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The isolation of a Pseudomonas putida DTB bacterium capable of utilizing N,N-diethyl-m-toluamide (DEET) as sole carbon and energy source and hydrolyzing DEET into 3-methylbenzoate and diethylamine is reported.

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. A demonstration that the ptsN gene product [IIA.sup.Ntr] participates in this repression is presented.

A [sigma.sup.54] dependent two-component system, the AauR-AauS that controls the uptake and metabolism of acidic amino acids in Pseudomonas putida was identified. The results confirmed that the aau system is a major regulator of acidic amino acid uptake and utilization.

Results indicate that depending on the carbon source used, supplied interms of aromatic hydrocarbons, bacteria exhibit different carbon and hydrogen stable isotope fractionation during substrate degradation as shown by Pseudomonas putida, strain mt-2, strain F1, and strain NCIB9816 and Ralstonia picketti strain PK01.

Results show that 2-aminophenoxazin-3-one-7-carboxylate is formed via oxidative dimerization of 4-amino-3-hydroxybenzoate, which undergoes replacement of the 4-amino group by a hydroxyl to form protocatechuate. Data indicate that 4-hydroxylaminobenzoate lyase converts 4-hydroxylaminobenzoate to 3,4-dihydroxybenzoate (protocatechuate).

Research has been conducted on hydroxyectoine. Results suggest that for Pseudomonas putida hydroxyectoine is superior to trehalose as a desiccation protectant and can be a drying excipient for the live microorganisms which are recalcitrant to trehalose.

Research has been conducted on the isolated toluene-tolerant strain Pseudomonas putida DOT-T1E. Results indicate that it can grow on high p-hydroxybenzoate concentrations.

Mutant analyses point out that phenylacetyl-CoA induces the central catabolism pathway involved in the aerobic degradation of phenylacetic acid in Pseudomonas putida U and not any other structurally or catabolically related aromatic compounds.

Microbial mercury removal using a mercury-resistant Pseudomonas putida strain, Spi3, is a potential biological treatment for chloralkali electrolysis wastewater. Treatment of actual chloralkali electrolysis factory effluents with a mercury-resistant P. putida strain isolated from polluted river sediments has been studied using a stepwise approach. Chloralkali wastewater from several plants in Europe was studied so that a system could be tailored to each condition. NaCl was the most common copollutant and interferes with mercuric reductase activity.

A high-expression clone for todC1 has been created while the purification and properties of TodC1 have been studied. The electron transfer between Ferredoxin(sub TOL) and TodC1 has been examined while TDO activity from purified TodC1 and C2 subunits have been reconstituted in vitro. TodC1 and C2 are the alpha and beta subunits of the oxygenase component of toluene dioxygenase from Pseudomonas putida F1. Reductase(sub TOL) is a flavoprotein reductase that transfers electrons from NADH to the Rieske protein, Ferrodoxin(sub TOL).

The metabolism of 2-chloro and 3-chlorotoluene by Burkholderia sp. strain PS12 and Pseudomonas putida F1 was analyzed to determine the biochemical characteristics of the doixygenolytic pathway. The toluene dioxygenase of Pseudomonas putida F1 and the chlorobenzene dioxygenase of Burkholderia sp. PS12 strain catalyzed the conversion of 2-, 3- and 4-chlorotoluene to dihydrodiols. The synthetic process was characterized by the formation of minor products such as methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes from 2- and 3-chlorotoluene.

The effects of toluene on Pseudomonas putida mt-2(pWW0) strain were analyzed to determine the biochemical characteristics of the microbe's stress response to carbon starvation. Protein synthesis was inhibited in Pseudomonas putida mt-2(pWW0) strain that was cultured in a medium in which toluene was the sole source of carbon. Toluene also elicited carbon starvation response in Pseudomonas putida which produced 26 new proteins during starvation. Furthermore, carbon supplementation of toluene-exposed cells rapidly suppressed the production of these proteins.

Naphthalene dioxygenase (NDO), expressed by Pseudomonas putida, catalyzes regio- and stereo-specific dihydroxylation of 9,10-dihydroanthracene (DHA) and 9,10-dihydrophenanthrene (DHP). The dihydroxylation produces previously undescribed homochiral cis-diene diols of R configuration at the bridgehead carbon atom-adjacent benzylic center. The location of the benzylic carbons affects the type of reaction that NDO catalyzes in benzocyclic substrates. The regio- and stereo-chemistry of the DHP and DHA cis-diols is consistent with the cis-diols of naphthalene, anthracene and phenanthrene.

Naphthalene degradation is not only maintained in starved (without substrate) Pseudomonas (P.) putida and Alcaligenes cultures, but the rate is higher than in the stationary-phase cultures. This indicates continuation of naphthalene-degrading enzymes during starvation. Naphthalene degradation activity in stationary-phase culture is low in both the bacteria, but that of Alcaligenes is considerably lower than P. putida. Induction of a high rate of naphthalene degradation activity by exposure to naphthalene takes a brief time for P. putida while it takes a longer time for Alcaligenes.

Pseudomonas putida M10 is capable of dissimilating morphine to morphinone by a highly specific NADP-dependent morphine dehydrogenase. Further characterization of the morphine utilization pathway by M10 showed the presence of a second enzyme involved in the dissimilation of morphine, an NADH-dependent reductase that reduces morphinone to hydromorphone, an important analgesic, and hydrocodone an antitussive. This enzyme has potential applications as a biocatalyst for the synthesis of these therapeutic agents.

The growth and respiration of Pseudomonas putida PpG7 was monitored in three field soils supplemented with the exotic carbon source, salicylate. Based on evolved carbon dioxide, the results showed that population density and activity are not neccessarily correlated. The addition of salicylate could selectively increase the growth and metabolism of PpG7, but the increase in growth was not concomitant with increase in activity. These results emphasize the importance of using an ecological kinetic approach when studying the population dynamics of a particular organism.

The effects of salicylate amendment and soil fumigation on the growth of Pseudomonas putida PpG7 introduced in agricultural field soils planted with tomato were investigated. The results showed that soil fumigation could significantly increase the population densities of PpG7. Other factors that affected population densities were inoculum level, field site and soil depth. Salicylate selectively increased PpG7 levels, and coupled with soil fumigation, caused a significant reduction in population densities of fungal salicylate users.

Pseudomonas putida strain Idaho, which has the ability to grow on concentrated solutions of p-xylene, toluene and other alkylbenzenes as sole carbon and energy sources, was characterized physiologically. The results showed that the bacterium's ability to grow on organic solvents was correlated with the logarithm of the octanol-water partition coefficient. Morphological changes during growth included the convolution and shedding of the outer membrane, and the invagination and vesicle-formation of the cytoplasmic membrane. Hybridization experiments showed the presence of sequences homologous to the upper operon in the TOL plasmid pWWO.

The effects of phenol on the lipid constituents of Pseudomonas putida P8 membranes were studied. Chromatographic and spectrometricexperiments showed that phenol induces cis-to-trans isomerization of fatty acids. One particular fatty acid identified was 9-trans hexadecanoic acid. Thiscis-to-trans isomerization was shown to be independent of lipid biosynthesis, and is catalyzed by a yet unknown cis-trans isomerase. Such cis-trans isomerization may serve to reduce membrane fluidity and permeability, as a protective mechanism against phenolic compounds.

Pseudomonas putida KT2442 is a strain that can make use of simple carbohydrate substrates such as glucose, fructose and glycerol as its carbon and energy sources. Poly(3-hydroxyalkanoates) or PHAs are usually accumulated by bacteria as their carbon and energy reserves utilizing related substrates for PHA synthesis. The related substrates usually used are fatty acids. This study proves that PHA synthesis is possible using nonrelated substrates. These substrates enable PHA formation subject to cultivation temperature. Saturated and unsaturated monomers of PHA form as intermediate products of de novo fatty acid biosynthesis with the degree of unsaturation depending on the temperature. PHA is synthesized from a common intermediate product in the carbohydrate substrate metabolism.

Pseudomonas putida S12 adapts to greater concentrations of styrene and therefore develops on styrene in a two-phase styrene-water system. P. putida S12 also adapts to greater concentrations of acetate, and these cells develop when supersaturating concentrations of toxic nonmetabolizable solvents such as toluene, a result of decreased membrane-fluidity of acetate-grown cells, are present. P. putida S12 cells which are precultured at enhanced concentrations of acetate or at subsaturating concentrations of toxic styrene become resistant to the toxic solvent.

The screening of 16,000 synthetic compounds for induction and inhibition and inhibition of quorum sensing in a Pseudomonas putida N-acylated L-homoserine lactone (AHL) sensor strain engineered with the LasR transcriptional activator is reported. High-throughput screening identified several compound with ability to inhibit and induce LasR activity which is important for controlling virulence gene expression in the opportunistic pathogen Pseudomonas aeruginosa and could be applied as therapeutic target.

The molecular analysis of two bacterioferritin genes, bfr[alpha] and bfr[beta] isolated from the model rhizobacterium Pseudomonas putida KT2440 was conducted to gain insight into the regulatory systems controlling the expression of these genes and the roles of these proteins in iron homeostasis. The data provide insight into the interaction-dependent function of bfr[alpha] and bfr[beta] and the primary benefit of the bacterioferritins to P. putida KT2440 for the enhancement of its survival in the environment by strengthening its tolerance to iron starvation.

A novel tightly regulated gene expression system is developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. Several Escherichia coli strains utilized in production and expression studies have shown high expression yields of target protein, tight regulation, rheostatic control and a homogeneous high-expression bacterial culture.

A bioscreen instrument was used to monitor growth dynamics of exposed cultures and effects of the chiral pharmaceuticals atenolol and propranolol on Pseudomonas putida, Pseudomonas aeruginosa, Micrococcus luteus, and Blastomonas matatoria. The study offers insigth into the different metabolic responses in the organisms on exposure to the different enantiomers and active pharmaceutical ingredients.

The phenomenon and application of using extracytosolic enzymes and medium conditions like pH are described that can catalyze metabolic pathways beyond their intracellular and extracytosolic catalysis. The method is termed as integrated bioprocessing as it has integrated intracellular and extracytosolic catalysis and it is applied to a lactonization reaction in Pseudomonas putida for the economical and high-titer biosynthesis of 4-valerolactone from the renewable source levulinic acid.

The Pseudomonas putida F1 todC1C2BA genes code for toluene dioxygenase, which initiates the degradation of toluene, as well as the catalysis of indan oxidation to (-)-(1R)-indanol and trans-1,3-indandiol. The todC1C2BA genes were transferred to Escherichia coli and the indan oxidation reaction catalyzed in this system was compared to that in P. putida. The results showed that E. coli oxidized indan to (-)-(1R)-indanol (83% R), while the indanol product of P. putida was (-)-(1R)-indanol (96% R). This difference was shown to be due to the presence of 1-indanol dehydrogenase in P. putida, which preferentially oxidizes (+)-(1S)-indanol.

The metabolic flux analysis of the solvent-tolerant Pseudomonas putida strains growing at butanol concentrations as high as 6% based on glucose-butanol consumption lowered cell maintenance costs is reported. The results provide insight into the butanol-tolerant P. putida strains as promising candidates for production hosts which overcomes the principal limitation of biobutanol production-product inhibition at low concentrations.

The amplification, cloning, functional expression and characterization of a 4-hydroxyacetophenone (HAPMO) from Pseudomonas putida JD1 oxidizing a broad range of aromatic ketones and further substrates are described. The studies have shown that the enzyme has oxidized ketones such as 3-phenyl-2-butanone with excellent enantioselectivity.

A modified and enhanced version of two solvent-tolerant Pseudomonas putida 12 strain producing the low-value compound phenol was employed for efficient production of the toxic value-added p-hydroxystyrene from glucose. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM was obtained in the 1-decanol phase.

A study is conducted to examine the activity of the three 4S route enzymes in the recombinant biocatalyst Pseudomonas putida CECT5279 and understand the biodesulfurization (BDS) process at molecular level. The results showed that the optimal BDS process in a recombinant P. putida strain is controlled by the availability of an active DszB enzyme.

A newly developed dual luciferase reporter (DLR) system is applied to strain KT2440 of Pseudomonas putida to assay its intracellular iron status and three putative bacterioferritin gene (Bfr, bfr[alpha], bfr[beta]) expressions. The results obtained from sensitive and reproducible DLR revealed that the expression of bfr (PP4856) is inversely related to the iron concentration and hence is more likely to encode a Dps-like protein rather than a bacterioferritin.

A study is conducted to identify and sequence a nicA gene involved in nicotine degradation by Pseudomonas putida strain S16. The results showed that the molecular mechanism for nicotine degradation in strain S16 that involves the pyrrolidine pathway is similar to one observed in pseudooxynicotine producing mammals, the direct precursor of a potent tobacco-specific lung carcinogen.

A study is conducted to examine the suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO). The simple and highly efficient method developed for production, extraction, and precipitation of PHO yield highly purified PHO, which consisted mainly of 3-hydroxyoctanoic acid.

The in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, have increased their activity on small alkanes. The in vivo evolution system can be used for directed evolution of enzymes that has functioned efficiently to hydroxylate small alkanes in engineered hosts.

A set of old yellow enzyme (OYE) homologs of Pseudomonas putida is utilized to study the potential complementary activities of multiple enzymes belonging to the same family within a single microorganism. The in vitro analysis of six OYE homologs identified in P. putida KT2440 offer insight into the catalytic activity of enzymes against 2,4,6-trinitrotoluene (TNT), which serves as a nitrogen source for microorganisms in vivo.

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to use xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The findings from the Pseudomonas putida S12 strain that is capable of utilizing glucose, xylose, and arabinose could be applied as an optimized host strain for efficient, green production of chemicals from renewable lignocellulose-derived feedstock.

A study was conducted to investigate the accumulation of polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) by the bacterium Pseudomonas putida CA-3 under aerobic growth conditions to determine whether increased polyP biosynthesis could lead to increased PHA production. The observation shows that polyP is not the rate-limiting step for mclPHA accumulation in Pseudomonas strains and it is possible to increase the strains by exposing the cells to other environmental signals or stresses.

Pseudomonas putida strain was engineered with two heterologous pathways to hydrolyze paraoxon and mineralize the hydrolysis products and use it as a sole carbon (C) and phosphorus (P) source. It was observed that there was no need for selection pressure to maintain the heterologous pathway, since the organism could utilize paraoxon as the sole C and P source and one of the intermediates in the pathways was toxic at high concentrations.

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida strain. It was demonstrated that alkane hydroxylase activity was induced by both the gases and the same enzyme system used for the oxidation of n-octane was used for oxidation of both the gases.

Phenylserine aldolase which catalyzes the cleavage of L-threo-3-phenylserine to yield benzaldehyde and glycine was purified and characterized to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. It was observed that in the reverse reaction the yield of the threo form was about two fold higher than that of the erythro form which reflects the binding property of the general base.

The global regulators involved in catabolite repression of the TOL plasmid promoters is identified and the o-xylene induction of TOL catabolic promoters in batch cultures in the presence and in the absence of one percent yeast extract (YE) are analyzed. It is found that in the early step of the regulatory process, catabolite repression control (Crc) acts by sensing the presence of YE.

Research has been conducted on conjugative plasmid transfer from Pseudomonas putida to the indigenous bacterial community of alfalfa sprouts cultivated from the commercially available seeds. The authors have demonstrated that alfalfa spouts provide environment which allows efficient conjugant gene transfer between the colonizing bacteria, and that the tranconjugant formation occurs later.

Research has been conducted on solvent-tolerant strain Pseudomonas putida DOT-T1E. The strain has been genetically engineered to biotransform toluene into 4-hydroxybenzoate in a catalyzed by toluene dioxygenase reaction.

The authors discuss their study of the green and red fluorescent proteins. They describe the dual-labeling technique used for in situ monitoring of the proteins' horizontal gene transfer via conjugation.

The authors have investigated periplasmic cytochrome c from the lupanine-utilizing Pseudomonas putida strain. The aim of the study has been the cloning, sequencing and heterologously expressing this cytochrome, and the determination of the cytochrome's molecular size via the use of electrospray mass spectrometry.

Research has been conducted on 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1. Results demonstrate that this compound is affected by dehydrogenation of its substrate and produces quinone methide which is then hydrated to alcohol.

Research has been conducted on naphthalene catabolic plasmid pCg1 carried by Pseudomonas putida Cg1. The authors report the identification of putative genes on this plasmid.