v54i0012&article=1857_tuobdaaerat&search_term=%28refkey%3D%28Lapota%232007%231857%231867%23D%29volkey%3D%280025326x%2354%231857%2312%29%29 2. Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. 2007. The use of bioluminescent dinoflagellates as an environmental risk assessment tool. Marine Pollution Bulletin 54: 1857-1867]<br />
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[http://www.jstor.org/sici?sici=0027-8424%282005%29102%3A40%3C14181%3ADOABMS%3E2.0.CO%3B2-P 3. Miller, S.D., S.H.D. Haddock, C.D. Elvidge, and T.F. Lee. 2005. Detection of a bioluminescent milky sea from space. Proceedings of the National Academy of Sciences 102: 14181-14184.]<br />
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[http://aem.asm.org/cgi/content/full/72/4/2295 4. Nealson, K.H. and J.W. Hastings. 2006. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Applied and Environmental Microbiology 72:2295-2297]<br />
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[http://biology.kenyon.edu/slonc/Micro/D489Mbio.pdf 5. Slonczewski, Joan, and John W. Foster. Microbiology an Evolving Science. 1st ed. New York: W.W. Norton and Company, Inc., 2009.]<br />
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[http://www3.interscience.wiley.com/journal/119558442/abstract?CRETRY=1&SRETRY=0 6. Sweeney, B.M. 2004. Microsources of bioluminescence in <i>Pyrocystis fusiformis</i> (Pyrrophyta). Journal of Phycology 18:412-416]<br />
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[http://www.jstor.org/sici?sici=0006-3185%281982%29162%3A3%3C423%3ADOSBSI%3E2.0.CO%3B2-Q 7. Widder, E.A. J.F. Case. 1982. Distribution of subcellular bioluminescent sources in a dinoflagellate <i>Pyrocystis fusiformis</i>. Biological Bulletin 162:423-448.] <br />
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[http://www.ucmp.berkeley.edu/protista/dinoflagellata.html 8. Williams, G. and A. MacRae. 1998. Introduction to the Dinoflagellata. www.ucmp.berkeley.edu (May 1st, 2009).]<br />
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Written by student Kim Miller<br />
Edited by student of [mailto:slonczewski@kenyon.edu Joan Slonczewski] for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238 Microbiology], 2009, [http://www.kenyon.edu/index.xml Kenyon College].</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Talk:Feline_Immunodeficiency_virus&diff=43022Talk:Feline Immunodeficiency virus2009-04-20T23:29:35Z<p>Millerk: </p>
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<div>You provide a very comprehensive and interesting discussion of FIV. After reading it, though, I did have some questions. When was the genome of FIV sequenced? Are there any implications in new sequencing technologies today? The idea that FIV can mutate between species, however, is so intriguing! Again, when did all these discoveries occur? Is it work that is funded today? I think you can include all this information to make the topic more applicable to today's readers. Also, it would be cool if you could expand your conclusion a bit--find out what research is being done to apply these discoveries to the development of drugs that can affect all immunodeficiency viruses!<br />
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General<br />
<br>Overall it’s very good and thorough. A few things could be explained in more detail- think as if someone was reading this who didn’t have a good background in molecular biology and genomics. I really like that you include information from a variety of species of cats, as opposed to just the domestic cat. There are a few typos- just some commas needed here and there. I suggest reading it outloud and seeing if the sentences make sense. <br />
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<br>Introduction<br />
<br>Once the capsid has been uncoated the RNA is converted to double-stranded DNA through the reverse transcriptase.<br />
<br>-stick a comma in after “uncoated”<br />
<br>Picture caption- “This was a photo ever taken of the virus, by Michael Podell, at Ohio State University.” Not sure what you’re trying to say here- first photo ever taken?<br />
<br> “At first the viral genome is able to replicate slowly only creating a few virions and thus not directly affecting the host.”<br />
<br>-comma after “slowly”<br />
<br> “After sometime the host cell can then generate a large amount of virions, thus causing the host to show symptoms of immunodeficiency.”<br />
<br>-separate “sometime” into “some time”<br />
<br> “The genome of FIV reveals important viral proteins and thus potential vaccine targets, as well as showing how the virus evolved..”<br />
<br>-there are two periods at the end of the sentence<br><br />
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<br> The genome of FIV<br />
<br>” Open reading frame A or orf A is induces host cell arrest at the second gap phase of the cell cylce [3]. Only gag and pol are the genes are in the primary viral genome and it is only after splicing that the other viral genes are expressed.”<br />
<br>-there are a few typos in these sentences<br />
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<br> Genomic Variation of FIV<br />
<br>” These mutations are typically occur in genes other than the pol gene”<br />
<br>-typo<br />
<br>” This gene is highly conserved in all lentiviruses because it contains the gene for reverse transcriptase a protein unique to all retroviruses [4]”<br />
<br>-I’m not sure what you’re trying to say here.<br />
<br>” For example each species of cat may have different T-cell receptors and thus the virus would have to mutate to bind to the new T-cell receptors [9]”<br />
<br> -need a comma after ‘For example’<br />
<br> “The strains of FIV would also have to mutate to avoid each species conventional immune responses.”<br />
<br>-I’m confused by this sentence as well. Do you mean to say “avoid each species’ conventional immune response” (with the ‘ after species)?<br />
<br> “The genomic variation of each strain of FIV not only reveals how FIV is able to mutate to avoid different immune systems of feline species, but it also reveals the evolution of FIV throughout felines in particular the Felidae”<br />
<br>- the end of this sentence is confusing. Maybe you just need a comma. So, it would say “but also reveals the evolution of FIV throughout felines, in particular the Felidae” or maybe say “felines, particularly the Felidae”<br />
<br><br />
<br>Evolution of feline immunodeficiency virus<br />
<br> “FIV in Puma concolor have long branching revealing that the viral divergence occurred quite a long time ago [6]. FIV in Panthera leo has the most variation revealing that the virus probably began in Africa.”<br />
<br>-you need a comma before “revealing” in both of these sentences<br />
<br> “Some strains of FIV appear to be transmitted interspecies, however for this to occur serious obstacles must be overcome.”<br />
<br>-what kinds of serious obstacles? You may want to mention a few. I see you mention them later, but they should be introduced when you first mention these issues or you need to state that they will be discussed later.<br />
<br> “Also lions incorporate biting and fighting in their everyday lifestyle, for example male lions are constantly taking over new prides.”<br />
<br>-this sentence is confusing<br />
<br>” these stains are highly divergent from the other FlV-Pco B.”<br />
<br>-should be strains instead of stains<br />
<br>” It is believed that FIV-Pco was first present in South American Puma concolor, which then re-colonized North America.”<br />
<br>-need to italicize Puma concolor<br />
<br>” However FIV-Pco has two distinct strains one that is just found in North America and one strain that is found in North, Central and South America.”<br />
<br>-confusing/awkward sentence… maybe it just needs a colon in between strains and one? <br />
<br><br />
<br>References<br />
<br>-link the references to the site- use the example website to see the format of how to link something directly<br />
<br />
<br />
<br>Kim Miller's comments:<br />
<br> -I think the scanning electron micrograph picture caption has a typo.<br />
<br> - In the "evolution of feline immunoficiency virus" section, the sentence "FIV-Fce to be greatly divergent from all other strains of FIV" doesn't make sense<br />
<br> -it is really interesting that FIV is host-specific and makes small mutations to evolve for each species of cat it infects. I did not know that.<br />
<br> -overall, this is a really interesting topic. I've heard of FIV, but I've always wanted to know more about the virus. I really liked how your conclusion linked to HIV and implications for research. Great job!</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Talk:Snottites&diff=43021Talk:Snottites2009-04-20T23:28:20Z<p>Millerk: /* Comment 1 */</p>
<hr />
<div>== Comment 1 ==<br />
<br />
I found your page on snottites particularly interesting, especially the part regarding the structural formation of the snottites as a result of their oxidation process. I find it intriguing that the gypsum is able to coat the walls and ceilings of the caves in layers that are so deep and thick. I was also curious to see the SEM images of some of the species involved. It seems that maybe some of the structure of these biofilms is not as organized as some of the other biofilm structures we have seen, which is interesting. Where in the United States can snottites be found? I have learned a lot about these interesting structures from your paper, and I think you did a very good job of organizing the information.<br />
<br />
<br />
<br> Kim Miller's comments:<br />
<br> - this is an amazing page. Good job! It is really well written and organized. <br />
<br> - I've always wanted to know more about snotties (ever since I saw Planet Earth), so I think this topic is really interesting. <br />
<br> - I thought that including the SEM images of the microbes was a great idea. I think the pictures are really interesting. <br />
<br> - I didn't notice any typos or things to change, again your page is fantastic.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Talk:Snottites&diff=43020Talk:Snottites2009-04-20T23:27:52Z<p>Millerk: /* Comment 1 */</p>
<hr />
<div>== Comment 1 ==<br />
<br />
I found your page on snottites particularly interesting, especially the part regarding the structural formation of the snottites as a result of their oxidation process. I find it intriguing that the gypsum is able to coat the walls and ceilings of the caves in layers that are so deep and thick. I was also curious to see the SEM images of some of the species involved. It seems that maybe some of the structure of these biofilms is not as organized as some of the other biofilm structures we have seen, which is interesting. Where in the United States can snottites be found? I have learned a lot about these interesting structures from your paper, and I think you did a very good job of organizing the information.<br />
<br />
<br />
Kim Miller's comments:<br />
- this is an amazing page. Good job! It is really well written and organized. <br />
- I've always wanted to know more about snotties (ever since I saw Planet Earth), so I think this topic is really interesting. <br />
- I thought that including the SEM images of the microbes was a great idea. I think the pictures are really interesting. <br />
- I didn't notice any typos or things to change, again your page is fantastic.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=Talk:Feline_Immunodeficiency_virus&diff=43017Talk:Feline Immunodeficiency virus2009-04-20T23:07:03Z<p>Millerk: </p>
<hr />
<div>You provide a very comprehensive and interesting discussion of FIV. After reading it, though, I did have some questions. When was the genome of FIV sequenced? Are there any implications in new sequencing technologies today? The idea that FIV can mutate between species, however, is so intriguing! Again, when did all these discoveries occur? Is it work that is funded today? I think you can include all this information to make the topic more applicable to today's readers. Also, it would be cool if you could expand your conclusion a bit--find out what research is being done to apply these discoveries to the development of drugs that can affect all immunodeficiency viruses!<br />
<br><br />
<br><br />
<br><br />
General<br />
<br>Overall it’s very good and thorough. A few things could be explained in more detail- think as if someone was reading this who didn’t have a good background in molecular biology and genomics. I really like that you include information from a variety of species of cats, as opposed to just the domestic cat. There are a few typos- just some commas needed here and there. I suggest reading it outloud and seeing if the sentences make sense. <br />
<br><br />
<br>Introduction<br />
<br>Once the capsid has been uncoated the RNA is converted to double-stranded DNA through the reverse transcriptase.<br />
<br>-stick a comma in after “uncoated”<br />
<br>Picture caption- “This was a photo ever taken of the virus, by Michael Podell, at Ohio State University.” Not sure what you’re trying to say here- first photo ever taken?<br />
<br> “At first the viral genome is able to replicate slowly only creating a few virions and thus not directly affecting the host.”<br />
<br>-comma after “slowly”<br />
<br> “After sometime the host cell can then generate a large amount of virions, thus causing the host to show symptoms of immunodeficiency.”<br />
<br>-separate “sometime” into “some time”<br />
<br> “The genome of FIV reveals important viral proteins and thus potential vaccine targets, as well as showing how the virus evolved..”<br />
<br>-there are two periods at the end of the sentence<br><br />
<br />
<br> The genome of FIV<br />
<br>” Open reading frame A or orf A is induces host cell arrest at the second gap phase of the cell cylce [3]. Only gag and pol are the genes are in the primary viral genome and it is only after splicing that the other viral genes are expressed.”<br />
<br>-there are a few typos in these sentences<br />
<br><br />
<br> Genomic Variation of FIV<br />
<br>” These mutations are typically occur in genes other than the pol gene”<br />
<br>-typo<br />
<br>” This gene is highly conserved in all lentiviruses because it contains the gene for reverse transcriptase a protein unique to all retroviruses [4]”<br />
<br>-I’m not sure what you’re trying to say here.<br />
<br>” For example each species of cat may have different T-cell receptors and thus the virus would have to mutate to bind to the new T-cell receptors [9]”<br />
<br> -need a comma after ‘For example’<br />
<br> “The strains of FIV would also have to mutate to avoid each species conventional immune responses.”<br />
<br>-I’m confused by this sentence as well. Do you mean to say “avoid each species’ conventional immune response” (with the ‘ after species)?<br />
<br> “The genomic variation of each strain of FIV not only reveals how FIV is able to mutate to avoid different immune systems of feline species, but it also reveals the evolution of FIV throughout felines in particular the Felidae”<br />
<br>- the end of this sentence is confusing. Maybe you just need a comma. So, it would say “but also reveals the evolution of FIV throughout felines, in particular the Felidae” or maybe say “felines, particularly the Felidae”<br />
<br><br />
<br>Evolution of feline immunodeficiency virus<br />
<br> “FIV in Puma concolor have long branching revealing that the viral divergence occurred quite a long time ago [6]. FIV in Panthera leo has the most variation revealing that the virus probably began in Africa.”<br />
<br>-you need a comma before “revealing” in both of these sentences<br />
<br> “Some strains of FIV appear to be transmitted interspecies, however for this to occur serious obstacles must be overcome.”<br />
<br>-what kinds of serious obstacles? You may want to mention a few. I see you mention them later, but they should be introduced when you first mention these issues or you need to state that they will be discussed later.<br />
<br> “Also lions incorporate biting and fighting in their everyday lifestyle, for example male lions are constantly taking over new prides.”<br />
<br>-this sentence is confusing<br />
<br>” these stains are highly divergent from the other FlV-Pco B.”<br />
<br>-should be strains instead of stains<br />
<br>” It is believed that FIV-Pco was first present in South American Puma concolor, which then re-colonized North America.”<br />
<br>-need to italicize Puma concolor<br />
<br>” However FIV-Pco has two distinct strains one that is just found in North America and one strain that is found in North, Central and South America.”<br />
<br>-confusing/awkward sentence… maybe it just needs a colon in between strains and one? <br />
<br><br />
<br>References<br />
<br>-link the references to the site- use the example website to see the format of how to link something directly<br />
<br />
<br />
<br>Kim Miller's comments:<br />
<br> -I think the scanning electron micrograph picture caption has a typo.<br />
<br> -Table 2 is a little hard to read, could you make it a bit bigger?<br />
<br> - In the "evolution of feline immunoficiency virus" section, the sentence "FIV-Fce to be greatly divergent from all other strains of FIV" doesn't make sense<br />
<br> -it is really interesting that FIV is host-specific and makes small mutations to evolve for each species of cat it infects. I did not know that.<br />
<br> -overall, this is a really interesting topic. I've heard of FIV, but I've always wanted to know more about the virus. I really liked how your conclusion linked to HIV and implications for research. Great job!</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Table_3a.jpg&diff=41735File:Table 3a.jpg2009-04-15T21:33:02Z<p>Millerk: </p>
<hr />
<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Figure_1.jpg&diff=41725File:Figure 1.jpg2009-04-15T21:21:57Z<p>Millerk: </p>
<hr />
<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Figure_4.png&diff=41703File:Figure 4.png2009-04-15T21:05:20Z<p>Millerk: </p>
<hr />
<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Latz_p1.jpg&diff=40816File:Latz p1.jpg2009-04-14T00:54:42Z<p>Millerk: </p>
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<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Dinos.jpg&diff=40811File:Dinos.jpg2009-04-14T00:51:40Z<p>Millerk: </p>
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<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=File:Bluebl1.jpg&diff=40781File:Bluebl1.jpg2009-04-14T00:33:04Z<p>Millerk: </p>
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<div></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Project_Index_2009&diff=40765BIOL 238 Project Index 20092009-04-14T00:20:11Z<p>Millerk: /* Title and Author */</p>
<hr />
<div>== Title and Author ==<br />
<br />
<br> J.T. Knight [[(+) Sense RNA Virus: West Nile Virus]]<br />
<br />
<br> [[Microbial Infection of Burn Wounds]]<br />
<br> Erin Pienciak<br />
<br />
<br>[[Evolution of Thermophilic Archaea]].<br />
<br> Julia DeNiro<br />
<br />
<br> [[Snottites]]<br />
<br> Paige Roberts<br />
<br />
<br>[[Chemotrophy Along Seafloor Hydrothermal Vents]]<br />
<br> Pamela Moriarty<br />
<br />
<br> [[Origins of a Homochiral Microbial World]]<br />
<br> Maggie Taylor<br />
<br />
<br> [[Antibacterial Surfaces]]<br />
<br> Charlie Halsted<br />
<br />
<br> [[Chlorobium FMO antenna complex characterisation]]<br />
<br> Khalid Eldahan<br />
<br />
<br> [[Prion Transmission]]<br />
<br> Kenny Farabaugh<br />
<br />
<br> [[Dental Plaque Biofilms]]<br />
<br> Anna Frutiger<br />
<br />
<br> [[Hospital-acquired Methicillin Resistant Staphylococcus Aureus (MRSA)]]<br />
<br> Anthony Alexander<br />
<br />
<br> [[Diagnosis and Prevention of Neisseria meningitides Induced Meningitis ]]<br />
<br> Emily Staudenmaier<br />
<br />
<br> [[Bioluminescence in pyrocystis fusiformis]]<br />
<br> Kim Miller</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Project_Index_2009&diff=40762BIOL 238 Project Index 20092009-04-14T00:14:59Z<p>Millerk: </p>
<hr />
<div>== Title and Author ==<br />
<br />
<br> J.T. Knight [[(+) Sense RNA Virus: West Nile Virus]]<br />
<br />
<br> [[Microbial Infection of Burn Wounds]]<br />
<br> Erin Pienciak<br />
<br />
<br>[[Evolution of Thermophilic Archaea]].<br />
<br> Julia DeNiro<br />
<br />
<br> [[Snottites]]<br />
<br> Paige Roberts<br />
<br />
<br>[[Chemotrophy Along Seafloor Hydrothermal Vents]]<br />
<br> Pamela Moriarty<br />
<br />
<br> [[Origins of a Homochiral Microbial World]]<br />
<br> Maggie Taylor<br />
<br />
<br> [[Antibacterial Surfaces]]<br />
<br> Charlie Halsted<br />
<br />
<br> [[Chlorobium FMO antenna complex characterisation]]<br />
<br> Khalid Eldahan<br />
<br />
<br> [[Prion Transmission]]<br />
<br> Kenny Farabaugh<br />
<br />
<br> [[Dental Plaque Biofilms]]<br />
<br> Anna Frutiger<br />
<br />
<br> [[Hospital-acquired Methicillin Resistant Staphylococcus Aureus (MRSA)]]<br />
<br> Anthony Alexander<br />
<br />
<br> [[Diagnosis and Prevention of Neisseria meningitides Induced Meningitis ]]<br />
<br> Emily Staudenmaier<br />
<br />
<br> [[Bioluminescence in Pyrocystis fusiformis]]<br />
<br> Kim Miller</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40458BIOL 238 Review 20092009-04-01T01:40:11Z<p>Millerk: /* Chapter 15 */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
<br><br><br />
<br />
Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
<br />
<br><br><br />
<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
<br><br><br />
<br />
In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
<br />
<br><br><br />
<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
<br><br><br />
<br />
Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
<br />
<br><br><br />
<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
<br><br><br />
<br />
A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
<br />
<br><br><br />
<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
<br><br><br />
<br />
Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
<br />
<br><br><br />
<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br />
The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
<br />
All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
<br />
Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br />
There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br />
Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br />
The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
<br />
<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br> <br />
<br />
The four types of control for gene expression are alterations of DNA sequence, control of transcription and mRNA stability, translational control and posttranslational control. Alterations of DNA sequence is when microbes program mutation, one way this happens is through phase variation or flipping of a DNA segment to turn off or on a gene. This method is the most irreversible and is the slowest to revert. Control of Transcription includes proteins binding DNA and inhibiting RNA polymerase or bending DNA at the promoter. This type of control is more reversible and takes less time than DNA segment but not as fast as translational and post translational. Translational method occurs when the ribosome is inhibited, for example attenuation. Post translational is the fastest where the made proteins are modified. <br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br />
Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br />
The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
NADH carries 2 or 3 times as much energy as ATP, depending on cell conditions. In high-energy reactions, NADH is used. NAD+/NADH accept and donate electrons, therefore if a cell needs to balance its electrons NADH is used in the reaction. NADH is also useful when a substrate needs to be reduced. ATP is used when less energy than NADH is required to fuel the reaction. ATP is formed when a reaction can provide enough energy to form ATP from ADP, but not enough energy to form NADH. Some reactions need a redox change. Always need to balance the redox levels in a cell. <br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
There are large quantities of starting materials in the soil (ex: landfill and methanogenesis) so rxn proceeds even though delta G is near zero. Polymers such as lignin exist in the soil, which take a long time to break down. Breaking down lignin has a small delta G b/c lignin is a stable, aromatic molecule. A lot of energy is required to break it down, so not much energy is produced at the end of the reaction, yielding a small delta G. <br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
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<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
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<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
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<br />
<br />
<br><br><br />
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<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
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<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
It seems that it would be difficult for an organism to switch between lithotrophy and organotrophy unless the organism is switching between an environment where organic and inorganic materials are present. If the organism contained both sets of enzymes that were required, then it would be necessary to use these enzymes as to avoid reductive evolution and lose the ability to use both lithotrophy and organotrophy. <br />
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<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
<br />
<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
Fatty acid biosynthesis is a cyclic cycle using acetyl-CoA with rounds of the same synthesis over and over again. The cycle feeds its products back repeatedly as substrates for further synthesis, so large polymers can be made using a limited number of enzymes. This pathway requires fewer enzymes in comparison to AA biosynthesis because it is a smaller pathway. Amino acid biosynthesis substrates are derived from the glycolysis pathways and the TCA cycle. AA biosynthesis requires more reduction because there are many different pathways used to form AA’s, while there is a single pathway to form fatty acids. AA biosynthesis requires also requires a greater number of different enzymes due to the many different pathways used to form AA’s. AA’s are complex, asymmetrical molecules that require many different conversions to synthesize. Each conversion is mediated by a different enzyme, therefore AA biosynthesis requires a greater number of different enzymes in comparison to fatty acid biosynthesis. <br />
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<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
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The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br />
<br />
<br />
Nitrogen sources include: organic amines (NH3→ NH4+), N2 gas from the atmosphere, and NO3- (nitrate, which is then made into ammonia). The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). Most bacteria can acquire nitrate (NO3-) or nitrite (NO2-) for reduction to ammonium ion, although they repress these E-expensive pathways when ammonium ion is present. While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated. Worldwide, factories fix enormous amts of N2 into ammonia for agricultural fertilizers. Industrial nitrogen fixation uses the Haber process, in which N gas is hydrogenated by methane under extreme heat and pressure. When nitrogen fertilizer is used, much of it is washed from the soil into streams when it rains. The streams take the nitrogen fertilizer to the oceans, where marine phytoplankton phototrophs fix the nitrogen and overgrow, causing algal blooms. The massive amounts of algal phototrophs eventually die, and sink to the bottom layers of the ocean. They deplete the oxygen available there by decomposing, causing eutrophication, where the lower layers of a body of water are depleted of oxyen as a result of overgrowth of microbial producers. The lack of oxygen causes a “dead zone” where no other aerobic organisms can survive. This has been a really big problem because it essential eliminates marine habitats, and has killed many marine organisms. <br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
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<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<br />
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<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
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<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
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<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
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<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
<br />
<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
<br />
<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
<br />
<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
<br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
<br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
<br><br><br />
<b><i>Sinorhizobium meliloti</i></b><br />
<br><br><br />
<b><i>Staphylococcus epidermidis</i></b><br />
<br><br><br />
<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
<br />
<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40457BIOL 238 Review 20092009-04-01T01:39:45Z<p>Millerk: /* Chapter 15 */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
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The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
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All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
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This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
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<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
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Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
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<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
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Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
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<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
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There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
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<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
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Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
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<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
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Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
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==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
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The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
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<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
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<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
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<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
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Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
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<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
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If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
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<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
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<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
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Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
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<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
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The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
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<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
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The four types of control for gene expression are alterations of DNA sequence, control of transcription and mRNA stability, translational control and posttranslational control. Alterations of DNA sequence is when microbes program mutation, one way this happens is through phase variation or flipping of a DNA segment to turn off or on a gene. This method is the most irreversible and is the slowest to revert. Control of Transcription includes proteins binding DNA and inhibiting RNA polymerase or bending DNA at the promoter. This type of control is more reversible and takes less time than DNA segment but not as fast as translational and post translational. Translational method occurs when the ribosome is inhibited, for example attenuation. Post translational is the fastest where the made proteins are modified. <br />
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<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
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Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
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<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
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Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
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<b>3. Where in the body do biofilms form infections? Why?</b><br />
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The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
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<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
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Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
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<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
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<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
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==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
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NADH carries 2 or 3 times as much energy as ATP, depending on cell conditions. In high-energy reactions, NADH is used. NAD+/NADH accept and donate electrons, therefore if a cell needs to balance its electrons NADH is used in the reaction. NADH is also useful when a substrate needs to be reduced. ATP is used when less energy than NADH is required to fuel the reaction. ATP is formed when a reaction can provide enough energy to form ATP from ADP, but not enough energy to form NADH. Some reactions need a redox change. Always need to balance the redox levels in a cell. <br />
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<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
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The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
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<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
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A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
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<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
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There are large quantities of starting materials in the soil (ex: landfill and methanogenesis) so rxn proceeds even though delta G is near zero. Polymers such as lignin exist in the soil, which take a long time to break down. Breaking down lignin has a small delta G b/c lignin is a stable, aromatic molecule. A lot of energy is required to break it down, so not much energy is produced at the end of the reaction, yielding a small delta G. <br />
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<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
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<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
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Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
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<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
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In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
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<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
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The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
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<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
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<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
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<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
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<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
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<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
It seems that it would be difficult for an organism to switch between lithotrophy and organotrophy unless the organism is switching between an environment where organic and inorganic materials are present. If the organism contained both sets of enzymes that were required, then it would be necessary to use these enzymes as to avoid reductive evolution and lose the ability to use both lithotrophy and organotrophy. <br />
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<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
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<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
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<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
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Fatty acid biosynthesis is a cyclic cycle using acetyl-CoA with rounds of the same synthesis over and over again. The cycle feeds its products back repeatedly as substrates for further synthesis, so large polymers can be made using a limited number of enzymes. This pathway requires fewer enzymes in comparison to AA biosynthesis because it is a smaller pathway. Amino acid biosynthesis substrates are derived from the glycolysis pathways and the TCA cycle. AA biosynthesis requires more reduction because there are many different pathways used to form AA’s, while there is a single pathway to form fatty acids. AA biosynthesis requires also requires a greater number of different enzymes due to the many different pathways used to form AA’s. AA’s are complex, asymmetrical molecules that require many different conversions to synthesize. Each conversion is mediated by a different enzyme, therefore AA biosynthesis requires a greater number of different enzymes in comparison to fatty acid biosynthesis. <br />
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<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
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The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br />
Nitrogen sources include: organic amines (NH3→ NH4+), N2 gas from the atmosphere, and NO3- (nitrate, which is then made into ammonia). The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). Most bacteria can acquire nitrate (NO3-) or nitrite (NO2-) for reduction to ammonium ion, although they repress these E-expensive pathways when ammonium ion is present. While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated. Worldwide, factories fix enormous amts of N2 into ammonia for agricultural fertilizers. Industrial nitrogen fixation uses the Haber process, in which N gas is hydrogenated by methane under extreme heat and pressure. When nitrogen fertilizer is used, much of it is washed from the soil into streams when it rains. The streams take the nitrogen fertilizer to the oceans, where marine phytoplankton phototrophs fix the nitrogen and overgrow, causing algal blooms. The massive amounts of algal phototrophs eventually die, and sink to the bottom layers of the ocean. They deplete the oxygen available there by decomposing, causing eutrophication, where the lower layers of a body of water are depleted of oxyen as a result of overgrowth of microbial producers. The lack of oxygen causes a “dead zone” where no other aerobic organisms can survive. This has been a really big problem because it essential eliminates marine habitats, and has killed many marine organisms. <br />
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<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
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<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
<br />
<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
<br />
<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
<br />
<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
<br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
<br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
<br><br><br />
<b><i>Sinorhizobium meliloti</i></b><br />
<br><br><br />
<b><i>Staphylococcus epidermidis</i></b><br />
<br><br><br />
<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
<br />
<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40456BIOL 238 Review 20092009-04-01T01:38:51Z<p>Millerk: /* Chapter 15 */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
<br><br><br />
<br />
Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<br><br><br />
<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
<br><br><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
<br />
<br><br><br />
<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
<br><br><br />
<br />
Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
<br />
<br><br><br />
<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
<br><br><br />
<br />
A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
<br />
<br><br><br />
<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
<br><br><br />
<br />
Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
<br />
<br><br><br />
<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br />
The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
<br />
All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
<br />
Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br />
There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br />
Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br />
The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
<br />
<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br> <br />
<br />
The four types of control for gene expression are alterations of DNA sequence, control of transcription and mRNA stability, translational control and posttranslational control. Alterations of DNA sequence is when microbes program mutation, one way this happens is through phase variation or flipping of a DNA segment to turn off or on a gene. This method is the most irreversible and is the slowest to revert. Control of Transcription includes proteins binding DNA and inhibiting RNA polymerase or bending DNA at the promoter. This type of control is more reversible and takes less time than DNA segment but not as fast as translational and post translational. Translational method occurs when the ribosome is inhibited, for example attenuation. Post translational is the fastest where the made proteins are modified. <br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br />
Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br />
The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
NADH carries 2 or 3 times as much energy as ATP, depending on cell conditions. In high-energy reactions, NADH is used. NAD+/NADH accept and donate electrons, therefore if a cell needs to balance its electrons NADH is used in the reaction. NADH is also useful when a substrate needs to be reduced. ATP is used when less energy than NADH is required to fuel the reaction. ATP is formed when a reaction can provide enough energy to form ATP from ADP, but not enough energy to form NADH. Some reactions need a redox change. Always need to balance the redox levels in a cell. <br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
There are large quantities of starting materials in the soil (ex: landfill and methanogenesis) so rxn proceeds even though delta G is near zero. Polymers such as lignin exist in the soil, which take a long time to break down. Breaking down lignin has a small delta G b/c lignin is a stable, aromatic molecule. A lot of energy is required to break it down, so not much energy is produced at the end of the reaction, yielding a small delta G. <br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
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<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br />
<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
<br />
<br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
It seems that it would be difficult for an organism to switch between lithotrophy and organotrophy unless the organism is switching between an environment where organic and inorganic materials are present. If the organism contained both sets of enzymes that were required, then it would be necessary to use these enzymes as to avoid reductive evolution and lose the ability to use both lithotrophy and organotrophy. <br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
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<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br />
Nitrogen sources include: organic amines (NH3→ NH4+), N2 gas from the atmosphere, and NO3- (nitrate, which is then made into ammonia). The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). Most bacteria can acquire nitrate (NO3-) or nitrite (NO2-) for reduction to ammonium ion, although they repress these E-expensive pathways when ammonium ion is present. While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated. Worldwide, factories fix enormous amts of N2 into ammonia for agricultural fertilizers. Industrial nitrogen fixation uses the Haber process, in which N gas is hydrogenated by methane under extreme heat and pressure. When nitrogen fertilizer is used, much of it is washed from the soil into streams when it rains. The streams take the nitrogen fertilizer to the oceans, where marine phytoplankton phototrophs fix the nitrogen and overgrow, causing algal blooms. The massive amounts of algal phototrophs eventually die, and sink to the bottom layers of the ocean. They deplete the oxygen available there by decomposing, causing eutrophication, where the lower layers of a body of water are depleted of oxyen as a result of overgrowth of microbial producers. The lack of oxygen causes a “dead zone” where no other aerobic organisms can survive. This has been a really big problem because it essential eliminates marine habitats, and has killed many marine organisms. <br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
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<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
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<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
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<b><i>Paramecium</i> sp.</b><br />
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<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
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<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
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<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40451BIOL 238 Review 20092009-03-31T22:44:25Z<p>Millerk: /* Chapter 13 */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
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<br />
==Chapter 7==<br />
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<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
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The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
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All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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==Chapter 8==<br />
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<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
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This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
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<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
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Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
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<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
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Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
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<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
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There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
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<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
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Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
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<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
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Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
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==Chapter 9 and 10==<br />
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<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
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The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
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<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
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<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
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<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
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Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
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<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
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If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
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<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
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<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
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Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
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<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
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The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
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<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
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<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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==Wozniak lecture on Biofilms==<br />
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<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
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Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
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<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
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Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
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<b>3. Where in the body do biofilms form infections? Why?</b><br />
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The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
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<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
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Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
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<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
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<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
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==Chapter 13==<br />
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<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
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NADH carries 2 or 3 times as much energy as ATP, depending on cell conditions. In high-energy reactions, NADH is used. NAD+/NADH accept and donate electrons, therefore if a cell needs to balance its electrons NADH is used in the reaction. NADH is also useful when a substrate needs to be reduced. ATP is used when less energy than NADH is required to fuel the reaction. ATP is formed when a reaction can provide enough energy to form ATP from ADP, but not enough energy to form NADH. Some reactions need a redox change. Always need to balance the redox levels in a cell. <br />
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<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
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The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
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<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
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A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
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<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
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There are large quantities of starting materials in the soil (ex: landfill and methanogenesis) so rxn proceeds even though delta G is near zero. Polymers such as lignin exist in the soil, which take a long time to break down. Breaking down lignin has a small delta G b/c lignin is a stable, aromatic molecule. A lot of energy is required to break it down, so not much energy is produced at the end of the reaction, yielding a small delta G. <br />
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<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
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<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
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<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
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<br><br><br />
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<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
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<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
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<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
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<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
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<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
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<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
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<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
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<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
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<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
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<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
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<br />
The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
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<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
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<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
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<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
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<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
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<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
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<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
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<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
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<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
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<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
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<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
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<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
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<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
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<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
<br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
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<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
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<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
<br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
<br />
<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40450BIOL 238 Review 20092009-03-31T22:42:36Z<p>Millerk: /* Chapter 13 */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
<br><br><br />
<br />
Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<br><br><br />
<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
<br><br><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
<br><br><br />
<br />
Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
<br />
<br><br><br />
<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
<br><br><br />
<br />
A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<br><br><br />
<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
<br><br><br />
<br />
Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
<br />
<br><br><br />
<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br />
The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
<br />
All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
<br />
Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br />
There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br />
Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br />
The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
<br />
<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br />
Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br />
The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
NADH carries 2 or 3 times as much energy as ATP, depending on cell conditions. In high-energy reactions, NADH is used. NAD+/NADH accept and donate electrons, therefore if a cell needs to balance its electrons NADH is used in the reaction. NADH is also useful when a substrate needs to be reduced. ATP is used when less energy than NADH is required to fuel the reaction. ATP is formed when a reaction can provide enough energy to form ATP from ADP, but not enough energy to form NADH. Some reactions need a redox change. Always need to balance the redox levels in a cell. <br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br />
<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
<br />
<br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
<br />
<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
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<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
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<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
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<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
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<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
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<b><i>Paramecium</i> sp.</b><br />
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<b><i>Plasmodium falciparum</i></b><br />
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<b><i>Prochlorococcus</i> sp.</b><br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
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<b><i>Rhodobacter</i> sp.</b><br />
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Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
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<b><i>Rhodospirillum rubrum</i></b><br />
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<b><i>Rickettsia</i> sp.</b><br />
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<b><i>Saccharomyces cerevesiae</i></b><br />
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<b><i>Salmonella enterica</i></b><br />
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<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
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<b><i>Streptococcus </i>sp.</b><br />
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<b><i>Streptomyces</i> sp.</b><br />
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<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
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<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40449BIOL 238 Review 20092009-03-31T22:40:54Z<p>Millerk: /* Wozniak lecture on Biofilms */</p>
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<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
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==Chapter 7==<br />
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<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
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The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
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All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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==Chapter 8==<br />
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<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
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This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
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<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
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Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
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<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
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Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
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<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
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There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
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<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
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Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
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<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
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Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
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==Chapter 9 and 10==<br />
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<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
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The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
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<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
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<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
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<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
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Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
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<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
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If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
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<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
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<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
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Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
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<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
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The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
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<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
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<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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==Wozniak lecture on Biofilms==<br />
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<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
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Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
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<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
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Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
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<b>3. Where in the body do biofilms form infections? Why?</b><br />
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The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
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<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
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Twitching motility is a form of solid surface translocation of a biofilm. Individual microbes in a biofilm move by retracting their type IV fimbriae (the pilus). Twitching motility does not involve flagellum. Twitching motility is required to colonize new surfaces, and required for biofilm development. It is required for biofilm development because the movement of the microorganisms in the biofilm allows for the formation of columns and complex structures seen in many types of biofilms. Flagellar motility uses flagella as helical propellers that drive the cell forward like the motor of a boat. Twitching motility uses the “grappling hook model”, moving across a surface through retraction of a pilus. The flagellum are rotated by means of a motor driven by the cell’s transmembrane proton current. Twitching motility is ATP driven, not driven by the cell’s transmembrane proton current. <br />
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<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
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<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
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==Chapter 13==<br />
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<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
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<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
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The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
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<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
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A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
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<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
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<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
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<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
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Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
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<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
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In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
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<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
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The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
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<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
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<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
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<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
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Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
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<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
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<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
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<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
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Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
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==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
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<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
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<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
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The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
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When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
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<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
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<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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==Nitrogen fixation and nodulation==<br />
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==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
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<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
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<b><i>Anabaena</i> sp.</b><br />
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<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
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<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
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<b><i>Bacillus anthracis</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
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<b><i>Bacillus subtilis</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
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<b><i>Bacillus thuringiensis</i></b><br />
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Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
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<b><i>Bacteroides thetaiotaomicron</i></b><br />
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Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
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<b><i>Borrelia burgdorferi</i></b><br />
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Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
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<b><i>Chlamydia</i> sp.</b><br />
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Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
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<b><i>Clostridium botulinum</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
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<b><i>Escherichia coli</i></b><br />
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Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
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<b><i>Geobacter metallireducens</i></b><br />
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Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
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<b><i>Halobacterium</i> sp.</b><br />
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Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
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Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
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Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
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Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
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<b><i>Paramecium</i> sp.</b><br />
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<b><i>Plasmodium falciparum</i></b><br />
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<b><i>Prochlorococcus</i> sp.</b><br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
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<b><i>Rhodobacter</i> sp.</b><br />
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Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
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<b><i>Rhodospirillum rubrum</i></b><br />
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<b><i>Rickettsia</i> sp.</b><br />
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<b><i>Saccharomyces cerevesiae</i></b><br />
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<b><i>Salmonella enterica</i></b><br />
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<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
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Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
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<b><i>Streptococcus </i>sp.</b><br />
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<b><i>Streptomyces</i> sp.</b><br />
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<b><i>Vibrio cholerae</i></b><br />
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Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
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<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40448BIOL 238 Review 20092009-03-31T22:40:17Z<p>Millerk: /* Wozniak lecture on Biofilms */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br />
The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
<br />
All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
<br />
Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br />
There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br />
Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br />
The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
<br />
<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
Bacterial biofilms display cell differentiation and specialization, as do multicellular organisms. Biofilms can also display resistance to antibiotics, which multicellular organisms do as well. Biofilms and muticellular organisms differ because most multicellular organisms are more specialized, forming organelles and organs. Biofilms are specialized and can form channels and columns, but are not as specialized as mutlicellular organisms. <br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br />
Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br />
The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br />
<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
<br />
<br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
<br />
<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
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<b><i>Paramecium</i> sp.</b><br />
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<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
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<b><i>Rhodospirillum rubrum</i></b><br />
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<b><i>Rickettsia</i> sp.</b><br />
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<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
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<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40447BIOL 238 Review 20092009-03-31T22:34:50Z<p>Millerk: /* Chapter 9 and 10 */</p>
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<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
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<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
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The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
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All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
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This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
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<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br> <br />
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Mycoplasma genitalium lost its genes to produce amino acids through degenerate evolution and now gain those amino acids through from the host. The M. genitalium would most likely have protein channels to let amino acids inside the cell since it does not contain any amino acids and channels are the easiest way to transport molecules that are not permeable to the membrane. The first M. genitalium could have gotten its protein channels from the host. The host could have synthesized the protein channels for the M. genitalium. <br />
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<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
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Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
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<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
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There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
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<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
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Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
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<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
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Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
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==Chapter 9 and 10==<br />
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<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
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The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
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<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
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<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
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<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
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Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
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<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
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If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
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<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
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<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
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Nonreplicative transposons jumps from one DNA site to another without replicating. Attachment of the element onto the protruding ends of the staggered cut produces duplicated sequences at either end of the new insertion. Every time the transposable element “jumps”, a new duplication is created in the target DNA. Replicative transposons copy to a new site, while the original copy remains at the old site. The target sequence is also duplicated and flanks the replicative transposon. Transposons spread within a cell via replicative transposition. Transposons spread among organisms if the transposon is found in a plasmid, and that plasmid is spread to a neighboring microorganism via horiztonal transfer. <br />
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<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
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The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
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<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
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<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
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==Wozniak lecture on Biofilms==<br />
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<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
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<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
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Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
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<b>3. Where in the body do biofilms form infections? Why?</b><br />
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The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
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<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
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<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
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<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
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==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
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<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
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The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
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<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
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A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
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<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
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<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
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<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
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Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
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<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
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In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
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<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
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==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
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The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
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<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
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Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
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<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
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<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
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Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
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<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
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<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
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<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
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Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
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==Chapter 15==<br />
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<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
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<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
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<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
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The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
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When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
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<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
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<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
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<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
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==Nitrogen fixation and nodulation==<br />
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==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
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<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
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<b><i>Aeromonas hydrophila</i></b><br />
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Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
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<b><i>Anabaena</i> sp.</b><br />
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<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
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<b><i>Aspergillus</i> sp.</b><br />
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Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
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<b><i>Bacillus anthracis</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
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<b><i>Bacillus subtilis</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
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<b><i>Bacillus thuringiensis</i></b><br />
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Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
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<b><i>Bacteroides thetaiotaomicron</i></b><br />
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Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
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<b><i>Borrelia burgdorferi</i></b><br />
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Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
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<b><i>Chlamydia</i> sp.</b><br />
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Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
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<b><i>Clostridium botulinum</i></b><br />
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Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
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<b><i>Escherichia coli</i></b><br />
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Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
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<b><i>Geobacter metallireducens</i></b><br />
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Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
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Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
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<b><i>Halobacterium</i> sp.</b><br />
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Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
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<b><i>Lactococcus</i> sp.</b><br />
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Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
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<b><i>Methanococcus</i> sp.</b><br />
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Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
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<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
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Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
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<b><i>Paramecium</i> sp.</b><br />
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<b><i>Plasmodium falciparum</i></b><br />
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<b><i>Prochlorococcus</i> sp.</b><br />
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<b><i>Pseudomonas aeruginosa</i></b><br />
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<b><i>Rhodobacter</i> sp.</b><br />
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Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
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<b><i>Rhodospirillum rubrum</i></b><br />
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<b><i>Rickettsia</i> sp.</b><br />
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<b><i>Saccharomyces cerevesiae</i></b><br />
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<b><i>Salmonella enterica</i></b><br />
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<b><i>Serratia marcescens</i></b><br />
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<b><i>Sinorhizobium meliloti</i></b><br />
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<b><i>Staphylococcus epidermidis</i></b><br />
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<b><i>Staphylococcus aureus</i></b><br />
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Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
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<b><i>Streptococcus </i>sp.</b><br />
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<b><i>Streptomyces</i> sp.</b><br />
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<b><i>Vibrio cholerae</i></b><br />
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Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
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<b><i>Vibrio fischeri</i></b><br />
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Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40445BIOL 238 Review 20092009-03-31T22:28:39Z<p>Millerk: /* Chapter 7 */</p>
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<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
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==Chapter 7==<br />
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<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
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Bidirectional replication is much more efficient for large genomes, because as both the leading strands and lagging strands are replicated at the same time<b> (and in both directions)</b>, it does not take quite as long. Replicating one strand and then the other in large genomes would take an unnecessarily long time. However, rolling circle replication is ideal for small, circular genomes, such as plasmids and bacteriophage genomes, because in these cases, large numbers of copies need to be made quickly <b>(in large numbers, using simpler enzymes, perhaps more error-prone)</b>. Bacteriophages, of course, need to produce as many copies of their genomes as possible in order to either destroy the host cell or incorporate themselves into the cell's DNA. As plasmids may contain genes that are advantageous under certain conditions--conferring antibiotic resistance, for example--it is important that each daughter cell receives these genes; much of the time, large numbers of copies of plasmids are needed, and rolling circle replication is the most efficient way to produce them.<br />
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<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
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In the Caulobacter mutant that lacks the XerD protein, the bacterial cells filament (keep getting longer instead of dividing). The nucleoids failed to separate, even though the DNA has replicated. In DNA replication and cell division, the cell must segregate in order to form new copies. This is seen when the cell DNA is labeled with GFP. The Bowman article showed other mutants, such as the PopZ mutant that kept filamenting because PopZ is needed for polar localization of DNA. Other mutants lead to minicells, such as the FDSZ protein mutant. DNA replication mutants display repeated sectored colonies. These mutants have mutations in cell division and repair apparatus’ that do not allow the cells to separate.<br />
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<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
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Each complex contains 2 DNA Pol III enzymes, which together form the replisome. One copy of the enzyme synthesizes the leading strand, while the other does the lagging strand. The synthesis of the lagging strand goes away from the replication fork. A new RNA primer is synthesized about every thousand bases, meaning it is synthesized in pieces, called Okazaki fragments. The DNA Pol III then uses the primer to synthesize a complementary DNA strand until it bumps into the beginning of the previous fragment. RNase cleaves the RNA primers to allow DNA Poly I to synthesize DNA patches using the 3’ OH end of the preexisting fragment as a primer. The pieces are joined by DNA ligase.<br><br />
<b>Yes, that sounds right.</b><br />
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<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
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A major mutation in either the XerC or XerD proteins, which recognize the <i>dif</i> site on each DNA molecule and catalyze a series of cutting and rejoining steps that result in catenane resolution, would cause a major mutation affecting the entire genome. A mutation in either of these proteins would cause the two replicated DNA genomes to not separate and thus the cells would filament, nucleoids would fail to separate, and the cell would not properly replicate. The mutation could also possibly lead to one double-length genome that contains the entire genome twice, or it could lead to the wrong ends in the Holiday junction being joined, and thus may result in two genomes that are quite different from the original. This type of mutation could possibly be prevented by employing multiple proofreading steps for the replicated genome, particularly in the sequences which encode XerC and XerD. By preventing this mutation, you would prevent improper cell replication, which would could possibly lead to further mutated replication or even cell death. <br />
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<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
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Once DNA replication has started, the process of cell division has to finish. If the replication complex is physically blocked, the DNA will not be able to replicate, but the cell will start to divide into 2 daughter cells anyway. As the septum forms, it will kill both daughter cells since the DNA will not be separated. <br />
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<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
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<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
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The sites at which the restriction enzymes cleave the DNA, also known as the restriction sites, are palindromic: the top and bottom strands are read the same in a 5' to 3' direction. For example, TAACGT would pair with AATGCT.<br><br />
<b>A good idea, but there are far more "ends" than there are different restriction sites; so how would these tell you how to line up all the genomic pieces correctly?</b><br />
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All fragments of DNA can be clones so that there are overlapping fragments (this is known as “shotgun” cloning). Each fragment can then be sequenced using dideoxy sequencing. The overlapping areas can be matched using a computer until the genome is reconstructed. <br><br />
<b>Yes, that will do it.</b><br />
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==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
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This can be done in cell free systems. The parts <b>(subunits)</b> from a RNA polymerase from a sensitive strain and a resistant strain are separated. The RNA polymerase is then reassembled using all parts from the sensitive strain, but one, which comes from the resistant strain. If RNA transcription proceeds in the presence of the antibiotic then the subunit from the resistant strain is targeted by the antibiotic. <br />
<b>Yes, that's how it works.</b><br />
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<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
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<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
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Ordinary bases of the tRNA are modified by specific enzymes, and are turned into rarer RNA bases such as wybutosine. It seems unlikely that such a diverse set of modified bases would arise simply to add functionality and extended half-life to tRNA molecules. These unusual bases were probably very prevalent a long time ago, where the variety of different bases could allow a wide range of catalytic RNAs that had a larger significance in cell function. This would be before the "rise" of amino acid proteins.<br><br />
<b>Yes, that's our best current view. Who knows what happened 4-billion years ago--never trust a geologist. ;)</b><br />
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<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
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There are many possibilities. A pharmaceutical could be designed that binds to the -35 and -10 promoters, thereby preventing RNA polymerase from binding there, in an effect similar to repressors. Other antibiotics could simply change the shape of or otherwise denature RNA polymerase, so that it cannot bind to the promoters. <br />
A third possible pharmaceutical agent could somehow bind to the sigma factor, preventing this protein from recognizing the promoter sequences.<br><br />
<b>Good ideas.</b><br />
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<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br />
Some proteins often prove useful to these other bacteria. The proteins might be able to digest certain food sources, for example. Also, genetic material might be exported by bacteria, genetic material that might contain resistance to viruses or antibiotics. Naturally, any bacteria that absorbed this material would have a distinct advantage over any bacteria that did not.<br />
<br><b>The genetic information might be useful. What else about protein and DNA molecules might be useful to a heterotroph?</b><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
Bioinformatics allows us to predict a cell’s physiology and evolutionary development by comparing known genomes of other cells. Computer analysis can be used to find all possible protein sequences that could be formed in all reading frames. These can then be compared to see if the protein predicted resembles other proteins in databases or proteins of known functions.<br><br />
<b>Bioinformatics implies you have sequence data already. How do you get the sequence data for a microbe that you cannot grow in culture?</b><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br />
The genes moved in conjugation are located on the F plasmid, what is commonly known as the "fertility factor". This plasmid is transferred by pilus from bacterium to bacterium; it is nicked at the 5' end and unwound. As this strand moves to the recipient bacterium, the remaining strand in the donor bacterium is replicated. Whether genes are moved individually or in groups depends on how many genes are on this plasmid. Part of a gene could be moved if the plasmid in the donor bacterium is nicked in the wrong place, or if not all is transferred to the recipient bacterium.<br />
<br />
<br><br><br />
<b>How is the strand "moved"? If the plasmid has twenty genes, will they all be moved?</b><br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
Each cell in the population exports CF until the concentration increased to a level high enough to induce formation of the translocasome in all the cells. In order for the population to be induced together all the cells must be able to sense the CF, which means that it must be exported. <br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
If one base is missing and the phosphate-sugar backbone is intact the replisome will fill in a base opposite the apurinic site. However, this could be any base, not necessarily the correct one. <br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
The ara operon can repress or activate gene expression. In the presence of arabinose, AraC binds araI1 and araI2 and interacts with RNA polymerase to permit transcription of araBCD. In the absence of arabinose, AraC becomes elongated and binds aria and araO2 causing the DNA to form a loop so that it cannot interact with RNA polymerase stopping transcription of araBCD. The lac operon is different in that it is only repressed by glucose because glucose inhibits cAMP production. When glucose levels are low and lactose is present, lactose is rearranged to form allolactose, which binds the repressor and releases the operator. <br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br />
Biofilms confer resistance to antibiotics, predatorial cells, and harmful chemicals. Bacteria in close contact with each other in biofilms are more likely to survive these stresses, because they are easily able to transfer genetic material to each other.<br />
<br><b>This is a good start. How do biofilms confer resistance to each thing? Genetics? Physiology and form of the biofilm? Secreted molecules?</b><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br />
The most common location on the body for biofilms that people think of is your teeth. Dark, hard to reach places are ideal for biofilms, as they are hard to clean and clear. The body has a strong immune system- cells attack infections within the body and the surface of the body is effective in keeping bacteria out. So, biofilms first need something to latch on to so they can grow, and dark holes in the body are the best opportunities. The ears, nose, throat, sweat glands, gaps around the teeth and gums, hair follicles, lungs, and tonsils are just a few examples of prime biofilm locations. Once biofilms establish themselves and build up into large enough groups protected by thick slime, they survive a better chance against the immune system and in the bloodstream, where they can spread the infection.<br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
The steps are reversed for gluconeogensis by incorporating a different catabolic enzyme. The irreversible steps are points of regulation along the pathway that maintains a consistent level of intermediates. The reversal of a step must be catalyzed by an entirely different enzyme. In this way the forward and reverse direction is like an on/off switch, where one enzyme is inhibited and the other is activated and vice versa usually by allosteric regulation by key intermediates, such as ADP and ATP.<br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
A cell may switch among these 3 pathways depending on the type of substrate that needs to be catabolized and on whether intermediates are needed to be redirected toward anabolism and biosynthesis. If glucose is available, the Embden-Meyerhof-Parnas (EMP) pathway, glucose 6-phosphate isomerizes to fructose 6-phosphate, eventually yielding 2 moc. of pyruvate. Net gain of Energy= 2NADH + 2ATP. The Entner-Doudoroff (ED)pathway offers a new way to catabolize sugars, especially sugar acids, which can be phosphorylated to 6-phosphogluconate. Th 6C substrate is split into 2 3C products, one of which is glyceraldehyde 3-phosphate and can enter the 2nd part of glycolysis. This pathway allows enteric bacteria to be able to colonize the intestinal epithelium, since gluconate is the sugar acid in intestinal mucus. The net gain of Energy= 1NADH + 1NADPH + 1ATP. Finally the pentose phosphate shunt is a way for glucose or sugar acids to eventually form ribulose 5-phosphate via the 6-phosphogluconate intermediate. The PPS is able to generate complex series of intermediates that can be shunted toward biosynthesis, where pairs of sugars can exchange short carbon chains to yield various sugar phosphates differing in lengths. The net gain of Energy= 2NADPH + 1ATP. <br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
Bacteria have to transfer the hydrogens from NADH + H+ back onto pyruvate because the NADH must be recycled. During fermentation, no oxygen is available to accept electrons, and NAD+ cannot serve as the terminal electron acceptor in place of oxygen, because there would be no way for reforming NAD+ so that it can act as the electron acceptor in another metabolic cycle. <br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
In complex environments, organisms can choose the preferred substrates depending on availability and the efficiency of Energy production. This can occur through regulating gene expression. An example is the sugar lactose in E. coli, which induces transcription of genes encoding beta-galactosidase and lactose permease. Glucose is a preferred carbon source, and its presence inhibits lac transcription. Amino acid catabolism can occur by two routes: (1) deamination or (2) decarboxylation. In a very acidic environment (low pH) decarboxylation is favored because the ammonia product is an amine that can buffer against the rising acidity level. This is essential for anaerobic soil or animal digestive tracts that undergo fermentation that many times results in acid production through lactic acid formation. <br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br />
The electron acceptors used by bacteria and archaea depend on what elements are available in the environment. For example, if the environment is rich in oxygen and the prokaryotes can perform aerobic respiration, they will use oxygen as an electron acceptor and a less efficient NADH Dehydrogenase. On the other hand, if the microbes live in an anaerobic environment, other electron acceptors such as nitrogen and sulfur compounds can be used, as these tend to accept electrons easily as well. <br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
Both transmembrane pH and electrical potential can be used to store energy in the proton motive force in two distinct but equivalent forms. The transmembrane pH difference can contribute to the proton motive force even if there is no charge gradient. This could occur if there was the same number of positive charged ions inside and outside of the membrane, but the ions differed in identity (Na+, H+, or K+); specifically, a higher concentration of H+ ions are on the outside than the inside, creating a force that tends to drive protons inward. A charge difference results when there is a greater number of positive charge on the outside of the membrane than the inside, and the excess cations outside are something other than H+ to result in only a charge gradient with no pH gradient. This creates a charged potential along the membrane that tends to pull positive charge in. At low pH, the form of energy probably used is the transmembrane pH, since the concentration of H+ rises sharply to create the drive for protons to enter. In high pH, the energy form used is likely to be the electrical potential, because there is a lack of H+ on the outside and other cations must be used to generate the electrical gradient for the proton to enter. <br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br />
<br />
Watery environments favor oxygenic photosynthesis, as water, H20, serves as the electron donor for the ETS. Evidently this is why cyanobacteria and other microbes that perform oxygenic photosynthesis first developed underwater.<br />
<br />
<br />
<br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
Environments favoring methanogenesis are landfills and the digestive systems of cattle or humans. This because methanogenesis must occur in the presence of adequate carbon dioxide and hydrogen, which are the gaseous fermentation products of bacteria undergoing anaerobic respiration that can be trapped in these habitats. Methanogens are so widespread despite their weak electron acceptors of carbon dioxide and water and strong electron donors of hydrogen and methane due to the fact that the availability of the carbon and hydrogen substrates is so abundant.<br />
<br />
<br><br><br />
<br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
The only form of nitrogen that microbes can directly assimilate into biomass is ammonium ion (NH4+ protonated from NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can be assimilated.<br />
<b>What are the other oxidized forms that bacteria and plants take up and reduce to ammonia and ammonium ion?<br />
What about N from reduced organic compounds?</b><br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. Plants can take up the nitrates and reduce them (with energy input), but a large excess runs into streams and water supplies. These high concentrations of nitrates in water form nitrites that can combine with hemoglobin (in the blood) to create a form of hemoglobin that is not able to take up oxygen. This is a problem for babies trying to breathe. <br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
<br>Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
<br>Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
<br>Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
<br>Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
<br>Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
<br>Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
<br>Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
<br>Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
<br>Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
<br>Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids: pxO1 and pxO2. These plasmids encode main virulent factors.<br />
<br>Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
<br>Habitat: Live in soils world-wide and is the main habitat.<br />
<br>Disease: Anthrax disease. Infectious endospores harm host by germinating for vegetative growth. During growth the bacteria produce toxins in the body of humans and animals. The slime capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
Broader categories: rod-shaped, halophilic, candidate for life on Mars. Genome: 1 large chromosome and 2 plasmids (3 circular replicons). Metabolism: aerobic, but not glucose degradation. Habitat: highly saline lakes. Disease: non-pathogenic<br />
<br />
<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
Broader categories: spherical, gram positive. Genome: 1 circular chromosome with 2, 365, 589 bp, where 86 % of the genome code for protein, 1.4 % for RNA, and 12.6 % for noncoding region. 64.2 % of the genes code for known functional proteins, and 20.1 % of the genes for known protein with unknown function. Metabolism: aerobic or anaerobic, often use lactic acid fermentation. Habitat: plant surfaces, digestive tract of cows. Disease: non-pathogenic.<br />
<br />
<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
Broader categories: gram-negative, cocci-shaped, archaea domain, thermophilic and mesophilic. Genome: 1 circular chromosome with 1 large extra-chromosomal element and 1 small extra-chromosomal element. Metabolism: autotrophic, anaerobic, reduces carbon dioxide with hydrogen gas to generate methane. Habitat: can grow up to pressures of 200 atm, temperature ranges between 48-94 degrees C, with optimal growth at 85 degrees C. Disease: non-pathogenic.<br />
<br />
<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
Broader categories: spherical without a cell wall, highly pathogenic--osmotic instability. Genome: 1 small circular chromosome. Metabolism: major nutrients come from mucosal epithelial cells of the host, many proposed metabolic pathways. Habitat: not found in the environment but can be cultured in medium-rich agar. Disease: highly pathogenic; parasitizes epithelial cells in the respiratory tract. <br />
<br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
Broader categories: quorum-sensing bacteria, flagella motility. Genome: unique complexities-2 chromosomes, 1 large and 1 small, circular chromosomes. Metabolism: photosynthesis mostly, also capable of lithotrophy, aerobic, and anaerobic respiration pathways. Habitat: aquatic and marine environments. Disease: non-pathogenic.<br />
<br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
<br><br><br />
<b><i>Sinorhizobium meliloti</i></b><br />
<br><br><br />
<b><i>Staphylococcus epidermidis</i></b><br />
<br><br><br />
<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
Broader categories: gram positive, spherical, immobile. Genome: 1 circular genome, resistance for antibiotics are encoded by a transposon. Metabolism: EMP and Pentose-Phosphate pathways. Lactate is the end product of anaerobic glucose metabolism. Acetate and carbon dioxide are the end products of aerobic metabolism. Habitat: skin, mucous membranes of animals. Disease: skin infections, invasive diseases, toxic shock syndrome (TSS). <br />
<br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, bent rod shaped, one polar flagellum. Genome: Two circular chromosomes. Metabolism: Fermentative and respiratory. Habitat: Aquatic environments. Disease: Responsible for cholera in humans, which is characterized by diarrhea and vomiting leading to dehydration. The bacteria secrete a toxin that ultimately causes an increase in cyclic AMP levels that stimulates ion transport in the cells lining the intestine. This is followed by water leaving the intestinal cells to compensate for the change in osmolarity. <br />
<br />
<b><i>Vibrio fischeri</i></b><br />
<br><br><br />
Broader Categories: Rod shaped, gram-negative. Genome: Two circular chromosomes with 2,284,050 bp and a plasmid. The lux operon controls bioluminescence. Metabolism: Heterotrophic. Habitat: Found in marine environments in a symbiotic relationship or as free-living. It can also be parasitic and saprophytic. Often found in the Hawaiian squid, Euprymna scolopes, where the bacteria bioluminesce in the light organ. The squid protects the bacteria from predators and provides nutrients, while the bacteria’s light eliminates the squid’s shadow, protecting it from predators. Disease: Other Vibrio species can cause human infections.</div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40364BIOL 238 Review 20092009-03-24T03:05:48Z<p>Millerk: /* Species to know for Test */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
The only form of nitrogen that is available to microbes for assimilation is ammonia (NH3). While there are other forms of nitrogen available in the environment, these other forms have to be reduced to ammonia before they can assimilate.<br />
<br />
When fertilizer is spread on farmland to nourish crops, lithotrophic microbes cause a problem by oxidizing ammonia fertilizer to nitrates and nitrites. These high concentrations of nitrites in water can combine with hemoglobin to create a form that is not able to take up oxygen. This means that nitrogen fixation cannot occur, as the environment is now aerobic, and the anerobic respirers cannot convert nitrate and nitrite to nitrogen gas. <br />
<br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids made of d.s. DNA: pxO1 and pxO2 encode main virulent factors.<br />
Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
Habitat: Live in soils world-wide and is the main habitat.<br />
Disease: Anthrax disease. Infectious endospores harms host by producing toxins in the body of humans and animals. The slimy capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. P. aeruginosa can metabolize on hundreds of other things besides arginine, and can respire on nitrate (although it does much better on O2).Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. P. aeruginosa forms biofilms in the lung of cystic fibrosis patients; it is the major cause of death in these individuals.<br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
<br><br><br />
<b><i>Sinorhizobium meliloti</i></b><br />
<br><br><br />
<b><i>Staphylococcus epidermidis</i></b><br />
<br><br><br />
<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
<b><i>Vibrio fischeri</i></b><br />
<br><br></div>Millerkhttps://microbewiki.kenyon.edu/index.php?title=BIOL_238_Review_2009&diff=40362BIOL 238 Review 20092009-03-22T21:00:12Z<p>Millerk: /* Species to know for Test */</p>
<hr />
<div>This page provides review questions for [http://biology.kenyon.edu/courses/biol238/biol238syl09.html BIOL 238] (Spring 2009). Answers may be posted by students.<br />
<br><br />
<br />
==Chapter 7==<br />
<br><br />
<b>1. What are the relative advantages and limitations of bidirectional replication versus rolling circle replication? What kind of genetic entities are likely to favor one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. During rapid growth, why would a bacterial cell die if the antibiotic drug “forms a physical barrier in front of the DNA replication complex.”?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. What are the relative advantages and limitations of bidirectional versus rolling-circle replication of DNA? Explain in terms of different genome sizes, types, and cell situation when replication occurs.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. When you sequence a genome, how do you know where the base pairs in the genome are located since the DNA used to sequence the genome is in fragments?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 8==<br />
<br><br />
<b>1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. If <i>Mycoplasma genitalium</i> cannot synthesize its own amino acids, does it have extensive/multiple protein channels (ABC pumps) to let amino acids pass its membrane? If proteins are made of amino acids, though, how did the first M. genitalium’s protein channels come into existence? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. In tRNA, there are "unusual" bases not found in mRNA How are these bases generated? Do you think they arise from a recently-evolved aspect of tRNA, or do you think they are an ancient phenomenon of the original RNA world? Explain.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How could you sequence the genome of an unculturable microbe?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 9 and 10==<br />
<br><br />
<b>1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. How are microbial species defined? What is the role of vertical phylogeny; and the role of horizontal gene exchange? Explain why species definition is a problem.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Why is competence factor exported out of the cell to bind to ComD externally in transformation of Streptococcus? Why doesn't the molecule bind internally? Doesn't exporting CF waste energy? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. If a spontaneous mutation occurs to form an apurinic site, transcription and replication are hindered, but what actually happens when the replisome gets to the hole where the base should be? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. Explain how the <i>ara</i> operon works, and how it differs from the lac operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>8. Explain how different mechanisms acting at different levels on DNA and RNA can modulate gene expression over a range of time scales, from multiple generations to within a few seconds.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>9. Explain the roles of thermodynamic and kinetic effects in attenuation control of the <i>trp</i> operon.</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Wozniak lecture on Biofilms==<br />
<br><br />
<b>1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. Where in the body do biofilms form infections? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the <i>psl</i> operon in bioflim development?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How was it proved that <i>psl</i> encodes PSL polysaccharide? How does PSL compare in structure with alginate?</b><br />
<br><br><br />
<br />
<br><br><br />
==Chapter 13==<br />
<br><br />
<b>1. ATP and NADH are both energy carriers: What are the advantages of using one over the other?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. When cells need to make glucose (gluconeogenesis), they "reverse glycolysis" because most steps are reversible. However, there are a couple of steps that are not reversible. How do you think they get reversed for gluconeogenesis? </b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. There are 3 main pathways to form pyruvate- EMP, ED and PPS. How and why might a cell switch among these?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. Explain why most soil bacteria grow using energy-yielding reactions with very small delta-G.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Why are glucose catabolism pathways ubiquitous, despite the fact that most bacterial habitats never provide glucose? Explain several reasons.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. In glycolysis, explain why bacteria have to return the hydrogens from NADH back onto pyruvate to make fermentation products. Why can't NAD+ serve as a terminal electron acceptor, like O<sub>2</sub>?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. Why do environmental factors regulate catabolism? Give examples. Why are amino acids decarboxylated at low pH, and under anaerobiosis?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>8. Why does catabolism of benzene derivatives yield less energy than sugar catabolism? Why is benzene-derivative catabolism nevertheless widespread among soil bacteria?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Chapter 14==<br />
<br><br />
<b>1. Explain how bacteria and archaea switch among various electron acceptors depending on environmental conditions.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Explain how cell processes such as ATP synthesis can be powered by either the transmembrane pH difference or by the charge difference across the membrane. Which form of energy is likely to be used at low external pH? At high external pH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. For phototrophy, discuss the relative advantages and limitations of using PS I versus PS II.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. What environments favor oxygenic photosynthesis, versus sulfur phototrophy and photoorganotrophy? Explain.</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. Explain why certain lithotrophs acidify their environments, to more extreme levels than fermentation. What are some practical consequences for human industry?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. Is it surprising that an organism may switch between lithotrophy and organotrophy? What enzymes would have to be replaced, and what enzymes could be used in common for both kinds of metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>7. What kind of environments favor methanogenesis? Why are methanogens widespread, despite the low delta-G of their energy-yielding metabolism?</b><br />
<br><br><br />
<br />
<br><br><br />
==Chapter 15==<br />
<br><br><br />
<b>1. Why does biosynthesis need both ATP and NADPH? Why couldn't biosynthetic pathways use just ATP, or just NADPH?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>2. Compare and contrast fatty acid biosynthesis and amino acid biosynthesis. Which pathway requires more reduction? Which requires a greater number of different enzymes? Why?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>3. What forms of nitrogen are available to microbes for assimilation? When fertilizer is spread on farmland to nourish crops, what problem is caused by microbes?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>4. How are the pathways of amino acid biosynthesis organized? What common routes flow from which core pathways?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>5. How and why do bacteria make "secondary products"? What are their functions?</b><br />
<br><br><br />
<br />
<br><br><br />
<b>6. How can we manipulate bacterial secondary product formation to develop new pharmaceutical agents?</b><br />
<br><br><br />
<br />
<br><br><br />
<br />
==Nitrogen fixation and nodulation==<br />
<br><br><br />
==Chapter 17==<br />
<br><br><br />
==Species to know for Test==<br />
<br><br><br />
<b>For each species, state one or two broader categories of organism (such as gram-positive endospore-forming bacteria), the type of genome, type(s) of metabolism, habitat, and disease caused (if any).</b><br />
<br><br><br />
<b><i>Aeromonas hydrophila</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic<br />
Genome: Genes that contribute to its toxicity are cytotoxic enterotoxin gene (act), heat labile enterotoxins (Alt), and heat-stable cytotonic enterotoxins (Ast).<br />
Metabolism: Heterotrophic, ferments glucose, digests gelatin, hemoglobin, and elastin.<br />
Habitat: Exists in aerobic and anaerobic environments: aquatic environments, fish guts, food, human bloodstream and organs.<br />
Disease: Causes many diseases in fish and amphibians, since it exists in aquatic environments. Can cause disease in humans, such as septiticemia, meningitis, pneumonia, and gastroenteritis.<br />
<br />
<b><i>Anabaena</i> sp.</b><br />
<br><br><br />
Broader Categories: Barrel-shaped cells. Filamentous cyanobacteria (blue-green algae) found as plankton.<br />
Genome: 1 circular chromosome with 5368 protein-coding regions and 6 plasmids (from sequenced PCC 7120 strain).<br />
Metabolism: Photoautotrophic, perform oxygenic photosynthesis. Form heterocysts (specialized nitrogen-fixing cells that convert nitrogen to ammonia) during nitrogen starvation.<br />
Habitat: Freshwater and damp soil. Form symbiotic relationships with certain plants.<br />
Disease: Produce neurotoxins, such as anatoxins (neuromuscular poisons), that are harmful to wildlife and farm animals.<br />
<br />
<b><i>Aspergillus</i> sp.</b><br />
<br><br><br />
Broader Categories: Over 185 species of this genus<br />
Genome: Largely incomplete<br />
Metabolism: Highly aerobic. Pathogenic species obtain nutrients from host, while non-pathogenic species obtain nutrients from soil, wood, and plant detritus.<br />
Habitat: Grow as molds in oxygen-rich environments and carbon-sources. Some species are capable of living in nutrient-depleted environments as well.<br />
Disease: About 20 species are pathogenic in humans and animals. Aspergillus fumigatus and Aspergillus flavus cause invasive pulmonary aspergillosis and is often fatal.<br />
<br />
<b><i>Bacillus anthracis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form endo-spores and biofilms<br />
Genome: 1 circular chromosome with over 5 million b.p. 2 circular plasmids made of d.s. DNA: pxO1 and pxO2 encode main virulent factors.<br />
Metabolism: Facultative anaerobe and must grow in medium with essential nutrients including C and N sources. Upon nutrient deprivation, endospores form (requires oxygen to form) and can live in inhospitable envrionments for many years. Can grow into vegetative cells in aqueous environment with adequate nutrients.<br />
Habitat: Live in soils world-wide and is the main habitat.<br />
Disease: Anthrax disease. Infectious endospores harms host by producing toxins in the body of humans and animals. The slimy capsule enables it to resist phagocytosis. 3 main forms of the disease: cutaneous, pulmonary, and gastrointestinal. Can cause death in 2-48 hours.<br />
<br />
<b><i>Bacillus subtilis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, form stress-resistant endospores<br />
Genome: 1 circular chromosome with 4100 genes coding for proteins.<br />
Metabolism: Can grow in aerobic and anaerobic conditions. Uses fermentation and nitrate ammonification to make ATP in the absence of oxygen.<br />
Habitat: Soil and vegetation at mesophilic temperatures.<br />
Disease: Non-pathogenic. Responsible for spoilage of food, since contamination often results in decomposition, but it rarely causes food-poisoning.<br />
<br />
<b><i>Bacillus thuringiensis</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, spore-forming, rod-shaped<br />
Genome: 1 circular chromosome with 5.2-5.8 Megabases. Contains many plasmids.<br />
Metabolism: Facultative anaerobe (makes ATP by aerobic respiration if oxygen is present, but can switch to fermentation).<br />
Habitat: Soil. It is used in 90% of pesticides. Fends off insects by producing crystal proteins (Cry proteins).<br />
Disease: Species-specific, non-pathogenic to humans, making it an environmentally-friendly insecticide. Cry toxin grows and sporulates in alkaline gut o finsect, which aids in its ability to infect the insect gut. The gut breaks down and the insect eventually dies.<br />
<br />
<b><i>Bacteroides thetaiotaomicron</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, anaerobic, human-bacterial symbiosis model<br />
Genome: 1 circular chromosome made of d.s. DNA, consists of 4776 proein-coding genes, 90% of which are essential in the binding and import of various polysaccharides.<br />
Metabolism: Starch (all 3 forms) is primary carbohydrate used as its source of C and Energy. Polysaccharides bind to the cell surface before undergoing hydrolysis.<br />
Habitat: Adult intestine-allows humans to degrade plant polysaccharides<br />
Disease: Serious infections include intra-abdominal sepsis and bacteremia. It is resistant to many antimicrobial agents.<br />
<br />
<b><i>Borrelia burgdorferi</i></b><br />
<br><br><br />
Broader Categories: Sprial-shaped with 2 flagella<br />
Genome: A linear chromosome with 910,725 b.p. with 853 genes. 17 linear and circular plasmids. <br />
Metabolism: Require specific nutritional requirements making it difficult to culture in vitro.<br />
Habitat: Live extracellularly and adapts to various host animals (tick, rodents, birds) by regulating various lipoproteins on thier surface.<br />
Disease: Lyme disease and recurring fever.<br />
<br />
<b><i>Chlamydia</i> sp.</b><br />
<br><br><br />
Broader Categories: Gram-negative, aerobic, coccoid or rod-shaped<br />
Genome: C. trachomatis-1,042,519 b.p. with 894 protein-coding sequences (70 genes are not homologous to sequences on the C. pneumoniae genome). C. pneumoniae- 1,230,230 b.p. (186 genes are not homologous to sequences on the C. trachomatis genome)<br />
Metabolism: Cannot synthesize its own ATP, so they cannot be grown on artificial medium and require growing cells to remain viable.<br />
Habitat: C. trachomatis-human host cells, C. suis- swine host cells, C. muridarum- mice and hamster host cells. Infectious elementary body form induces endocytosis upon contact with host cell. Once inside, the elementary body germinates to vegetative form, and divides every 2-3 hrs. It then reverts back to the elementary form and is released by the cell through exocytosis.<br />
Disease: C. trahomatis causes chlamydia and is the most common STD in the world. C. pneumoniae causes pneumonia and bronchitis<br />
<br />
<b><i>Clostridium botulinum</i></b><br />
<br><br><br />
Broader Categories: Gram-positive, rod-shaped, anaerobic, spore-former<br />
Genome: Genome size of 4039 kbp, which is larger than most Gram-positive genomes, indicating the extra genomic requirements needed for sporulation and pathogenic toxin production<br />
Metabolism: Lie dormant in very adverse environments. Spores can begin to grow in favorable conditions: non-halophilic salinity and anaerobic conditions. Grows at mesophilic temps.<br />
Habitat: Soils and improperly canned food products<br />
Disease: A-G subtypes produce different botulin toxin- al except C and D subtypes are human pathogens. The toxin prevents propagation of action potentials to the muscle fibers- causing paralysis by inhibiting muscle contraction. Fatalities usually occur due to asphyxiation<br />
<br />
<b><i>Escherichia coli</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, aerobic<br />
Genome: 1 circular chromosome, (4300 coding sequences) with 1800 known proteins. Some contain circular plasmid.<br />
Metabolism: Facultative anaerobe. Uses mixed-acid fermentation in anaerobic conditions. Growth driven by aerobic or anaerobic respiration using a large variety of redox pairs: oxidation of pyruvic acid, formic acid, hydrogen, and amino acids and reduction of oxygen, nitrate, dimethyl sulfoxide, trimethylamine N-oxide.<br />
Habitat: Lower intestines of human and mammals, where it aids in digestion processes: vitamin K production, food breakdown and absorption<br />
Disease: E. coli O157:H7 (enterohemorrhagic strain) causes food poisoning- leading to bloody diarrhea and kidney failure due to its production of Shiga-like toxin. Also can cause urinary tract infections by ascending infections of the urethra.<br />
<br />
<b><i>Geobacter metallireducens</i></b><br />
<br><br><br />
Broader Categories: Gram-negative, rod-shaped, possesses flagella, and pili<br />
Genome: 1 circular chromosome encoding 3621 genes. Plasmid encodes 13 genes, 1 of which is addiction module toxin (gives resistance to bacteria and another encoding plasmid stabilization system protein (allows bacteria to adapt to new environmental conditions)<br />
Metabolism: First organism with the ability to oxidize organic compounds and metals (iron, radioactive metals like Uranium, and petroleum compounds) into environmentally benign carbon dioxide while using iron oxide and other available metals an electron acceptors.<br />
Habitat: anaerobic conditions in soils and aquatic sediments<br />
Disease: Non-pathogenic<br />
<br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
Broader categories: Gram-negative, rod-shaped, does not produce spores. Habitat: due to its capability to synthesize arginine, P. aeruginosa proliferates in anaerobic conditions. It can be found in environments such as soil, water, humans, animals, plants, sewage, and hospitals. Metabolism: Aerobic respiration. Genome: a single and supercoiled circular chromosome in the cytoplasm. Disease(s) caused: Causes disease in immuno-compromised patients, such as those with cystic fibrosis, cancer, or AIDS. It further weakens the patient, allowing the patient to become more susceptible to other diseases. Usually this kills the patient. <br />
<br />
<b><i>Halobacterium</i> sp.</b><br />
<br><br><br />
<b><i>Lactococcus</i> sp.</b><br />
<br><br><br />
<b><i>Methanococcus</i> sp.</b><br />
<br><br><br />
<b><i>Mycoplasma pneumoniae</i> sp.</b><br />
<br><br><br />
<b><i>Paramecium</i> sp.</b><br />
<br><br><br />
<b><i>Plasmodium falciparum</i></b><br />
<br><br><br />
<b><i>Prochlorococcus</i> sp.</b><br />
<br><br><br />
<b><i>Pseudomonas aeruginosa</i></b><br />
<br><br><br />
<b><i>Rhodobacter</i> sp.</b><br />
<br><br><br />
<b><i>Rhodospirillum rubrum</i></b><br />
<br><br><br />
<b><i>Rickettsia</i> sp.</b><br />
<br><br><br />
<b><i>Saccharomyces cerevesiae</i></b><br />
<br><br><br />
<b><i>Salmonella enterica</i></b><br />
<br><br><br />
<b><i>Serratia marcescens</i></b><br />
<br><br><br />
<b><i>Sinorhizobium meliloti</i></b><br />
<br><br><br />
<b><i>Staphylococcus epidermidis</i></b><br />
<br><br><br />
<b><i>Staphylococcus aureus</i></b><br />
<br><br><br />
<b><i>Streptococcus </i>sp.</b><br />
<br><br><br />
<b><i>Streptomyces</i> sp.</b><br />
<br><br><br />
<b><i>Vibrio cholerae</i></b><br />
<br><br><br />
<b><i>Vibrio fischeri</i></b><br />
<br><br></div>Millerk