Pseudomonas putida

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A Microbial Biorealm page on the genus Pseudomonas putida

Contents

Classification

Higher order taxa

Domain: Bacteria

Phylum: Proteobacteria

Class: Gamma proteobacteria

Order: Pseudomonadales

Family: Pseudomonadaceae

Genus: Pseudomonas

Species: Pseudomonas putida

Species

Pseudomonas putida

Also known as: Pseudomonas ovalis, Pseudomonas arvilla, Arthrobacter siderocapsulatus, Pseudomonas striata, Pseudomonas rugosa, Pseudomonas incognita, Pseudomonas convexa, Pseudomonas eisenbergii, Bacillus putidus, Bacillus fluorescens putidus, and Arthrobacter siderocapsulatus.

NCBI: Taxonomy

Description and significance

Pseudomonas putida is a rod-shaped, flagellated, gram-negative bacterium that is found in most soil and water habitats where there is oxygen. It grows optimally at 25-30 C and can be easily isolated. Pseudomonas putida has several strains including the KT2440, a strain that colonizes the plant roots in which there is a mutual relationship between the plant and bacteria. The surface of the root, rhizosphere, allows the bacteria to thrive from the root nutrients. In turn, the Pseudomonas putida induces plant growth and protects the plants from pathogens. Because Pseudomonas putida assist in promoting plant development, researchers use it in bioengineering research to develop biopesticides and to the improve plant health. [17]

Pseudomonas putida has a very diverse aerobic metabolism that is able to degrade organic solvents such as toluene and also to convert styrene oil to biodegradable plastic Polyhydroxyalkanoates (PHA). This helps degrade the polystyrene foam which was thought to be non-biodegradable. Due to the bacteria’s strong appetite for organic pollutants, researchers are attracted to using Pseudomonas putida as the “laboratory ‘workhorse’ for research on bacteria-remediated soil processes”. [4] This bacteria is unique because it has the most genes involved in breaking down aromatic or aliphatic hydrocarbons which are hazardous chemicals caused by burning fuel, coal, tobacco, and other organic matter. There is great interest in sequencing the genome of Pseudomonas putida due to its strong effect in bioremediation. [3]

Aside from aiding in bioremediation, Pseudomonas putida is very helpful in the research of different species in the genus Pseudomonas, especially Pseudomonas aeruginosa, a pathogenic bacterium that is one of the leading fatal diseases in humans. Researchers find that Pseudomonas putida, although saprophytic, can aid in the research on cystic fibrosis, an inherited disorder caused by a defective CFTR chloride transporter, which leads to recurrent opportunistic infections by Pseudomonas aeruginosa. The two bacteria are very closely related and share similar sequenced genomes (approximately 85% are shared), except Pseudomonas putida lack the genes that determine virulence. Because of its nonpathogenic nature, many researchers find Pseudomonas putida very beneficial to research due to its versatility and ease of handling. [3,4]

Genome structure

In 1995, the scientists at The Institute for Genomic Research in Germany decoded the first complete genome sequence of Pseudomonas putida. Thirty microbial strains have been completed and fully sequenced, while another seventy-five are in the process of being sequenced. [4] Through the genome analysis, Pseudomonas putida is found to have approximately 6.2 million DNA base pairs. Among the Pseudomonas putida, the strain F1 is 5,959,964 nucleotides long and contains 61% guanine and cytosine content and 39% adenine and thymine content. While another important strain, KT2440 is 6,181,863 nucleotides long. [2] Pseudomonas putida has a circular genome where at least eighty genes in oxidative reductases, a family of enzymes, are involved in decomposing substances in the environment. Moreover, the majority of the genes are for detecting chemical signals in the surroundings so it can quickly respond to toxins. [3] This bacterium also has many important plasmids, such as the sequenced TOL and OCT plasmid, which play an important role in the degradation of pollutants. [5, 6] Nonetheless, not all plasmids are helpful in bioremediation. Some create a disadvantage for Pseudomonas putida because it reduces the growth rate and is useless in function such as the plasmid R68-45. [7]

Cell structure and metabolism

Pseudomonas putida is a rod-shaped, nonsporeforming, gram-negative bacteria that utilizes aerobic metabolism. This bacterium also has multiple polar flagella for motility. The flagella have a waveform that is usually 2 to 3 wavelengths long. Pseudomonas putida is sensitive to the environment and suppresses the changes in the direction of flagella rotation upon sensing chemoattractants. This is very helpful in guiding the Pseudomonas putida to propel towards the seeds of the plants which provides nutrients to the bacterial cells. [1]

Pseudomonas putida is able to tolerate environmental stresses due to its diverse control of proteins including protein and peptide secretion and trafficking, protein modification and repair, protein folding and stabilization, and degradation of proteins, peptides, and glycopeptides. [8] Some important proteins include the global regulatory proteins which link the pathway genes to the cell status. Pseudomonas putida exercises a very complex metabolism, the proteins control a particular pathway that not only depends on the signal received, but also the specific promoters and regulators in the pathway. And in turn, once the signals are received, it informs the cell of the oxygen and nutrient availability. Another important protein is the Crc protein which is part of the signal transduction pathway moderating the carbon metabolism. It also functions in biofilm production. [10]

Pseudomonas putida has metabolism functions in biodegradable plastics. Styrene degradation in Pseudomonas putida CA-3 degrades styrene in two pathways 1) vinyl side chain oxidation and 2) attack on the aromatic nucleus of the molecule. [14] Pseudomonas putida also has sideospores, an iron chelating compound that allows the bacteria to enhance levels of iron and promote the active transport chain. [11] Strains of Pseudomonas putida have outer membrane receptor proteins that help transport the iron complex to the sideospores, specifically known as pyoverdines, which are found in the bacterial cell. From there the iron is used in metabolic processes where oxygen is the electron acceptor. [12] Oxygen serves as a good electron acceptor. The oxygen byproducts, however, are toxic to the bacteria including superoxide and hydrogen peroxide. In response, Pseudomonas putida produces catalase to protect the cell from the reactive properties of the byproducts. [13]

In addition, Pseudomonas putida has important lipids that are developed as an adaptation mechanism to respond to physical and chemical stresses. The bacteria is able to change its degree of fatty acid saturation, the cyclopropane fatty acids formation, and the cis-trans isomerization. In different phases, the cell changes its characteristics to better respond to the environment. During the transition from growth to stationary phase, there is a higher degree of saturation of fatty acid and a higher membrane fluidity which improves substrate uptake, thus regulating the cell. [9] All these characteristics allow Pseudomonas putida to survive deadly toxins in the soil and allow it to thrive in contaminated areas. Its metabolism allows these bacteria to convert harmful organic solvents to nontoxic composites which are so essential to bioremediation.

In addition to the ability for P. putida to degrade synthetic compounds, it can also use an alternative metabolic pathway such as the Entner-Doudoroff pathway. In this pathway, P. putida degrades common hexoses, such as glucose and gluconate, to yield one net ATP for every glucose molecule degraded. This is in contrast to the two net ATP produced for every glucose molecule degraded in the classic glycolysis pathway.

The Entner-Doudoroff pathway begins by converting glucose to gluconate-6-phosphate through two intermediates. The first intermediate is gluconate which is then converted to 2-ketogluconate. 2-ketogluconate is then converted to gluconate-6-phosphate. It should be noted that in some cases, gluconate-6-phosphate can be produced directly via phosphorylation of gluconate. The gluconate-6-phosphate is converted to 2-Keto-3-deoxy-gluconate-6-phosphate (KDGP). Finally, KDGP is converted to triosephosphate and pyruvate. Interestingly, P. putida has many alternative pathways that it can utilize to produce energy, yet it does not use them and mainly relies on the Entner-Doudoroff pathway outlined above. [26]

Ecology

Pseudomonas putida are significant to the environment due to its complex metabolism and ability to control pollution. There is a high versatility of bacterial communities towards contaminations which is further increased by certain catabolic sequences on the TOL plasmids in the cell. [7] Even the plasmids are important in sensing the environmental stress. Some of the environmental stresses are caused by benzene, xylene, and toluene, the main components of gasoline and are major sources of water contamination. Pseudomonas putida can degrade the hydrocarbons of these organic solvents through oxidative reactions therefore placing Pseudomonas putida as one of the most important microbes in bioremediation. [15]

Pseudomonas putida also interacts with other organisms in the soil. One such interaction with Saccharomyces cerevisiae in the rhizosphere led to beneficial effects on the state of the Pseudomonas putida. Fungi Saccharomyces cerevisiae produced the necessary glucose and also maintained the pH which was both favorable to the bacteria Pseudomonas putida. [16] The complex interaction of Pseudomonas putida and Saccaromyces cerevisiae together regulate plant health. Moreover, the bacteria itself is a great maintainer of abundant plant life. The production of the siderophores, such as pyoverdine and pyochelin, protect the plants from fungal pathogens. The mutual relationship benefits both partners. While Pseudomonas putida is able to reside in the plant seed and rhizosphere, the plant is, in turn, protected from plant pathogens and able to obtain vital nutrients from the bacteria. [17]

Pathology

In 1982, the US National Institutes of Health designated Pseudomonas putida a safety strain which meant it could be used to clone genes from other soil-inhabiting bacteria. Certain strains of Pseudomonas putida are not pathogenic due to lack of certain genes including those for enzymes that digest cell membranes and walls of humans and plants.

However, several cases were found where Pseudomonas putida was dubbed pathogenic. In one case, Pseudomonas putida was recovered from ten patients with chronic sinusitis. The bacteria was found in the ear, nose and throat. Further investigation found Pseudomonas putida in a commercial anti-fog product and also in StaKleer unopened stock solutions. Because these products were not sterilized correctly, it was infected with the bacteria. [18] In another case, Pseudomonas putida was the cause of the disease outbreak in rainbow trout where it caused ulceration on the dorsal of the fish. [19]

Although Pseudomonas putida is saprophytic and deemed a safe bacteria, other species in the genus are opportunistic pathogens such as Pseudomonas aeruginosa and Pseudomonas syringae. Pseudomonas aeruginosa has been isolated from the human body through many different locations; it also infects the urinary and respiratory tracts. This bacteria is associated with pneumonia, enteritis, vaginitis, and mastitis. Both opportunistic pathogens have the ability and enzymes to release toxins and degrade cell membranes, something that Pseudomonas putida lacks.

Application to Biotechnology

Pseudomonas putida has the ability to produce Poly-3-hydroxyalkanoates (PHA) from the aromatic hydrocarbon styrene. Styrene, a major environment toxic pollutant, is released in millions of kilograms a year from industrial sites and is known to cause spinal tract irritation, muscle weakness, and narcosis in humans and other mammals. The conversion to PHA allows the cure of styrene pollution.

Because PHA is beneficial to society, it serves in medical applications such as tissue engineering and also as antibiotics and vitamins. PHA is also very environmental friendly, oil and grease resistant, and has a long shelf life therefore it is also used in everyday items such as plastic utensils and other disposable items. Unlike styrene, PHA can readily break down in soil or water. Commonly used styrofoam, aka polystyrene foam, is transformed into biodegradable plastic through the Pseudomonas putida’s complex metabolism.

Styrofoam is at first converted to styrene oil where it is introduced to Pseudomonas putida to convert to PHA. [20] Within Pseudomonas putida, PHA accumulates under unbalanced growth conditions as a means of intracellular storage, storing excess carbon and energy. These PHA polymers are synthesized by enzyme PHA synthase which is bound to the surface of the PHA granules and uses coenzyme A thioesters of hydroxyalkanoic acids as substrates. [21]

Current Research

In a recent study, Pseudomonas putida strains were compared to each other to determine the phylogenetic relationships. Because the Pseudomonas putida strain KT2440 genome is fully sequenced it serves as a standard reference to compare with other Pseudomonas putida strains. They carried out a study that assessed the utility of Pseudomonas putida strain KT2440-based high-density DNA microrarrays for transcriptomics studies of DSM 6125, DSM 3931, DSM 291, and S12, along with other non-sequenced strains. Transciptomics allow researchers to gain careful insight into the complex metabolic and cellular systems of the bacteria. In conclusion, they were able to draw similarities between the different strains, for instance, proving that strains DSM 6125 was completely identical to that of DSM 3931. [22]

In another research study, Pseudomonas putida strain RB1500 and strain RB1501 (which were created from Pseudomonas putida strain KT2440) were tested in South Carolina to determine whether light will be produced in the strains in response to trinitrotoluene (TNT), and whether production of light means detection of TNT. The technology will then be used for land mine detection in soils. A concern during the experiment was if Pseudomonas putida will present an ecological hazard; due to the nonpathogenic characteristics of the soil bacteria, however, researchers were assured that it will not cause any hazards to the environment or to the society. [23]

A research proved that Pseudomonas putida N-acyl homoserine lactone quorum sensing regulation involves Lon protease. Screening of a transposon mutant bank of Pseudomonas putida was done to identify regulators that were concerned in negative regulation of acyl homoserine lactones quorum sensing. After the experiment, three Tn5 mutants were identified. One mutant had the transposon located in the Lon protease gene, which plays an important role in degradation of misfolded proteins. In brief, the Lon protease is a negative regulator of acyl homoserine lactone production. [24]

A 2005 research article stated that Pseudomonas putida is associated with clinical infections in pre-mature babies. Babies that were admitted into Special Care Nursery (SCN) were fed through catheters. These catheters went through a heparin flush procedure in order to keep the pathway clear for fluids to enter the body. During this procedure, the environment conditions within the tube allowed P. putida to grow. This growth of bacteria caused bloodstream infections within the baby. Although heparin has antibiotic activity, P. putida is resistant and is able to grow. This shows the importance of asceptic techniques and how preparing sterile products should be done on demand rather than being pre-prepared. [25]

References

1) Harwood, C.S., Fosnaugh K., Dispensa M. “Flagellation of Pseudomonas putida and analysis of its motile behavior”. Journal of Bacteriology. July 1989. Volume 171. p. 4063-4066. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=210162

2) NCBI. http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=21068

3) Marcus, A. “Versatile soil-dwelling microbe is mapped”. Genome News Network. January 2003. http://www.genomenewsnetwork.org/articles/01_03/soil_microbe.shtml

4) Kowalski, H. “U.S. – German Research Consortium Sequences Genome of Versatile Soil Microbe”. J.Craig Venter Archive. December 2002. http://www.tigr.org/news/pr_12_02_02.shtml

5) Vandenbergh, P.A., Wright, A. M. “Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida”. Applied and Environmental Microbiology. June 1983. Volume 45. p.1953-1955. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=242567

6) Muller, R. “Bacterial Degradation of Xenobiotics”. Microbial Control of Pollution. March 1992. Volume 48. p.52.

7) Reanney, D., Gowland, P., Slater, J. “Genetic Interactions Among Communities”. Microbes in Their Natural Environments. April 1983. Volume 34. p.408.

8) TIGR Comprehensive microbial resource. http://www.gem.re.kr/tigr-scripts/CMR2/gene_table_section.spl?db=gpp&main_role=Protein+fate&main_role_only=1&role_order=&nt_choice=tigr

9) Härtig, C., Loffhagen, N., Harms, H. “Formation of trans Fatty Acids Is Not Involved in Growth-Linked Membrane Adaptation of Pseudomonas putida”. Applied and Enbironmental Microbiology. April 2005. Volume 71. p.1915-1922. http://aem.asm.org/cgi/content/abstract/71/4/1915

10) Ruiz-Manzano, A., Yuste, L., Rojo, F. “Levels and Activity of the Pseudomonas putida Global Regulatory Protein Crc Vary According to Growth Conditions”. Journal of Bacteriology. June 2005. Volume 187. p.3678-3686. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1112034

11) Boopathi, E., Rao, K.S. “A sideophore from Pseudomonas putida type A1: structural and biological characterization”. November 1999. Volume 1435. p.30-40. http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=10561535&dopt=AbstractPlus

12) Lopez, J.E., Henkels, M.D. “Utilization of Heterologous Siderophores Enhances Levels of Iron Available to Pseudomonas putida in the Rhizosphere”. Applied and Environmental Microbiology. December 1999. Volume 65. p.5357-5363. http://aem.asm.org/cgi/content/abstract/65/12/5357

13) Miller, C.D., Kim Y.C., Anderson A.J. “Cloning and mutational analysis of the gene for the stationary-phase inducible catalase (catC) from Pseudomonas putida”. Journal of Bacteriology. August 1997. Volume 179. p.5241-5245. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=179388

14) O’Connor, K., Duetz, W., Wind, B., Dobson, A. D. W. “The Effect of Nutrient Limitation of Styrene Metabolism in Pseudomonas putida CA-3”. Applied and Environmental Microbiology. October 1996. Volume 62. p.3594-3599. http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=168165&blobtype=pdf

15) Otenio, M. H., Lopes da Silva, M. T., Marques, M., Roseiro, J., Bidoia, E. “Benzene, Toluene and Xylene Biodegradation by Pseudomonas putida CCMI 852”. Brazilian Journal of Microbiology. Volume 36. p.258-261. http://www.scielo.br/pdf/bjm/v36n3/arq10.pdf

16) Romano, J., Kolter, R. “Pseudomonas-Saccharomyces Interactions: Influence of Fungal Metabolism on Bacterial Physiology and Survival”. Journal of Bacteriology. February 2005. Volume 187. p.940-948. http://jb.asm.org/cgi/content/full/187/3/940

17) Espinosa-Urgel, M., Salido, A., Ramos, J. “Genetic Analysis of Functions Involved in Adhesion of Pseudomonas putida to Seeds”. Journal of Bacteriology. May 2000. Volume 182. p.2363-2369. http://jb.asm.org/cgi/content/full/182/9/2363

18) “Pseudo-Outbreak of Pseudomonas putida in a Hospital Outpatient Clinic Originating From a Contaminated Commercial Anti-Fog Solution”. Canada Communicable Disease Report. November 2000. Volume 26. p.21. http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/00vol26/dr2621eb.html

19) Altinok, I., Kayis, S., Capkin, E. “Pseudomonas putida infection in rainbow trout”. Aquaculture. December 2006. Volume 261. p.850-855. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T4D-4KWT6C1-4&_user=10&_coverDate=12%2F01%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1156f0e28f4bd4f21f8e877e7febb12e

20) Ward, P. G., de Roo, G., O’Connor, K. E. “Accumulation of Polyhydroxyalkanoate from Styrene and Phylacetic Acid by Pseudomonas putida CA-3”. Applied Environmental Microbiology. April 2005. Volume 71. p.2046-2052. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1082534

21) Ribera, R., Monteoliva-Sanchez, M., Ramos-Cormenzana, A. “Production of polyhidroxyalkanoates by Pseudomonas putida KT2442 harboring pSK2665 in wastewater from olive oil mills (alpechin)”. Electronic Journal of Biotechnology. August 2001. Volume 4. http://www.scielo.cl/scielo.php?pid=S0717-34582001000200010&script=sci_arttext

22) Ballerstedt, H., et al. “Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies”. Applied Microbiology and Biotechnology. July 2007. Volume 75. p,1133-1142. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1914237

23) “TSCA Experimental Release Application Approved for Pseudomonas putida Strains”. United States Environmental Protection Agency. April 2007. http://www.epa.gov/oppt/biotech/pubs/submissions/4-5dec.htm

24) Bertani, I., Rampioni, G., Leoni, L., Venturi, V. “The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation”. BMC Microbiology. July 2007. Volume 7. p. 71. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1949823

25) Perz, J., et al. "Pseudomonas putida Septicemia in a Special Care Nursery Due to Contaminated Flush Solutions Prepared in a Hospital Pharmacy" Journal of Clinical Microbiology, 2005. volume 43, p.5316-5318.

26) M. Vicente and J. L. Canovas. “Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants” Journal of Bacteriology, 1973. Volume 116. p. 908-914.

Edited by Mai-An Pham, student of Rachel Larsen

Edited by KLB

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