BIOL 238 Review 2009

This page provides review questions for BIOL 238 (Spring 2009). Answers may be posted by students.

Chapter 1

[Note: To answer a question in edit mode, please place your answer like this, inbetween two double-line breaks.]

1. What historical discoveries in microbiology, both medical and environmental, laid the foundation for the discovery by Rita Colwell and Anwar Huq of an inexpensive way for Bangladeshi villagers to prevent cholera?

Rita Colwell and Anwar Huq performed research on Vibrio cholerae and discovered that it was associated with environmental zooplankton (ie. the copepod). Because copepods are large (200 micrometers), they figured that these copepods could be filtered out of the water and thus reduce the incidence of cholera. They took this to Bangladesh where they taught women to filter their water through their saris, which greatly eliminated the presence of copepods from the water.

2. The Colwell interview depicts three different ways of visualizing microbes. What are the capabilities and limitations of each method? Which method(s) would have been available before Leeuwenhoek? By Leeuwenhoek? For Peter Mitchell and Jennifer Moyle?

The three methods of microbe visualization that are mentioned in the Colwell interview are scanning electron microscopy (SEM), light microscopy, and naked eye visualization. SEM is able to resolve microbes at a very detailed level and appears to give the microbes a 3-D appearance. The only shortcomings with SEM is that the microbe is killed in the process. Light microscopy is useful in that it is not necessary to kill the microbe. However, visualizing very small microbes is near impossible and it is hard to get much resolution. The naked eye visualization is not very helpful in that you can't see much of anything unless the microbes are cultured. Before Leeuwenhoek, only the naked eye visualization method was possible, as he was the first to observe unicellular microbes under a microscope. Leeuwekhoek would have been able to visualize the copeland zooplankton via light microscopy, but the SEM would not yet have been possible. For Peter Mitchell and Jennifer Moyle, they would have had the ability to use all three types of microbe visualization. Because they didn't develop their chemiosmotic hypothesis until 1961, and the modern electron microscope was developed in 1950, they would not have had the same limitations as Leeuwenhoek.

3. Compare the "family tree" of life as drawn by Herbert Copeland, Robert Whittaker, Lynn Margulis, and Carl Woese. How were they similar, and how did they differ? How did their differences relate to different tools available for study?

Herbert Copeland first proposed a classification system that divided the monera kingdom into two groups: eukaryotic protists and prokaryotic bacteria. This was an addition to the original 3 kingdom classification system by Ernst Haeckel which included animals, plants and monera. Robert Whittaker later added fungi onto Copeland's 4 kingdom hypothesis to make a 5th kingdom (bacteria, protists, fungi, multicellular plants, and multicellular animals). Neither Copeland or Whittaker had access to DNA information, which caused their classification system to be based solely on observation of cellular structure, biochemistry and behavior.

Margulis modified this system by suggesting the endosymbiosis theory, in which eukaryotes evolved by merging with bacteria by the idea that one cell internalized another. This was equated to the mitochondria (for example) in eukaryotic cells. This theory also suggested that these organisms had multiple ancestry of living species (polyphyletic), rather than having evolved from a common ancestor (monophyletic). DNA sequencing was later able to confirm this hypothesis.

Carl Woese utilized gene sequencing to determine the evolutionary origins of living things. His discoveries (mainly with archaea) suggested the need for a broader taxonomic classification than the kingdom. He named this taxa the domain, of which there are three: Bacteria, Eukarya, and Archaea. This differentiation was important because the archaea domain was shown to have genetic sequences which diverge equally from those of bacteria and eukaryotes.

4. Outline the different contributions to medical microbiology and immunnology of Louis Pasteur, Robert Koch, and Florence Nightingale. What methods and assumptions did they have in common, and how did they differ?

Nightingale used medical statistics to show that the mortality of soldiers increased with septic conditions in the summer months. Pasteur's finding that yeast can produce alcohol in the absence of oxygen led him to assume that Spallanzani's failure to find spontaneous generation was not due to a lack of oxygen. By using an unsealed flask with a "swan neck," which admitted air while keeping the boiled contents microbe-free, he found that the growth medium within the flask remained microbe-free for years. However, by tilting the flask and allowing the growth medium to contact the broth with dust containing microbes, growth occurred. Finally, Koch was responsible for developing the causative linke between a pathogen and a disease. Nightingale used statistical analysis to elucidate the causes of disease and found a positive correlation between a septic environment and disease frequency. Both Pasteur and Koch used a similar approach in that they both did experiments that allowed them to factor out confounds and focus on a causative link. After Pasteur found no growth even in the presence of oxygen, he then tilted the flask in order to establish the link between dust contanct and growth. Koch could have easily stopped after postulate 2, but in order to establish causation instead of correlation, he needed to introduce the isolated microbe into a healthy organism to see if the same symptoms occurred and reisolate it again. Pasteur's experiment was essentially trying extend Spallanzani's and disprove proponents claiming that non-spontaneous generation was a lack of oxygen, while Koch put Nightingale's statistics and Pasteur's theory that microbial growth requires preexisting microbes together to elucidate the chain of infection in organisms.

These are excellent insights. The one point I'm not sure about is that Koch probably did not know about Nightingale's statistics, but he knew about anecdotal evidence of disease associated with germs. His postulates were designed to give a "yes or no" answer, rather than a statistical probability. Koch was aware of Pasteur's work, and much of their experiments consisted of a scientific dialogue (often contentious).

5. Does the human immune system react similarly to both attenuated pathogens and more active pathogens?

Yes, if the weakened (attenuated) pathogens are introduced in great enough quantity. The weakened pathogens will have the same physical presence as the more active form - human antibodies should bind to both with equal tenacity and trigger the immune response. Furthermore, if the active form has increased resistance to phagocytosis or is capable of weakening the immune system, then human response to the attenuated type will be more favorable (for the human.

6. Outline the different contributions to environmental microbiology of Sergei Winogradsky and Martinus Beijerinck. Why did it take longer for the significance of environmental microbiology to be recognized, as compared with pure-culture microbiology?

Winogradsky was one of the first to study microbes in their natural environment. He developed a model of wetland microbial ecosystems, the Winogradsky column, which could be studied within a laboratory setting. He also discovered lithotrophic bacteria and utilized selective growth media in order to sustain them in the lab. His findings demonstrated the important role of bacteria in geochemical cycling.

Beijerinck was the first to discover the endosymbiotic relationship between nitrogen-fixing bacteria and leguminous plants.

7. It is always necessary to prepare a tissue culture to study viruses, as they can't grow without a host cell. Do certain bacteria need tissue in their cultures?

Two examples of bacteria that have yet to be grown out of tissue culture are Rickettsia prowazekii (the pathogen that causes Rocky Mt. fever) and chlamydia. It seems that these bacteria are so efficient at surviving in their respective environments that they have lost the ability to perform certain necessary functions on their own.

A question was raised in the review session today as to what, then, really differentiates these microbes from viruses...

8. How did Alexander Fleming's cultured plate of Staphylococcus become moldy with Penicillium notatum? Is it common for petri dishes to become moldy if left in the open air for too long?

Chapter 2

1. Explain what features of bacteria you can study by: light microscopy; fluorescence microscopy; scanning EM; transmission EM.

Light microscopy includes bright-field and dark-field. Bright-field allows you to determine the relative shape of individual microbes and how they associate with one another (chains, tetrads, etc.) Using dark-field, you can detect narrow microbial species and subparts (flagella). Fluorescence enables you to label specific parts of cells using antibody tags. It is good for the detection of microbes and subcellular structures and avoid seeing "dust" and non-living cells. Further, it is good for detection of organisms living in dilute environments. SEM enables you to examine the surface of microbes in detail and see smoothness/bulges that serve special functions such as pathogen attachment. TEM enables you to determine the intracellular structures of attachment sites and internal organelles as well as the shape of macromolecular complexes.

2. Explain the difference between detection and resolution. Explain how resolution is increased by magnification; why can't the details be resolved by your unaided eye? Explain why magnification reaches a limit; why can it not go on resolving greater detail?

Detection is the ability to perceive the presence of something, while resolution is the smallest distance between two objects at which they can be distinguished. Therefore, resolution refers to the detail in which one sees an object. Resolution with the human eye is limited by the distance between photoreceptors, which is about 500 times larger than the wavelengths of visible light. For an object to be resolved, light with a wavelength equal to or smaller than the object is needed. Magnification increases resolution in that it spreads these light rays needed to observe small objects so that they may be detected by the photoreceptors. This appears to make an object bigger. Magnification is limited due to interference at the focal point from converging light rays and the resulting Airy disks. This decreases the expansion of detail that accompanies increased magnification.

3. How does refraction enable magnification?

The light that pass through a refractive media are shaped in a way (convex lens) that spreads its rays, thus widening the wave front. The parallel light impinging on the lens intersect at the focal point and continue to expand outward on the other side of the lens. The refractive index of the lens is higher, thus the light slows down, thus causing a change in direction.

4. Explain why artifacts appear, even with the best lenses. Explain how you can tell the difference between an optical artifact and an actual feature of an image.

5. How can "detection without resolution" be useful in microscopy? Explain specific examples of dark-field observation, and of fluorescence microscopy.

6. Explain how the Gram stain works. What are its capabilities and limitations? How does the Gram stain relate to bacterial phylogeny?

The gram stain works by using a dye like crystal violet to bind to the bacteria. When iodine is added to the cells, the stain binds to the gram positive cells which have thicker cell walls. After the cells are washed, the stain remains bound to the gram-positive bacteria while it is washed away from the gram-negative bacteria, which have thinner cell walls. The thicker the cell wall of the bacteria, the more dye that is retained. A safranin counterstain is then added which colors the gram-negative cells pink. The gram stain is useful in that it can easily distinguish between two different groups of bacteria that each have very different cell wall structures. It can also improve detection of these bacteria. The only limitations of the stain are that the cell is killed during this process. Additionally, because light microscopy is used to view these cells, the resolution of them is limited. The gram stain relates to bacterial phylogeny as it differentiates between a key biochemical characteristic of the cell: the cell wall. It is likely that those cells who are gram-negative will have genetically similar traits, as well as the genetic traits of the gram-positive bacteria being similar as well.

7. If shapes of bacteria are common to many taxonomic groups, including spirochetes which cause Lyme disease as well as others, how accurately can different bacteria be identified just based on shape?

8. Why should we believe scanning probe microscopy (SPM) is accurate? If scientists should be concerned by possible artifacts in EM why wouldn‘t they be concerned about artifacts or even further complications in SPM?

9. When would you use TEM over SEM, or vice versa?

TEM would be used to view internal structures of a microorganism. In TEM, electrons pass through the sample, and the electron density is added through the depth of the section. For example, TEM could be used to view a cross section of a microorganism. SEM would be used to examine the outer surface and shape of a specimen, as the electron beams move over the sample to create a three-dimensional picture. Additionally, SEM is able to cover a very broad size range (much larger than TEM).

Chapter 3

1. For one of your card pathogens, explain the type of cell membrane, cell wall, and outer membrane if any. Explain how any particular components of the membrane and envelope contribute to pathogenesis.

2. Compare and contrast the structure and functions of the cell wall and the S-layer.

3. The antibiotic linezolid prevents the 50S ribosome subunit from binding the 30S subunit. If you isolate ribosomes by ultracentrifugation, how might the results in the tube look different with linezolid present?

One would witness fractions of the 30S and 50S subunits at their appropriate levels in the sucrose gradient. No 70S (entire ribosome complex) fraction should be seen. If linezolid is large enough (probably not?) then it may also have its own fraction in the centrifuge tube.

4. Explain how the FtsZ and MinD proteins function in cell division. What happens to a cell with a mutation in one of these genes?

5. In the laboratory, what selective pressure may cause loss of S-layers over several generations of subculturing? Similarly, why would subcultured bacteria lose flagella?

Loss of the S-layer is an example of reductive evolution. S-layers require many proteins/glycoproteins for construction, and are therefore energetically costly to maintain. In the lab culture there is no risk of predation, so those organisms that lose their s-layers and focus their resources on proliferation are the most successful. Loss of the flagella probably occurs because growth medium is abundant and the organisms have little need to move - the food is all around them.

6. For one of your card pathogens, explain what specialized structures it has, such as pili or storage granules. Explain how they might contribute to pathogenesis.

7. Why might a human cell have a protein complex that imports a bacterial toxin? How might such a situation evolve?

One way in which this may happen is that the the receptor for this bacterial toxin may be present in many species, but the protein toxin is really only "toxic" to certain species. An example of this is E. coli O157:H7, which is present in undercooked hamburger meat (thus lives in the cow naturally), however, ingestion of this strain in humans causes hemolytic uremic syndrome. The protein may evolve perhaps because of it benefiting certain species but is detrimental to other species due to other environmental/physiological factors.

8. What aspects of the outer membrane prevent phagocytosis, and how?

The LPS is the outermost covering on gram negative and gram positive cells and is essentially a slippery, mucousy capsule, which helps prevents phagocytosis by macrophages.

9. If the peptidoglycan cell wall is a single molecule, how does the cell expand and come apart to form two daughter cells?

10. What form of energy is used to drive the membrane-embedded ATP synthase, and the flagellar motor? Suppose a cell only makes ATP from glucose breakdown (not from the membrane complex). How could it use the membrane ATP synthase complex to drive flagellar rotation?

11. Explain two different ways that an aquatic phototroph might remain close to the light, or that an aerobe might remain close to the air surface.

Bowman et al., 2008

1. Compare and contrast the mechanisms of cell division and DNA replication in Caulobacter crescentus and in E. coli. What feature of C. crescentus cell division may explain the different organization of DNA replication?

2. Draw a diagram showing how Caulobacter replicates its DNA during cell division. Show the positions and movements of proteins MreB, FtsZ, ParB, MipZ, and PopZ.

3. Explain what is tested, and what the results show about cell division, in Figures 1, 2, 3, and 5. For each figure, explain what the panels show, and what remains to be shown.

Chapter 4

1. Suppose in Yellowstone Park, Mammoth Spring, a thermophilic bacterium (Bacillus steareothermophilus increases its population size by ten-fold in 40 minutes. What is the generation time, or doubling time? Why might these bacteria grow faster than Bacillus megaterium, in our laboratory at Kenyon?

2. Mycobacterium tuberculosis, the cause of tuberculosis (TB), has a generation time of 18 hours. How many days will it take to grow a colony containing a million cells? What is the consequence for research on TB?

3. Explain the different mechanisms that membrane protein complexes can use to transport nutrients: ABC transporters, group translocation, and ion cotransport (symport and antiport). Discuss the advantages and limitations of each mechanism.

4. Under what growth conditions do bacteria eat the contents of other bacteria? How do they manage do do this? What is the significance for medical research?

This phenomena can occur in the late stationary stages of batch cultures. When major nutrients in the broth have been depleted, bacteria may start killing each other in order to obtain these necessary materials.

Another interesting example is a strategy used by actinomyces (i.e. streptomyces). The back end of the bacterium undergoes senescence and leaks nutrients; this attracts other bacteria that would like to eat this particular bacterium. At the same time, the front end releases antibiotics that kill the approaching bacteria. The actinomyces then eats the remains of the others that had come to prey on it.

5. In the laboratory, why is it important to grow isolated colonies? What can occur in colonies that we might not notice? What research problems cannot be addressed with isolated colonies?

6. Compare and contrast the advantages and limitations of different responses to starvation: stationary phase; sporulation; and fruiting body formation.

7. Explain the differences between: phototrophy and chemotrophy; autotrophy and heterotrophy; literotrophy and organotrophy. Explain examples of metabolism combining aspects of these concepts.

Chapter 5

1. Look through a grocery store, inspecting the labels of packaged foods. What chemical preservatives do you recognize, and what is their mechanism for killing bacteria or inhibiting growth? For example, propionate and sorbate are membrane-permeant acids that depress cytoplasmic pH.

2. Explain the major difference between the effects of general sterilization and disinfectants, versus antibiotics such as penicillin or streptomycin. Why do antibiotics rapidly select for resistant strains, whereas disinfectants and sterilizing agents do not?

3. Explain which extreme environmental conditions select for membrane unsaturation. What is the advantage of unsaturated membranes for these conditions?

4. Explain how protein structure is modified during evolutionary adaptation to high temperatures, or to high pressure.

5. Suppose it takes a heat treatment 3 minutes to halve the population of bacteria in the food. How long will it take to decrease the bacteria content by 2D-values? Would you want to eat the food at this point? Explain.

6. What kind of habitats will show halophiles? What is the difference between moderate halophiles, extreme halophiles, and halotolerant organisms? Describe what will happen to halophile populations in a pool under the hot sun.

7. What is the mechanism of killing of organisms by ionizing radiation? Why is ionizing radiation less effective on frozen foods?

8. Explain the mechanism of action of Penecillin, and of Linezolid. How might bacteria evolve resistance to each antibiotic? Describe a form of resistance carried on a plasmid, and a form of resistance inherited on the genomic chromosome.

Chapters 6 and 11

1. Discuss the different functions of different structural proteins of a virion, such as capsid, nucleocapsid, tegument and envelope proteins. How do these functions compare and contrast with functions of cellular proteins?

2. Explain how viruses are cultured, and how a pure isolate of a virus can be obtained. How do the procedures differ from that of pure culturing bacteria? What special difficulty arises when defining genetically pure isolates of RNA viruses such as herpes and HIV?

3. Explain two different ways that viruses may cause cancer (oncogenesis). How can strongly oncogenic viruses be assayed in culture?

4. What are the relative advantages of the virulent phage life cycle of phage T4; the lysis-lysogeny options of phage lambda; and the slow-release life cycle of phage M13? Under what conditions might each strategy be favored over the others?

5. Compare and contrast the life cycles of polio virus and influenza virus. What do they have in common, and how do they differ?

6. RNA viruses and DNA viruses represent fundamentally different reproductive strategies. What are the relative advantages and limitations of each? How do their different strategies impact the immune response, and the development of antiviral agents?

7. Discuss the role of host-modulating viral proteins in smallpox, in herpes, and in papillomavirus. What various kinds of functions do these proteins serve for the virus; and what are their effects on the host?

Chapter 7

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?

2. What kinds of mutant phenotypes reveal aspect of the mechanism of DNA replication and cell division? Explain two specific examples.

3. Explain how it's possible for the replisome to replicate the leading and lagging strands simultaneously.

4. During resolution of a catenane, how might a major mutation occur affecting the entire genome? How do you think this mutation is prevented?

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.”?

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.

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?

Chapter 8

1. Explain how a biochemical experiment can demonstrate the specific protein targeted by a new antibiotic that impairs transcription.

2. If Mycoplasma genitalium 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?

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.

4. What kinds of pharmaceutical agents could you design to act on gene promoters? Explain using protein and/or RNA molecules.

5. Why do you think bacterial cells absorb protein and nucleic acids that are exported by other bacteria?

6. How could you sequence the genome of an unculturable microbe?

Chapter 9

1. In the process of conjugation, how are genes moved? Are genes moved individually or in groups? Could part of a gene be moved?

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.

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?

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?

5. Explain how a DNA sequence inverts during phase variation. Would you expect it to revert at the same rate? Why or why not?

6. Explain the different propagation strategies available to a replicative transposon. What are various ways the transposon could spread within a cell? Among organisms?

Wozniak lecture on Biofilms

1. What do bacterial biofilms have in common with multicellular organisms? How do they differ?

2. What are the advantages to bacteria of biofilm formation? What properties do biofilms confer?

3. Where in the body do biofilms form infections? Why?

4. Explain the basis of "twitching motility." Compare and contrast twitching with flagellar motility. How does twitching motility promote biofilm development?

5. How does the ara promoter work (pBAD)? How was pBAD used to test the role of the psl operon in bioflim development?

6. How was it proved that psl encodes PSL polysaccharide? How does PSL compare in structure with alginate?