Halobacterium NRC-1

Description and significance
Pseudomonas putida are Gram-negative rod-shaped bacteria. They are classified as Group 1 in Pseudomona. Other Pseudomonads are being re-evaluated to see if they truly fall into this category, while P. putida is firmly place in this group. P. putida are flourescent, aerobic, non sporeforming, oxidase positive bacteria. Having one or more polar flagella, they are motile organisms. They can be found in moist environments, such as soil and water, and grow optimally at room temperature. Certain strains have the ability to grow on and break down many dangerous pollutants and aromatic hydrocarbons such as toluene, benzene, and ethylbenzene. P. putida can also be used in petroleum plants to purify fuel. This bacterium is also capable of promoting plant growth after root colonization as well as simultaneously providing protection for the plant from pests and other harmful bacteria.

Genome structure
The genome of Pseudomonas putida was sequenced due to the many unique abilities that this bacterium possesses. Scientists are interested in which genes cause what function. So far, P. putida has the most genes of any microorganism that break down chemicals such as aromatic hydrocarbons. Research is being done on the difference in genome of P. putida and its relative Pseudomonas aeruginosa in relation to cystic fibrosis. While P. aeruginosa infects and kills those with the disease, P. putida lacks the genes that causes such destruction, like the genes that code for enzymes that digest cell membranes.

The Pseudomonas putida strain KT2440 genome was sequenced as a joint project between The Institute for Genomic Research and a German consortium in 1999. The way that they sequenced the genome was using the random shotgun method. They found that the one circular chromosome contains 6,181,863 base pairs. The total number of genes is 5,516, with 5,421 being protein coding. The total number of repeats, or stretches greater than 200 base pairs and almost identical, was 398. Interestingly, there was a high GC content in the genome, which created some difficulty in sequencing through the traditional methods. A significant amount of genes were found to code for enzymes that are used in the decomposition of matter. Most of the other genes are critical for Pseudomonas putida’s ability to recognize and react to external toxins and chemical signals. They also contain multiple accessory plasmids, including TOL and OCT plasmids, that aide the bacterium in breaking down environmental pollutants found in soil and water. Through sequencing the Pseudomonas putida genome, scientists were able to determine the biotechnological potential of the organism.

Cell structure and metabolism
Pseudomonas putida are aerobic oxidase positive bacteria, with one or more flagella. They can be found in moist environments, such as soil and water, and grow at a temperature of 25-30 degrees Celcius. Although Pseudomonas putida does not form spores, they are still able to withstand harsh environmental conditions. It is able to resist the severe effects of organic solvents that pollute the surrounding soil. In response to changes in its chemical surroundings and to help with membrane fluidity and cellular uptake, it can alter the degree of fatty acid saturation and even undergo cis-trans isomerization. P. putida are unique saprobes in that use a wide variety of non-living material as their source of nutrition, including multiple types of aromatic hydrocarbons. This allows them to be agents of bioremediation, one of the most differentiating and impressive features of Pseudomonas putida.

Ecology
Pseudomonas putida has an incomparable effect on the environment. They are able to protect plants from pests, promote plant growth, and clean up organic pollutants found in soil and water.

The surface of the root and the soil that surrounds it are loaded with nutrients released by the plant. This environment is optimal for microbial growth. Pseudomonas putida is attracted to this area, and in turn promotes plant growth and even protects the root against pathogens. Two key elements that allow P. putida to attach in the first place is that they are motile and chemotactic towards the root output. After the initial attraction and migration toward the root, the bacteria immediately begins to grow and divide, forming multiple colonies around the root. The maximum population size is directly related to root weight, and once it is reached the number of colonies will stay constant. All of this can happen in less than 48 hours!

Pseudomonas putida play a huge role in bioremediation, or the removal or naturalization of soil or water contaminants. They can degrade toluene, xylene, and benzene, which are all toxic components of gasoline that leak into the soil by accidental spills. Other strains can convert styrene, better known as packing peanuts, which do not degrade naturally, into the biodegradable plastic polyhydroxyalkanoate (PHA). Methods used to get rid of styrene include incinerating it, spreading it on land, and injecting it underground, all of which release the toxins into the environment. Styrene can cause muscle weakness, lung irritation, and may even effect the brain and nervous system. Due to the fact that P. putida can use styrene as its only source of carbon and energy, it can completely remove this toxic chemical. P. putida can also turn Atrizine, an herbicide that is toxic to wildlife, into carbon dioxide and water.

Pathology
In genetic terms, Pseudomonas putida is very similar to strains of Pseudomonas aeruginosa, an opportunistic human pathogen. Although there is a considerable amount of genome conservation, P. putida seems to be missing the key virulent segments that P. aeroginosa has. Being a non-pathogenic bacteria, there has been only a handful of episodes where P. putida has infected humans. For the most part, it has been with immunocompromised patients, causing septicaemia, pneumonia, urinary tract infections, nosocomial bacteremia, septic arthritis, or peritonitis. P. putida is also closely related to Pseudomonas syringae, an abundant plant pathogen, but again it lacks the gene that causes such disease.

Several cases of disease caused by Pseudomonas putida have been investigated, being that the bacterium rarely colonizes mucosal surfaces or skin. One case was a 43-year-old female who was receiving nightly peritoneal dialysis treatments following a laparoscopic ovarian cyst operation. She developed peritonitis due to infection by Pseudomonas putida. Through this case and others, it was determined that risk factors for developing such an infection include the insertion of catheters, intubation, and/or intravascular devices following a recent course in antibiotics.

Another case of Pseudomonas putida infection was found in ten patients in and ear, nose, and throat outpatient clinic during the summer of 2000. All ten patients had chronic sinusitis, making them more susceptible to infection due to their challenged immune systems. Through investigation, it was discovered that all of the patients shared the same examination room. The source of the bacteria was from a contaminated bottle of StaKleer found in that room. StaKleer is an anti-fog solution used on mirrors and endoscopes to prevent condensation from occurring, allowing for the proper visualization of tissues. Other unopened bottles of the solution at the clinic were found to be contaminated with Pseudomonas putida as well.

Application to Biotechnology
Pseudomonas putida is being used in conjunction with Escherichia coli for developing new drugs. This study focuses on myxochromide S, a compound produced by Stigmatella aurantiaca, but the method is revolutionary in that there is unprecedented expression of gene clusters. The beginnings of many new drugs are from natural sources, such as plants and microorganisms, but they are too expensive to harvest from the origin. Combinatorial biosynthesis has revolutionized drug development by allowing the structure of certain molecules to be changed within an organism. With this metabolic engineering, where genes are introduced and their expressions are tightly controlled, successful production of drugs is possible. Pseudomonas putida is unique in that it allows the expression of a large biosynthetic cluster, producing five times as much myxochromide S as Stigmatella aurantiaca. This will also permit scientists to connect multiple clusters of genes onto a single DNA fragment.

Pseudomonas putida is able to purify fuel, a capability that the petroleum industry has taken great interest in. As previously mentioned, P. putida is able to convert styrene, a toxic waste product, into a biodegradable plastic. The strain CA-3 turns styrene into a stored energy source, in the form of a plastic polymer called polyhydroxyalkanoate (PHA). Using styrene its only source of carbon and energy, the styrene is completely used up, creating an elastic type of polymer. This polymer can then be used in the production of drug carriers, plastic coating of cardboard, and medical implants.

"Benzene, Toluene, and Xylene Biodegradation by Pseudomonas putida CCMI 852"
Gasoline spills create a large amount of toxic pollution in the environment, being that the major components are benzene, toluene and xylene isomers. Catalogued by the U.S Environmental Protection Agency as “priority pollutants”, gasoline is a main cause of water well and spring contamination. Pseudomonas putida can successfully degrade these dangerous components of gasoline, and can aide in the cleanup of such pollutants. This article discusses the research being done in order to determine what environment and mixture of compounds will allow for the most degradation. The metabolic pathway that P. putida uses to break down these compounds is investigated. The TOL pathway does not utilize benzene as a substrate, while the TOD pathway does. Various combinations of these elements of gasoline were used during experimentation. Maximum degradation occurred when each compound was alone in solution. Once any other compound was introduced, the rate automatically dropped, but it dropped most drastically when benzene was introduced. Toluene was degraded at a rate twice as fast as xylene. Benzene concentration always remained unchanged, when alone or in a mixture. It is then obvious that P. putida did not degrade benzene, even when no other compounds were present. It is suggested that this P. putida CCMI 852 strain contains a TOL plasmid, therefore preventing the degradation of benzene.

"Diversity and activity of biosurfactant-producing Pseudomonas in the rhizosphere of black pepper in Vietnam"
Black pepper, a major crop and source of income for the country of Vietnam, is also the most important spice crop in the world. This ‘King of Spices’ is particularly susceptible to the pathogen Phytophthora capsici, which eats away at the roots of the plant and causes death and disease. It can potentially cause up to 40-50% of crop death, and in Vietnam an annual loss of around 20%. Pseudomonas putida was found to produce biosurfactants, which disrupt the membrane of the ''Phy. Capsici'' zoospores, causing death within minutes. Using P. putida as a method for controlling ''Phy. Capsici'' will be more effective than using pure biosurfactants created in a lab. One reason for this is that the chemical form may not be delivered efficiently to the roots, because they must fully penetrate the soil to the level of the rhizosphere. P. putida is chemotactic towards root output, and move via flagella directly toward the root, subsequently creating secure colonies. Using P. putida as a pesticide will also be a long-lasting form of treatment, because the colonies not will get washed away by a rainstorm like the chemical form could.

Rhizosphere samples were taken from three different districts in Vietnam, and various testing was done with different biosurfactanct producing organisms and bacteria. P. putida provided a significant amount of prevention of plant wilt, providing adequate protection from ''Phy. capsici''. This was greater then when contrasted with other bacteria tested, such as Bacillus spp., Trichoderma harzianum, and P. flourescens. When P. putida was introduced to plant root when no pathogen was present, there were more interesting results. This bacterium had considerably increased shoot height and weight, and also raised the number of roots grown. More research is being done in the area, as it further development of this idea will help control plant pathogens.

"Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3"
Pseudomonas putida has the unique ability to transform a toxic pollutant into a biodegradable plastic. Over 25 million kilograms of styrene, a compound that can cause respiratory tract infection, muscle weakness, and narcosis, is released into the environment every year in the U.S. alone. The product, polyhydroxyalkanoate, can be used as synthons for certain antibiotics, vitamins, and anti-cancer drugs. They are also used in other areas of medical applications such as tissue engineering and wound management. The pathway and mechanism for this transformation is investigated, where aromatic hydrocarbons are transformed into aliphatic PHA. During the growth cycle, there is the strict use of carbon and nitrogen with styrene, glucose, and phenylacetic acid by P. putida as the carbon and energy source. The effects of altering the carbon to nitrogen ratio and level are tested in relation to the accumulation of the product PHA.

The study showed that when nitrogen levels dropped, PHA production began. The effect of carbon supply was studied as well, and only when carbon to nitrogen ratios of 9:1 for cells on glucose, 10:1 for cells on phenylacetic acid, and 14:1 for cells grown on styrene were reached did PHA begin to accumulate. Next, nitrogen was manipulated while carbon was held constant, showing that the highest PHA content was found when nitrogen concentration was the lowest.

The manner in which styrene is converted to PHA is through fatty acid de novo biosynthesis, which is catalyzed by 3-hydroxy-acyl-ACP:CoA transacylase. The plastic polymer has a destruction point of 265 degrees Celcius, and a uniquely low molecular weight and high polydispersity. This is also the first time that an aromatic substrate is converted into aliphatic PHA. Further research will be done to investigate ways to increase PHA yield from styrene using P. putida.