Ps. putida

Biotechnol Bioeng, 2003 Mar 20, 81(6), 683 - 94
Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis; Buhler B et al.; The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde . Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids . A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied . On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde . Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol . The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels . Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour . The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general .

Arch Insect Biochem Physiol, 2003 Feb, 52(2), 71 - 80
The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta; Park Y et al.; The bacterium, Xenorhabdus nematophilus, is a virulent insect pathogen . We tested the hypothesis that this bacterium impairs insect cellular immune defense reactions by inhibiting biosynthesis of eicosanoids involved in mediating cellular defense reactions . Fifth instar tobacco hornworms, Manduca sexta, produced melanized nodules in reaction to challenge with living and heat-killed X . nematophilus . However, the nodulation reactions were much attenuated in insects challenged with living bacteria (approximately 20 nodules/larva for living bacteria vs . approximately 80 nodules/larva in insects challenged with heat-killed bacteria) . The nodule-inhibiting action of living X . nematophilus was due to a factor that was present in the organic, but not aqueous, fraction of the bacterial cultural medium . The nodule-inhibiting factor in the organic fraction was labile to heat treatments . The immunodepressive influence of the factor in the organic fraction was reversed by treating challenged hornworms with arachidonic acid . The factor also depressed nodulation reactions to challenge with the plant pathogenic bacteria, Pseudomonas putida and Ralstonia solanacearum . These findings indicate that one or more factors from X . nematophilus depress nodulation reactions in tobacco hornworms by inhibiting eicosanoid biosynthesis .

J Am Chem Soc, 2003 Jan 22, 125(3), 705 - 14
Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam; Bell SG et al.; Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings . (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida . The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene . Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations . All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene . Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type . The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam) . Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor . If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product . All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type . (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products . The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis . (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule . One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different . Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution . In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors . The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure . The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone . The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone . Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings . The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis . The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.

Biomacromolecules, 2003 Jan-Feb, 4(1), 46 - 51
Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS . 2 . Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water; Sato H et al.; This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP(8)EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) . The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP(8)EO as the sole carbon source and (18)O-labeled water as an incubation medium . In the MALDI-MS spectra of biodegraded samples, a series of OP(n)EO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OP(n)EC) were observed . The use of purified OP(8)EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates . Furthermore, the formation of OP(8)EC (the oxidized product of OP(8)EO) supported the notion that terminal oxidation is a step in the biodegradation process . When biodegradation study was carried out in (18)O-labeled water, incorporation of (18)O atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products . These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.

Protein Expr Purif, 2003 Jan, 27(1), 175 - 81
Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida; Thuesen MH et al.; The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida . High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy . The recombinant cytochrome c(4) protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry . Cyclic voltammograms and UV-vis electronic absorption spectra were indistinguishable from the equivalent data of native P . stutzeri cytochrome c(4) . Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c(4) were identical . Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2089 - 94
16S rDNA sequence analysis of environmental Bdellovibrio-and-like organisms (BALO) reveals extensive diversity; Snyder AR et al.; Bdellovibrio-and-like organisms (BALO) are Gram-negative, predatory bacteria that inhabit terrestrial, freshwater and salt-water environments . Historically, these organisms have been classified together despite documented genetic differences between isolates . The genetic diversity of these microbes was assessed by sequencing the 16S rRNA gene . Primers that selectively amplify predator 16S rDNA, and not contaminating prey DNA, were utilized to study 17 freshwater and terrestrial and nine salt-water BALO isolates . When the 16S rDNA sequences were compared with representatives of other bacterial classes, 25 of the 26 BALO isolates clustered into two groups . One group, supported 100% by bootstrap analysis, included all of the Bdellovibrio bacteriovorus isolates . Each member of this group was isolated from either a freshwater or terrestrial source . The genetic distance between these isolates was less than 12% . The other group, supported 94% by bootstrap analysis, includes Bacteriovorax starrii, Bacteriovorax stolpii and the salt-water isolates . The salt-water isolates form a subgroup (83% by bootstrap) and differ within the subgroup by less than 110% . This observation implies that the salt-water isolates arose from Bacteriovorax progenitors . The difference between isolates in different clades is over 17%, a quantity similar to differences between bacterial species in different classes . However, both the Bdellovibrio and Bacteriovorax clades were closest to other representatives of the delta-Proteobacteria using maximum-likelihood . One freshwater isolate, James Island, was distinct from all other BALO (> 19%), but differed from Pseudomonas putida, a member of the gamma-Proteobacteria, by only 3% . Thus, by 16S rDNA sequence analysis, the BALO appear to have multiple origins, contrary to the unified taxonomic grouping based on morphology and natural history . These observations are consistent with the need to review and revise the taxonomy of these organisms.

Invest Ophthalmol Vis Sci, 2003 Jan, 44(1), 190 - 6
Molecular analysis of Pseudomonas aeruginosa protease IV expressed in Pseudomonas putida; Traidej M et al.; PURPOSE: In this study, the protease IV gene of Pseudomonas aeruginosa was expressed in the nonocular pathogenic host, Pseudomonas putida, to elucidate the molecular properties and virulence contribution of the enzyme . Recent determination of the protease IV gene sequence suggests that the protein of 463 amino acids contains a signal sequence, a propeptide domain, and a mature protease . The only form of this protein that has been detected previously is the extracellular mature protease . METHODS: The protease IV gene was cloned and expressed in a protease IV-negative Pseudomonas species, P . putida . The cloned protease IV gene product was analyzed to identify biochemical, enzymatic, and immunologic properties and its contribution to corneal virulence . RESULTS: P . putida expressing the cloned protease IV gene had significantly greater extracellular enzyme activity than P . aeruginosa . These P . putida cell extracts produced a protein with the same molecular mass as mature protease IV and two other polypeptides representing larger precursors, all of which were recognized by protease IV-specific antibodies . P . putida producing protease IV, relative to P . putida with the vector alone, caused a threefold increase in ocular inflammation and tissue damage when intrastromally injected into rabbit corneas . CONCLUSIONS: The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease . The results also confirm a significant correlation between production of protease IV and corneal virulence.

J Gen Appl Microbiol, 1999 Jun, 45(3), 105 - 113
A new type of flagellin gene in Pseudomonas putida; Senapin S et al.; Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida . The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins . We propose that P . putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb) . Type I and type II flagellin genes have been reported . The new 0.8-kb type III gene was expressed in E . coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin . The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots . This antiserum was determined to be functional in a motility inhibition assay . Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P . putida has been found . Preliminary electron microscopic study revealed that P . putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.

Biotechnol Bioeng, 2003 Feb 20, 81(4), 405 - 20
Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry; Amanullah A et al.; The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains . This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains . Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made . Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection . The toxic concentrations of the substrate, product, and by-products for each strain have been determined . The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested . The Gram-negative P . putida (421-5 and GM 730) and E . coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms . P . putida GM 730 and the E . coli TDO 123 strains responded similarly to toxic effects, and the E . coli TDO 123 strain was more resistant than the P . putida 421-5 strain . In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches .

J Bacteriol, 2003 Jan, 185(1), 184 - 95
Transcriptional organization of the Pseudomonas putida tol-oprL genes; Llamas MA et al.; Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria . In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA . Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2 . We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA . On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein . Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of sigma(70)-recognized promoters . The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited sigma(70) -10/-35 recognition sequences . The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions . In addition, analyses of the beta-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).

J Gen Appl Microbiol, 2001 Aug, 47(4), 193 - 200
Induction of 2-amino-D2-thiazoline-4-carboxylic acid hydrolase and N-carbamoyl-l-cysteine amidohydrolase by S-compounds in Pseudomonas putida AJ3865; Tamura Y et al.; The induction of 2-amino-Delta(2)-thiazoline-4-carboxylic acid hydrolase (ATCase) and N-carbamoylcysteine amidohydrolase (NCCase), both of which are involved in the conversion step of 2-amino-Delta(2)-thiazoline carboxylic acid (ATC) to cysteine, was studied with Pseudomonas putida AJ3865 . We found that L-ATC induced L-ATCase and L-NCCase, but that D-ATC induced only L-NCCase, whereas L- or D-NCC and thiazoline derivatives did not induce both enzymes . The bacterium showed neither D-ATCase nor D-NCCase activities, indicating that the role of L-ATC and D-ATC was different in the enzyme induction . We also found new inducers, d- and l-methionine, S-methyl-L-cysteine, cysteic acid, and 2-aminoethane sulfonic acid . However, the induction level of both enzymes by new inducers was much lower than those by L-ATC and D-ATC . Furthermore, the induction rate of both enzymes was synergistically increased only under a combination of D,L-ATC and new inducers . S-Compounds, however, such as new inducers except S-methyl-L-cysteine, inhibited both enzyme activities . This is the first report on the new inducers, synergistic induction, and the new inhibitors of L-ATCase and L-NCCase.

J Gen Appl Microbiol, 2001 Oct, 47(5), 269 - 277
Molecular cloning and transcriptional analysis of the alkyl hydroperoxide reductase genes from Pseudomonas putida KT2442; Fukumori F et al.; Pseudomonas putida KT2442TOL (formerly designated TOL), a toluene-resistant variant of strain KT2442 constitutively overexpressed several proteins . The most abundantly produced 24-kDa soluble protein was found to be similar to AhpC, the small subunit of alkyl hydroperoxide reductase . Molecular cloning of the P . putida ahpC based on the N-terminal sequence allowed cloning of closely located ahpF, the large subunit of alkyl hydroperoxide reductase . The deduced amino acid sequences of these genes showed high similarity with corresponding bacterial homologues . Results of RNA transcriptional analyses suggested that P . putida ahpC and ahpF were co-transcribed . A lower level expression of the ahpF may result from an attenuation of transcription by stem-and-loop structures located between two genes . oxyR, the known expression regulatory gene of ahpC-ahpF, was separately cloned and a point mutation that rendered an amino acid change (Phe(106) to Ile) in OxyR was observed . Reverse mutation of the oxyR gene by allelic exchange in P . putida KT2442TOL revealed that this mutation was the cause of the overexpression . About 50% of the reverse mutated cells lost colony-forming ability under toluene, indicating the mutation of oxyR that contributes to overexpression of the oxyR-regulated genes has some relationship with the solvent resistance, but their contribution was not significant.

J Hazard Mater, 2003 Jan 3, 96(1), 15 - 27
Solubilization and mineralization of polycyclic aromatic hydrocarbons by Pseudomonas putida in the presence of surfactant; Doong RA et al.; The solubilization and mineralization of polycyclic aromatic hydrocarbons (PAHs) in a soil system amended with different surfactants was examined . Mineralization experiments were conducted with the addition of {14C}pyrene . An inoculum of the PAH-degrading microorganism, Pseudomonas putida, was investigated for its sensitivity towards four non-ionic and one anionic surfactants with different polyoxyethylene (POE) chain lengths . The addition of surfactant was found to enhance the bioavailability of naphthalene, phenanthrene and pyrene with efficiencies ranging from 21.1 to 60.6%, 33.3 to 62.8% and 26.8 to 70.9%, respectively . The enhanced efficiency followed the order of Brij 30, Triton X-100, Tween 80, and Brij 35, which is correlated with the polyoxyethylene chain of the surfactants . Brij 35 and Tween 80 inhibited the growth of P . putida . However, microorganisms can utilize Triton X-100 and Brij 30 as the sole carbon and energy sources at concentrations above CMC values . In the aqueous system without the addition of surfactants, microorganisms could mineralize {14C}pyrene to 14CO(2) which corresponds to 28% of mineralization . The addition of surfactants decreased the mineralization rate of pyrene . Also, the fraction of the micellar-phase pyrene that can be directly biodegraded decreased as the concentration of micelle increases . However, the mineralization rate can be enhanced by the amendment of Brij 30 when soil was applied to the cultures . This suggests that biodegradable surfactants can be applicable for increasing the bioavailability and mineralization of PAHs in soil systems.

J Am Chem Soc, 2002 Dec 11, 124(49), 14571 - 9
Roles of the proximal hydrogen bonding network in cytochrome P450cam-catalyzed oxygenation; Yoshioka S et al.; Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated . A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L . The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P . The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively . Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I . J . Inorg . Biochem . 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam) . The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1) . The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form . These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1) . The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond . We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron . Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.

Biochemistry, 2002 Dec 10, 41(49), 14499 - 508
Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam; Prasad S et al.; The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest . We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy . Previously {Prasad et al . (2000) FEBS Lett . 477, 157-160}, we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment . In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments . The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site . We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements . The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes . The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.

Ecotoxicology, 2002 Oct, 11(5), 349 - 55
Assessment of the influence of use on ecotoxicological characteristics of synthetic ester lubricants; Maxam G et al.; Synthetic ester lubricants need optimisation about their technical and their ecotoxicological characteristics . To determine the ecotoxicological potential the required examinations can be based on the procedure for a risk assessment of chemicals . At present risk classification of lubricant oils is carried out with new oil fluids that are normally prepared before application in aqueous bioassays . In order to improve the ecotoxicological characteristics of some lubricant oils, the quality of the preparation method has been optimised . The resulting preparation protocol leads to aqueous extracts of the oil fluids that can be tested using biological assays . The extent of the changes of the chemical composition caused by the use as well as the ecotoxicological effects caused by additives have to be taken into consideration . For this reason various used lubricants are tested in addition to new oil fluids . In this work various lubricant samples were examined with standardised bacterial growth assays with Vibrio fischeri and Pseudomonas putida, luminescence inhibition assay with V . fischeri, survival assay with Daphnia magna and algal growth inhibition assay with Scenedesmus subspicatus . The chemical characterisation of the aqueous extracts included the determination of pH, conductivity, heavy metals, the content of dissolved organic carbon, inorganic anions and the content of phosphorus . The results emphasize the thesis that environmentally acceptable lubricants can undergo a change of their ecotoxicological potential during the use . Some of the substances that are normally added to base fluids in order to enhance the applicability of the oils may possess a high toxicological potential.

Environ Microbiol, 2002 Nov, 4(11), 703 - 12
Streptomycin-resistant (rpsL) or rifampicin-resistant (rpoB) mutation in Pseudomonas putida KH146-2 confers enhanced tolerance to organic chemicals; Hosokawa K et al.; We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA) . The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7% . Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively . These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene . The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12 . The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit . Mutation points in Gen mutants still remain unknown . Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA . Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance . Uptake experiments using {14C}-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA . Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.

Environ Microbiol, 2002 Nov, 4(11), 676 - 82
Alkane hydroxylase homologues in Gram-positive strains; van Beilen JB et al.; We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene . Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2180 - 1 Epub 2002 Nov 23.
Preliminary crystallographic studies of the creatinine amidohydrolase from Pseudomonas putida; Ito K et al.; Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida has been overexpressed in Escherichia coli and crystallized by the hanging-drop method . The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 150.7, c = 167.1 A . Native data were collected to 1.8 A resolution by a rotation method at 100 K using an ADSC Quantum 4R CCD detector with synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2173 - 4 Epub 2002 Nov 23.
Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp . strain NCIMB 12038; Malik ZA et al.; The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp . strain NCIMB 12038 . The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized . These enzymes utilize a mononuclear non-heme iron centre to catalyze the addition of dioxygen to their respective substrates . In this reaction, two electrons, two protons and a dioxygen molecule are consumed . The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported . In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method . The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 A, alpha = beta = gamma = 90 degrees, and diffract to 2.3 A resolution.

Appl Environ Microbiol, 2002 Dec, 68(12), 5933 - 42
Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531; Whyte LG et al.; The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized . Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4) . In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB) . The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters . Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 {NRRL B-16531}; alkB2 and rubB {Q15}) was achieved in Escherichia coli and Pseudomonas expression systems . Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes . All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH{S/A}DHH and NYXEHYG{L/M}), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase . The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 617 - 24
Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat; Bakker PA et al.; Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat . Two genetically modified derivatives carried the phz or the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG) . In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied . A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected . Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied . Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured . All introduced strains decreased from 10(7) CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants . The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers . The phl genes were also stably maintained in the DAPG producing derivative of WCS358r . Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis . Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora . However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.

J Bacteriol, 2002 Dec, 184(24), 7062 - 7
Cross-regulation between a novel two-component signal transduction system for catabolism of toluene in Pseudomonas mendocina and the TodST system from Pseudomonas putida; Ramos-Gonzalez MI et al.; The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1 . Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found . The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster . The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P . putida . The tod operon is regulated by a two-component signal transduction system encoded by the todST genes . Two novel genes from P . mendocina KR1, tmoST, were rescued by complementation of a P . putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT . We show that transcription from P(tmoX) and P(todX) can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.

J Bacteriol, 2002 Dec, 184(24), 6957 - 65
Different spectra of stationary-phase mutations in early-arising versus late-arising mutants of Pseudomonas putida: involvement of the DNA repair enzyme MutY and the stationary-phase sigma factor RpoS; Saumaa S et al.; Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers . Mutational processes in starving cells are different from those occurring in growing bacteria . Here, we present evidence that changes in mutational processes also take place during starvation of bacteria . Our test system for selection of mutants based on creation of functional promoters for the transcriptional activation of the phenol degradation genes pheBA in starving Pseudomonas putida enables us to study base substitutions (C-to-A or G-to-T transversions), deletions, and insertions . We observed changes in the spectrum of promoter-creating mutations during prolonged starvation of Pseudomonas putida on phenol minimal plates . One particular C-to-A transversion was the prevailing mutation in starving cells . However, with increasing time of starvation, the importance of this mutation decreased but the percentage of other types of mutations, such as 2- to 3-bp deletions, increased . The rate of transversions was markedly elevated in the P . putida MutY-defective strain . The occurrence of 2- to 3-bp deletions required the stationary-phase sigma factor RpoS, which indicates that some mutagenic pathway is positively controlled by RpoS in P . putida.

J Mol Biol, 2002 Nov 29, 324(3), 519 - 33
Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases; Tanaka N et al.; Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family . The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor . PFDH can catalyze aldehyde dismutations without an external addition of NAD(H) . The structural basis of the tightly bound cofactor of PFDH is unknown . The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution . The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry . Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed . A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD(+) molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH . This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs . Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.

Appl Microbiol Biotechnol, 2002 Nov, 60(3), 293 - 9 Epub 2002 Oct 12.
Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations; Hofer H et al.; The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1 . Orcinol was used as an inducer for enzyme production . The growth medium was optimized in two steps . In the first step, screening for optimal glucose concentration was performed . In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium . After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium . Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods . In a subsequent step, the maximal orcinol-degradation rate was determined . The results obtained were used to find an optimal feeding profile for enzyme production . By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments . Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).

Anal Bioanal Chem, 2002 Nov, 374(5), 841 - 7 Epub 2002 Oct 16.
Whole-cell biosensing of 3-chlorocatechol in liquids and soils; Guan X et al.; A rapid and sensitive technique is needed to analyze water and soils for chlorocatechols, common environmental pollutants produced from wood pulp chlorination and other processes . The soil bacteria Pseudomonas putida, harboring plasmid pSMM50R-B', selectively express beta-galactosidase in response to 3-chlorocatechol in pure water samples . The objective of the study was to determine whether background matrices in fresh water, sea water, soils, and organic solvents interfered with 3-chlorocatechol analysis by use of a bacteria-sensing system and by high-performance liquid chromatography (HPLC) . Although 3-chlorocatechol detection by HPLC was not substantially affected by the background composition of aqueous or organic solvents, HPLC was ineffective in the analysis of contaminated soils due to irreversible contaminant sorption . Whereas detection by the bacteria-sensing system was reduced in the presence of aqueous and organic solvents, interferences could be reduced by sample dilution . 3-Chlorocatechol was detected when the bacteria were added directly to contaminated soils, suggesting that the organism enhanced desorption or had access to the sorbed compounds . Results indicate that the bacteria-sensing system has wide application for detection of 3-chlorocatechols in environmental samples, especially in soils where extraction and HPLC analysis are not efficient due to extensive contaminant sorption.

J Bacteriol, 2002 Dec, 184(23), 6581 - 91
Transposition of DEH, a broad-host-range transposon flanked by ISPpu12, in Pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing; Weightman AJ et al.; Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families . The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0 . DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats . The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase . A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively . The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout . Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients . In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria . Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P . putida PaW340 . Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P . putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing . Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes . The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.

J Bacteriol, 2002 Dec, 184(23), 6572 - 80
A third transposable element, ISPpu12, from the toluene-xylene catabolic plasmid pWW0 of Pseudomonas putida mt-2; Williams PA et al.; A 3,372-bp insertion sequence, ISPpu12, has been identified on the archetypal toluene-xylene TOL catabolic plasmid pWW0 from Pseudomonas putida mt-2 . The insertion sequence element is located on the plasmid between bases 84397 and 87768 in a region which also contains the termini and transposase genes of the catabolic transposons Tn4651 and Tn4653 (A . Greated, L . Lambertson, P . A . Williams, and C . M . Thomas, Environ . Microbiol., in press) . ISPpu12 has terminal inverted repeats of 24 bp with three mismatches and contains four open reading frames, a tnpA homologue and three open reading frames (lspA, orf1, and orf2) of undetermined function . After insertion in vitro of a Km(r) cassette into ISPpu12 either in the intergenic region between orf1 and orf2 or directly into the orf1 gene and ligation into a suicide vector, the modified ISPpu12-Km transposes at high frequency, often in multiple copies, into the chromosome of a P . putida recipient . Inactivation of lspA, orf1, and orf2 by introducing a 7-bp deletion into the 5' region of each gene had no major effect upon transposition, but a similar mutation of tnpA completely eliminated transposition . Analysis of the literature and of strains derived from the chlorobenzoate-degrading Pseudomonas sp . strain B13 suggests that the promiscuity of this element has played an important role in the history of plasmid pWW0 . Database comparisons and the accompanying paper (A . J . Weightman, A . W . Topping, K . E . Hill, L . L . Lee, K . Sakai, J . H . Slater, and A . W . Thomas, J . Bacteriol . 184:6581-6591, 2002) show that ISPpu12 is a transposable element also found in other bacteria.

Folia Microbiol (Praha), 2002, 47(4), 458 - 60
Influence of calcium hydroxide root-canal sealer on microbial growth in vitro; Pezelj-Ribaric S et al.; The calcium hydroxide-based filling material Apexit, which is often used in endodontic practice, was evaluated for its antibacterial and antifungal effects against microorganisms isolated from oral cavity (Serratia marcescens, Pseudomonas putida, Staphylococcus aureus, Candida albicans) . Two different quantitative techniques were employed--the direct-contact test was used to examine the efficacy of freshly mixed material while the broth-survival test was employed to check the antimicrobial properties of 5-d-old material . Apexit inhibited Gram-negative bacteria more effectively than Gram-positive ones but had none or a very weak inhibitory effect on C . albicans.

Microb Ecol, 2003 Jan, 45(1), 97 - 107 Epub 2002 Nov 06.
Simultaneous growth on citrate reduces the effects of iron limitation during toluene degradation in Pseudomonas; Dinkla IJ et al.; Rhizoremediation has been suggested as an attractive bioremediation strategy for the effective breakdown of pollutants in soil . The presence of plant root exudates such as organic acids, sugars, and amino acids that may serve as carbon sources or biosynthetic building blocks and the limited bioavailability of iron may influence the degradation of pollutants in the rhizosphere . To test the effect of such compounds on hydrocarbon degradation, trace concentrations of yeast extract or mixtures of organic acids and amino acids were added to continuous cultures of Pseudomonas putida mt2 and P . putida WCS358 (TOL) growing on toluene . By addition of these compounds increased growth yields and higher specific growth rates on toluene were obtained . The effects of iron limitation on the substrate utilization pattern of both strains were tested by growing the strains on a mixture of toluene and the readily degradable carbon source citrate while the iron concentration was varied . Simultaneous use of both substrates under carbon-limited as well as iron-limited conditions was observed . Growth yields were less reduced and iron requirement was lower during iron-limited growth in the toluene + citrate grown cultures compared to cultures in which toluene was used as the sole carbon source . The kinetic properties of the cells for toluene degradation were less hampered by the lack of iron when citrate was used as an additional carbon source . The results indicate that the availability of low concentrations of natural organic compounds, such as produced in the rhizosphere, may positively influence the degradative performance of hydrocarbon-degrading bacteria.

Curr Microbiol, 2002 Dec, 45(6), 410 - 4
Mechanism of copper resistance in a copper mine isolate Pseudomonas putida strain S4; Saxena D et al.; The mechanism of copper resistance in a multiple-metal-resistant natural isolate Pseudomonas putida strain S4 is based on inducible efflux . Active extrusion of copper ions occurs from the cytoplasm during the exponential phase of growth . Involvement of ATPase in the efflux of copper ions has been demonstrated by employing specific inhibitors . The effluxed copper is not thrown out of the cell, but remains in a bound form (to a protein) in the periplasm . Thus, a balance between the intracellular level, to fulfill the metabolic requirements, and the periplasmic sequestration, to evade toxicity, is maintained by this isolate.

Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1945 - 50
Changes in membrane fluidity and fatty acid composition of Pseudomonas putida CN-T19 in response to toluene; Kim IS et al.; A bacterial isolate, Pseudomonas putida CN-T19, could grow in a two-phase medium with toluene up to 50% (v/v) . Changes in fatty acid composition and membrane fluidity of the isolate were investigated to understand how this microorganism responds toluene . The changes in the ratios of unsaturated to saturated fatty acids were insignificant between cells grown with and without toluene . The changes in the ratio of cis- to trans-fatty acids of C16:1 and C18:1 was, however, significantly lower in cells grown with toluene than cells grown without toluene, giving approximately 1.3 and 9.7, respectively . Toluene had a fluidizing effect on the membrane of cells grown without toluene, resulting in decrease in membrane polarization ratio . Less fluidizing effect of toluene on the membrane of cells grown with toluene was observed, giving 11% of polarization percentage, which was significantly lower than 53% in cells grown without toluene . These results suggest that cis/trans isomeration of C16:1 and C18:1 makes cell membranes more rigid to respond toluene, and is an adaptive strategy allowing P . putida CN-T19 to grow in the presence of organic solvent.

Yakugaku Zasshi, 2002 Oct, 122(10), 805 - 11
{Structural and functional analysis of enzymes and their application to clinical analysis--study on Pseudomonas putida formaldehyde dehydrogenase}; Ito K; Formaldehyde dehydrogenase (PFDH) was isolated from the creatinine-decomposing bacterium Pseudomonas putida, and its gene has been cloned . PFDH is unique because it was the only enzyme that catalyzed the dehydrogenation of formaldehyde without glutathione . PFDH belongs to a zinc-containing alcohol dehydrogenase family . Quantitative analysis of the reaction products using NMR revealed that the enzyme is not simply a dehydrogenase but is an aldehyde dismutase catalyzing a simultaneous conversion of both aldehyde to carboxylate and aldehyde to alcohol . The enzyme contains a tightly bound cofactor of NAD+/NADH per subunit and is classified as a nicotinoprotein . The enzyme reaction can proceed without external addition of the nucleotide cofactor . The formaldehyde was crystallized using the hanging-drop vapor diffusion method with ammonium sulfate as a precipitant . The crystal structure was determined using the multiwavelength anomalous diffraction method with intrinsic zinc ions . The overall structure of PFDH is similar to that of a classic horse liver alcohol dehydrogenase . However, a comparison of these structures indicated that the insertion loop specifically found in PFDH may be responsible for the tight binding of the cofactor, thereby making PFDH a dismutase.

Biofizika, 2002 Sep-Oct, 47(5), 920 - 5
{Mathematical model of the interaction of components in a plant-rhizospheric microorganisms system at the higher level of carbon dioxide in atmosphere}; Pis'man TI et al.; A mathematical model describing the interaction of plants and rhizospheric microorganisms on complete mineral medium at a higher CO2 level in the atmosphere was constructed . The positive effect of CO2-enrichment on the system plant--rhizospheric microorganisms was shown . The effect of rhizospheric microorganisms on plant growth at normal and high level of carbon dioxide was demonstrated . It was shown that the biomass of plant in the system is smaller than the biomass of plant growing without microorganisms . It was experimentally demonstrated that a simple ecosystem wheat--Pseudomonas putida--artificial soil develops and functions differently than its individual constituents in the case of a wheat-artificial soil system . With unlimited nutrition and a higher CO2 level (0.06%), plants with roots inoculated with microorganisms have a smaller biomass than plants that were not inoculated with microorganisms.

Appl Biochem Biotechnol, 2002 Jul-Dec, 102-103(1-6), 337 - 47
Biodegradation of a medium-chain-length polyhydroxyalkanoate in tropical river water; Ho YH et al.; The medium-chain-length polyhydroxyalkanoate (PHA(MCL)) produced by Pseudomonas putida PGA1 using saponified palm kernel oil as the carbon source could degrade readily in water taken from Kayu Ara River in Selangor, Malaysia . A weight loss of 71.3% of the PHA film occurred in 86 d . The pH of the river water medium fell from 7.5 (at d 0) to 4.7 (at d 86), and there was a net release of CO2 . In sterilized river water, the PHA film also lost weight and the pH of the water fell, but to lesser extents . The C8 monomer of the PHA was completely removed after 6 d of immersion in the river water, while the proportions of the other monomers (C10, C12, and C14) were reversed from that of the undegraded PHA . By contrast, the monomer composition of the PHA immersed in sterilized river water did not change significantly from that of the undegraded PHA . Scanning electron microscopy showed physical signs of degradation on the PHA film immersed in the river water, but the film immersed in sterilized river water was relatively unblemished . The results thus indicate that the PHA(MCL) was degraded in tropical river water by biologic as well as nonbiologic means . A significant finding is that shorter-chain monomers were selectively removed throughout the entire PHA molecule, and this suggests enzymatic action.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 89 - 95
Analysis of the zwf-pgl-eda-operon in Pseudomonas putida strains H and KT2440; Petruschka L et al.; A 3.9-kb fragment of the genome of Pseudomonas putida H, containing the complete zwf-pgl-eda-operon, encoding glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 2-keto-3-deoxy-6-phosphogluconate-aldolase, respectively, and part of the divergently transcribed regulatory gene, hexR, was cloned and analyzed . The nucleotide sequences of these genes showed high similarities to the corresponding DNA sequences of P . putida KT2440 and also to sequences of Pseudomonas aeruginosa PAO1 . Derivatives of strains H and KT2440, containing transcriptional lacZ fusions to P(zwf) were generated and used to study the expression of these operons . In both strains, this operon was induced by carbohydrates such as glucose, gluconate, fructose and glycerol . The transcription rate of the zwf-pgl-eda-operon was found to be about three times higher in the KT2440 background than in strain H . In both strains the induction of the zwf-pgl-eda-operon by carbohydrates during growth on carboxylic acids was not affected by carbon catabolite repression.

Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 179 - 85 Epub 2002 Aug 22.
Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8; von Wallbrunn A et al.; The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced . The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents . Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin . As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids . Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production . Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis . As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.

Biochemistry, 2002 Oct 15, 41(41), 12313 - 9
Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding; Xu Y et al.; (S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids . Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH) . Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond {Lehoux, I . E., and Mitra, B . (2000) Biochemistry 39, 10055-10065} . In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair . Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized . Our results show that Arg165 has a role similar to, but less critical than, that of Arg277 . It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond . Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower . Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis . Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E . Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants . pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165.

Carbohydr Res, 2002 Sep 27, 337(17), 1589 - 91
Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867; Knirel YA et al.; The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->

Appl Environ Microbiol, 2002 Oct, 68(10), 5191 - 4
Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons; Morasch B et al.; 13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene . Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.

Appl Environ Microbiol, 2002 Oct, 68(10), 4965 - 70
Accumulation of 2-aminophenoxazin-3-one-7-carboxylate during growth of Pseudomonas putida TW3 on 4-nitro-substituted substrates requires 4-hydroxylaminobenzoate lyase (PnbB); Hughes MA et al.; During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a lambda(max) of 446 nm . The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC) . This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water . It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3 . Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC . This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.

Appl Environ Microbiol, 2002 Oct, 68(10), 4758 - 63
Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts; Barak JD et al.; Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts . We examined how S . enterica serovars, E . coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts . Growth on and adherence to sprouts were not significantly different among different serovars of S . enterica, but all S . enterica serovars grew on and adhered to alfalfa sprouts significantly better than E . coli O157:H7 . E . coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S . enterica remained attached per sprout . S . enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar . S . enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E . coli O157:H7; however, three of four other E . coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E . coli O157:H7 and as well as the Pseudomonas and Rahnella strains . Therefore, attachment to alfalfa sprouts among E . coli serotypes is variable, and nonpathogenic strains of E . coli to be used as surrogates for the study of pathogenic E . coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.

J Biol Chem, 2002 Nov 29, 277(48), 45860 - 5 Epub 2002 Sep 18.
Isonitrile hydratase from Pseudomonas putida N19-2 . Cloning, sequencing, gene expression, and identification of its active acid residue; Goda M et al.; Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides . Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene . Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da . Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain . The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101) . A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.

J Inorg Biochem, 2002 Sep 20, 91(4), 586 - 96
Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase; Schunemann V et al.; It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues . In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d(4), S=1) and one is located on the porphyrin (S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin pi-cation radical system . However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S'=1/2), via Mossbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent {Schunemann et al., FEBS Lett . 479 (2000) 149} . In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable . We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid . This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation . As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations . We were able to confirm earlier findings {Rutter et al., Biochemistry 23 (1984) 6809}, that an antiferromagnetically coupled Fe(IV)=O porphyrin pi-cation radical system is formed . We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time . In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.

Plant Physiol, 1993 Apr, 101(4), 1231 - 1237
Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis); Funk C et al.; (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures . In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells . Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light . Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold . A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata) . Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells . These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.

Can J Microbiol, 2002 Jul, 48(7), 635 - 42
Regulation of indoleacetic acid production in Pseudomonas putida GR12-2 by tryptophan and the stationary-phase sigma factor RpoS; Patten CL et al.; The phytohormone indole-3-acetic acid (IAA) accumulates in the culture medium of the plant growth-promoting bacterium Pseudomonas putida GR12-2 only when grown in the presence of exogenous tryptophan, suggesting that expression of indolepyruvate decarboxylase, a key enzyme in the IAA biosynthesis pathway in this bacterium, may be regulated by tryptophan . To test this hypothesis, we isolated the promoter region for the ipdc gene encoding indolepyruvate decarboxylase by inverse polymerase chain reaction (PCR) and inserted it upstream of the bioluminescent reporter gene luxAB on a plasmid in P . putida GR12-2 . Activity of the ipdc promoter, measured by quantifying light production, increased fivefold in the presence of L-tryptophan, confirming that ipdc expression is induced by tryptophan . In addition, transcription of ipdc is regulated by the stationary phase sigma factor RpoS: the ipdc promoter contains a sequence similar to the RpoS recognition sequence, and transformation of P . putida GR12-2 with a plasmid carrying rpoS under the control of a constitutive promoter induced promoter activity before the onset of stationary phase when RpoS is not normally produced and prolonged a higher level of transcription at the later stages of the cell cycle.

J Microbiol Methods, 2002 Nov, 51(3), 337 - 48
Multiple enzyme restriction fragment length polymorphism analysis for high resolution distinction of Pseudomonas (sensu stricto) 16S rRNA genes; Porteous LA et al.; Members of the genus Pseudomonas (sensu stricto) are important phytopathogens and agents of human infections, while other strains and species have beneficial bioremediation and biocontrol activities . Traditionally, these important species have been difficult to differentiate phenotypically; thus, rRNA lineage analyses have often been invoked . In this report, a newly developed approach is described to rapidly detect and distinguish fluorescent Pseudomonas isolates: PCR amplification of a Pseudomonas-specific 990-bp ribosomal RNA gene (rDNA) fragment {Appl . Environ . Microbiol . 64 (1998) 2545.} coupled with multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of AluI, HinfI, RsaI, and Tru9I incubated at 37 degrees C . The method distinguished 116 published sequences and 47 reference strains of authentic Pseudomonas representing 28 nomenspecies . A total of 55% (64/116) of the sequences analyzed by MERFLP were grouped into distinct phylogenetic clusters including Pseudomonas putida, P . syringae, P . aeruginosa, P . stutzeri, and P . fluorescens . The utility of the MERFLPs was confirmed when 100% (33/33) of the above named control reference strains were correctly placed into their phylogenetic clusters . The environmental relevance of the MERFLP method was confirmed when 67% of 28 forest and agricultural soil-derived presumptive Pseudomonas environmental clones and isolates were placed into the five major pseudomonad clusters, one clone fell into the P . agarici cluster, and five clones clustered near related pseudomonads . These data demonstrated that the PCR-MERFLP protocol provides an efficient and powerful tool for distinguishing isolates and rDNA gene libraries of environmental Pseudomonas species .

Bioorg Med Chem Lett, 2002 Oct 7, 12(19), 2673 - 80
Structural assignment of 2,6- and 2,7-disubstituted naphthalenes and prediction of (13)C nuclear magnetic resonance chemical shifts: applications of topology and two-dimensional NMR spectroscopy; Khadikar PV et al.; Unambiguous assignments of monocarboxymethylnapthalenes isolated as oxidation products of dimethylnaphthalenes by Pseudomonas putida, a bacterial strain, were made using two-dimensional nuclear Overhauser enhancement correlation spectroscopy (NOESEY) . The two-dimensional long-range heteronuclear correlation NMR technique was also utilized for the assignment of quaternary carbons in the naphthalene system . In addition, we describe methods for prediction of 13C NMR chemical shifts of 2,6- and 2,7-disubstituted naphthalenes using topological approach . The method involves computation of molecular descriptors from topological representation of molecule, namely Wiener (W) and Szeged (Sz) indices . The results have shown that W and Sz indices can be successfully used for predicting 13C NMR chemical shifts and that Sigma13Cn can be used as a molecular property which in turn can be modeled by both W and Sz indices successfully.

Biomacromolecules, 2002 Sep-Oct, 3(5), 1006 - 12
Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers; Asrar J et al.; In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) . The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104 . The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests . Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information . Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution . By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition . Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics . Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content . The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers . Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV) . However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.

Microbiology, 2002 Sep, 148(Pt 9), 2857 - 67
Molecular characterization of an operon, cueAR, encoding a putative P1-type ATPase and a MerR-type regulatory protein involved in copper homeostasis in Pseudomonas putida; Adaikkalam V et al.; The authors have characterized a chromosomally localized two-gene operon, cueAR, which encodes a putative P1-type ATPase, CueA, and a MerR-type metalloregulatory protein, CueR, in Pseudomonas putida PNL-MK25 . Disruption of cueAR by the insertion of mini-Tn5::gfp into the wild-type strain led to a mutant strain with a sixfold reduction in its tolerance to copper; however, the tolerance of this mutant strain to the other seven related transition metals tested was not affected . The sensitivity of the mutant strain was attributed to a higher level of accumulation of intracellular copper, suggesting the involvement of CueA in copper export . Insertion of the cloned cueAR operon into the copper-sensitive mutant strain fully restored its tolerance to copper . cueA::gfp expression studies confirmed that the cueAR operon was transcriptionally regulated by copper and CueR . Studies done on the mutant strain complemented with cueR and cueA revealed partial functional redundancy of cueA and cueR, respectively, in copper tolerance . Thus, the results of this study clearly suggest the involvement of cueAR in copper homeostasis in P . putida.

Electrophoresis, 2002 Jul, 23(14), 2233 - 41
Two-dimensional electrophoresis analysis of protein production during growth of Pseudomonas putida F1 on toluene, phenol, and their mixture; Reardon KF et al.; The protein profiles of Pseudomonas putida F1 during growth on toluene, phenol, and their mixture were examined by two-dimensional polyacrylamide gel electrophoresis . Although this bacterium uses the same catabolic pathway for both substrates, P . putida F1 produced specific sets of proteins in response to toluene and phenol as single or mixed substrates . Proteins associated with growth on these substrates could be classified into three categories: ten Group T proteins were associated with the degradation of toluene, seventeen Group P proteins were associated with the degradation of phenol, and one Group M protein was observed to be associated only with toluene-phenol mixture degradation . During growth on the mixture, the protein profile of the cells shifted from Group T proteins to Group P proteins . This correlated well with the substrate consumption pattern, in which toluene was consumed first and growth on phenol did not begin until the medium was nearly depleted of toluene . Individual Group T and Group P protein intracellular concentrations had different transients as the cells grew on the mixture; seven protein levels increased, four decreased, and sixteen reached a maximum and then declined . The Group M protein reached a concentration maximum near the time when growth on phenol began . Variations in the maintenance of these proteins were also noted . These results demonstrate that cells growing on a mixture of substrates undergo significant physiological changes . Further investigation of these changes is expected to shed light on the unusual biodegradation kinetics previously observed with this mixed-substrate system.

Mol Microbiol, 2002 Sep, 45(5), 1421 - 32
Multiple bacteria encode metallothioneins and SmtA-like zinc fingers; Blindauer CA et al.; Zinc is essential but toxic in excess . Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA . The dearth of other reported bacterial metallothioneins has been surprising . Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons . Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins . Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA . A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) . (1)H-NMR of proteins from P . aeruginosa, Anabaena PCC 7120 and E . coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA . These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA . We have identified 12 smtA-like genes in sequence databases including four of the gatA type.

Arch Environ Contam Toxicol, 2002 Oct, 43(3), 265 - 9
Bisphenol a degradation by bacteria isolated from river water; Kang JH et al.; Recently, there is increasing interest in the microbial degradation of endocrine disruptors . This study was conducted to show the isolation and property of bacteria having bisphenol A (BPA) biodegradability in river water and to identify the difference of BPA degradation under aerobic and anaerobic conditions . Three river water samples spiked with BPA (1 mg/L) were rapidly degraded under aerobic conditions . The half-life for BPA degradation ranged from 2 to 3 days, and BPA was below detection limit (less than 0.005 mg/L) on the 10th day . But a decrease of BPA under anaerobic conditions was hardly identified at 30 degrees C for 10 days (less than 10%) . Also, most bacteria (10 out of 11) isolated from three river waters had BPA biodegradability, but there were differences in removal rates of BPA (18% to 91%) . Moreover, two strains that had high BPA biodegradability (about 90%) were identified as a Pseudomonas sp . and a Pseudomonas putida strain.

Protein Eng, 2002 Jul, 15(7), 585 - 93
Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis; Lingen B et al.; Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme . After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone . From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated . The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center . BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme . L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity . Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter . Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product . The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.

J Biol Chem, 2002 Nov 8, 277(45), 42926 - 36 Epub 2002 Aug 27.
Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP:CoA transacylase, which diverts intermediates of fatty acid de novo biosynthesis; Hoffmann N et al.; The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis . PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His(6)) fusion protein in high yield . The His(6)-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established . Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His(6)-PhaG, gave K(0.5) values of 28 microm (ACP) and 65 microm (3-hydroxyacyl-CoA) considering V(max) values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively . A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity . Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol . Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase . A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz) . In addition, the alignment with the alpha/beta-hydrolase fold region indicated that PhaG belongs to alpha/beta-hydrolase superfamily . Accordingly, CD analysis suggested a secondary structure composition of 29% alpha-helix, 22% beta-sheet, 18% beta-turn, and 31% random coil . Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site . Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme . Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding . The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.

Anal Bioanal Chem, 2002 Apr, 373(8), 696 - 703 Epub 2002 Jul 04.
Pesticide toxicity assessment using an electrochemical biosensor with Pseudomonas putida and a bioluminescence inhibition assay with Vibrio fischeri; Farre M et al.; Two different toxicity tests, an electrochemical biosensor Cellsense and a bioluminescence inhibition assay ToxAlert were performed in order to establish and compare the acute toxicity responses of different types of raw and spiked water for a selected group of pesticides . The selected compounds were endosulfan, chlorfenvinphos, dimethoate, fenamiphos, ametryn, deltamethrin and alpha-cypermethrin; all of them are used in large quantities for agricultural purposes . In the first step, the study of the toxicity responses for each individual pesticide with Milli-Q water was carried out . Next, the toxic responses of different mixtures of these pesticides in different water matrices, i.e., Milli-Q water, surface water, groundwater and wastewater were studied in order to evaluate (i) device advantages and limitations for the toxicity evaluation of real environmental samples, (ii) antagonistic or synergistic effects and (iii) the influence of the water matrices . The survey of pesticides in real samples was carried out using a combined method involving both chemical analysis and toxicity bioassays . Chemical analysis involved the use of solid-phase micro-extraction (SPME) followed by gas chromatography with electron capture detection (GC/ECD) or thermoionic specific detection (GC/TSD) with mass spectrometric confirmation (GC/MS).

Curr Microbiol, 2002 Oct, 45(4), 250 - 4
Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacterial sensors; Degrassi G et al.; The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs) . This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria . The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs . In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358 . Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria . The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed.

Arch Microbiol, 2002 Sep, 178(3), 180 - 92 Epub 2002 Jun 18.
Aerobic metabolism of phenylacetic acids in Azoarcus evansii; Mohamed Mel-S et al.; The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii . Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented . 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase . The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase . Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA . Whole cells of A . evansii transformed {1-(14)C}PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e . Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus . A chromosomal region from A . evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized . The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E . coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria . RT-PCR analysis of the paa genes of A . evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons . The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.

Microbiology, 2002 Aug, 148(Pt 8), 2319 - 29
nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA; Park W et al.; In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism {Schell, M . A . & Poser, E . F . (1989) . J Bacteriol 171, 837-846} . The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P . putida NCIB 9816-4 was verified . Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source . The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P . putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers . The amplified nahR homologues and the intergenic regions were cloned and sequenced . Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates . The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5'-ATTCACGCTN(2)TGAT-3') . Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not . DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories . PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene . The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes . Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.

Mol Genet Genomics, 2002 Jul, 267(5), 656 - 63 Epub 2002 Jun 20.
Molecular analysis of aerobic phenylacetate degradation in Azoarcus evansii; Rost R et al.; The Azoarcus evansii gene which codes for phenylacetate-CoA ligase, an enzyme involved in the aerobic degradation of phenylacetate, was isolated from a genomic library, using as the probe a fragment of the gene which encodes the isoenzyme that is induced under anaerobic conditions . By this means both the gene and its flanking sequences were recovered . The gene is homologous to the phenylacetate-CoA ligase genes of Pseudomonas putida U and Escherichia coli W . Induction by phenylacetate under aerobic growth conditions was demonstrated using lacZ fusions . Western analysis showed that phenylacetate-CoA ligase is involved in the degradation of the aromatic amino acid phenylalanine . Genes coding for the phenylacetate-CoA ligase and for the putative hydroxylating enzyme were expressed in E . coli . Detection of 2-hydroxyphenylacetate in the recombinant E . coli strain indicated hydroxylation of phenylacetyl-CoA . The gene pacL, which codes for the putative ring-opening enzyme was mutated to enable the isolation of intermediates in aerobic phenylacetic acid degradation, which were characterized by GC-MS and NMR analyses.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 545 - 50 Epub 2002 Jul 03.
Cloning and characterization of a FAD-monooxygenase gene ( cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07; Trevino-Quintanilla LG et al.; A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07) . Several transposon insertion mutants unable to degrade chloranilic acid were selected . The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds . The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound . We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P . putidagenome that is encoded in a megaplasmid or in an unstable chromosomal region.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 449 - 54 Epub 2002 Jun 22.
P450(camr), a cytochrome P450 catalysing the stereospecific 6- endo-hydroxylation of (1 R)-(+)-camphor; Grogan G et al.; Rhodococcus sp . NCIMB 9784 accumulated 6- endo-hydroxycamphor 3 when grown on (1 R)-(+)-camphor 1 as sole carbon source . The structure of 3 has been unambiguously assigned for the first time using X-ray crystallography . A soluble cytochrome P450 hydroxylase, induced by growth on (1 R)-(+)-camphor and designated P450(camr), has been isolated from the bacterium Rhodococcus sp . NCIMB 9784 . Using authentic 6- endo hydroxycamphor as standard, a cell-free system consisting of pure P450(camr) and putidaredoxin and putidaredoxin reductase from Pseudomonas putida confirmed that the enzyme hydroxylates (1 R)-(+)-camphor specifically in the 6- endoposition, in contrast to the 5- exo hydroxylation catalysed by the well-studied P450(cam) from P . putida . P450(camr) has a molecular mass of approximately 44 kDa, and a pI of 4.8.

J Bacteriol, 2002 Sep, 184(17), 4757 - 66
Site-specific recombination system encoded by toluene catabolic transposon Tn4651; Genka H et al.; The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M . Tsuda, K.-I . Minegishi, and T . Iino, J . Bacteriol . 171:1386-1393, 1989) . Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site . The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS . In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination . Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution . It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule . Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.

FEMS Microbiol Lett, 2002 Aug 6, 213(2), 159 - 65
Interaction of NahR, a LysR-type transcriptional regulator, with the alpha subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4; Park W et al.; NahR, a LysR-type transcriptional regulator, is required for expression of naphthalene catabolic operons . However, detailed mechanisms of transcriptional activation by NahR are poorly understood . Many transcriptional activators make direct contact with RNA polymerase (RNAP) to initiate transcription . We investigated the hypothesis that direct contact between NahR and the alpha subunit of RNAP (alphaRNAP) may be involved in expression of the naphthalene catabolic operons in Pseudomonas putida NCIB 9816-4 . Interactions between the NahR and alphaRNAP in P . putida NCIB 9816-4 were analyzed using the yeast two-hybrid system . The results obtained indicate that protein-protein interactions occur between alphaRNAP and the NahR . Gene activation by NahR is consistent with the general transcriptional mechanism of class I transcription factors, which function by contacting alphaRNAP.

Water Res, 2002 May, 36(10), 2443 - 50
The enhancement of 2-chlorophenol degradation by a mixed microbial community when augmented with Pseudomonas putida CP1; Farrell A et al.; The effect of the introduction of Pseudomonas putida CP1 to a commercial mixed microbial community for the degradation of 1.56mM 2-chlorophenol was investigated . Degradation of 2-chlorophenol by the commercial mixture was via a meta-cleavage pathway leading to incomplete degradation, while P . putida CPI was shown to be capable of the complete degradation of 2-chlorophenol via an ortho-cleavage pathway . Augmentation of the commercial mixed culture with P . putida CP1 resulted in complete degradation of 2-chlorophenol via an ortho-cleavage pathway . The augmented mixed culture displayed increased degradative capabilities, with times of degradation reduced when compared to those achieved by P . putida CP1 in isolation . The ability of P . putida CP1 to degrade 2-chlorophenol was increased with the addition of increasing concentrations of the mixed culture . Increasing the mixed culture inoculum size added to P . putida CP1 decreased lag periods and increased rates of degradation, resulting in decreased times of degradation.

Appl Environ Microbiol, 2002 Aug, 68(8), 3795 - 801
Role of Pseudomonas putida indoleacetic acid in development of the host plant root system; Patten CL et al.; Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA) . While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion . To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis . The canola seedling primary roots from seeds treated with wild-type P . putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds . In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots . Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant . These results suggest that bacterial IAA plays a major role in the development of the host plant root system.

Annu Rev Microbiol, 2002, 56, 743 - 68 Epub 2002 Jan 30.
Mechanisms of solvent tolerance in gram-negative bacteria; Ramos JL et al.; Organic solvents can be toxic to microorganisms, depending on the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strains . The toxicity of a given solvent correlates with the logarithm of its partition coefficient in n-octanol and water (log Pow) . Organic solvents with a log Pow between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions . Several possible mechanisms leading to solvent-tolerance in gram-negative bacteria have been proposed: (a) adaptive alterations of the membrane fatty acids and phospholipid headgroup composition, (b) formation of vesicles loaded with toxic compounds, and (c) energy-dependent active efflux pumps belonging to the resistance-nodulation-cell division (RND) family, which export toxic organic solvents to the external medium . In these mechanisms, changes in the phospholipid profile and extrusion of the solvents seem to be shared by different strains . The most significant changes in phospholipids are an increase in the melting temperature of the membranes by rapid cis-to-trans isomerization of unsaturated fatty acids and modifications in the phospholipid headgroups . Toluene efflux pumps are involved in solvent tolerance in several gram-negative strains, e.g., Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa . The AcrAB-TolC and AcrEF-TolC efflux pumps are important for n-hexane tolerance in E . coli . A number of P . putida strains have been isolated that tolerate toxic hydrocarbons such as toluene, styrene, and p-xylene . At least three efflux pumps (TtgABC, TtgDEF, and TtgGHI) are present in the most extensively characterized solvent-tolerant strain, P . putida DOT-T1E, and the number of efflux pumps has been found to correlate with the degree of solvent tolerance in different P . putida strains . The operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive . Specific and global regulators control the expression of the efflux pump operons of E . coli and P . putida at the transcriptional level.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1356 - 8 Epub 2002 Jul 20.
Crystallization and preliminary crystallographic analysis of creatininase from Pseudomonas putida; Beuth B et al.; Creatininase (CrnA) from Pseudomonas putida is a homohexameric heat-stable enzyme composed of 259 amino acids per subunit . The molecular weight of each monomer is 28.4 kDa . The enzyme hydrolyses creatinine to yield creatine . Crystals of this protein have been grown from ethanol/PEG 8000 . They belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 A, alpha = gamma = 90, beta = 103.8 degrees . The diffraction limit is 2.5 A . The self-rotation function of the native data set is consistent with a CrnA hexamer in the asymmetric unit and suggests D(3) point-group symmetry of the enzyme.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1322 - 8 Epub 2002 Jul 20.
Structure of creatine amidinohydrolase from Actinobacillus; Padmanabhan B et al.; The crystal structure of Actinobacillus creatine amidinohydrolase has been solved by molecular replacement . The amino-acid sequence has been derived from the crystal structure . Crystals belong to space group I222, with unit-cell parameters a = 111.26 (3), b = 113.62 (4), c = 191.65 (2) A, and contain two molecules in an asymmetric unit . The structure was refined to an R factor of 18.8% at 2.7 A resolution . The crystal structure contains a dimer of 402 residues and 118 water molecules . The protein structure is bilobal, consisting of a small N-terminal domain and a large C-terminal domain . The C-terminal domain has a pitta-bread fold, similar to that found in Pseudomonas putida creatinase, proline aminopeptidases and methionine aminopeptidase . Comparison with complex crystal structures of P . putida creatinase reveals that the enzyme activity of Actinobacillus creatinase might be similar to that of P . putida creatinase.

Biochemistry, 2002 Jul 30, 41(30), 9611 - 26
Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase; Wolfe MD et al.; The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene . Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and Mossbauer spectroscopies . BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske {2Fe-2S} cluster and a mononuclear iron site . Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization . Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number . At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and Mossbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers . These results are in agreement with our previous study of naphthalene 1,2-dioxygenase {Wolfe, M . D., Parales, J . V., Gibson, D . T., and Lipscomb, J . D . (2001) J . Biol . Chem . 276, 1945-1953}, which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone . The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme . The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product . Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced . Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis . The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters . The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.

Mol Plant Microbe Interact, 2002 Jul, 15(7), 734 - 41
Pseudomonas putida strain PCL1444, selected for efficient root colonization and naphthalene degradation, effectively utilizes root exudate components; Kuiper I et al.; Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies . After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv . Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots . Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components . High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids . Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P . stutzeri . Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase . Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid . Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase . When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid . These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P . putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components . Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.

Arch Microbiol, 2002 Aug, 178(2), 149 - 60 Epub 2002 Jun 14.
The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P . oleovorans and of the enoyl-CoA hydratase genes phaJ from P . oleovorans and Pseudomonas putida; Fiedler S et al.; In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized . The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium . Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E . coli wild-type strain harboring a PHA synthase gene . However, PHA accumulation was strongly impaired in a recombinant E . coli fadB mutant, which harbored a PHA synthase gene . These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the ( R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis . However, the phaJ gene from P . oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified . The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E . coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate . These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P . oleovorans.

J Biol Chem, 2002 Oct 4, 277(40), 37519 - 26 Epub 2002 Jul 11.
Crystal structure of the F87W/Y96F/V247L mutant of cytochrome P-450cam with 1,3,5-trichlorobenzene bound and further protein engineering for the oxidation of pentachlorobenzene and hexachlorobenzene; Chen X et al.; We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J . P., O'Hare, E . J., and Wong, L . L . (2001) Eur . J . Biochem . 268, 1460-1467) . In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene . The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction . In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron . The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines . The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101)(-1) min(-1), 45 times faster than the F87W/Y96F/V247L parent mutant . The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min(-1) . Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms . In principle, the F87W/Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

J Bacteriol, 2002 Aug, 184(15), 4096 - 103
Reactivity of toluate dioxygenase with substituted benzoates and dioxygen; Ge Y et al.; Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates . The two components of this enzyme were hyperexpressed and anaerobically purified . Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe(2)S(2) centers . Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism . TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 +/- 1 microM, k(cat) = 3.9 +/- 0.2 s(-1), and K(m)O(2) = 16 +/- 2 microM (100 mM sodium phosphate, pH 7.0; 25 degrees C), where K(m)O(2) represents the K(m) for O(2) and KmA represents the K(m) for the aromatic substrate . The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate approximately 3-chlorobenzoate > p-toluate approximately 4-chlorobenzoate >> o-toluate approximately 2-chlorobenzoate . The transformation of each of the first five compounds was well coupled to O(2) utilization and yielded the corresponding 1,2-cis-dihydrodiol . In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O(2) utilization, with >10 times more O(2) being consumed than benzoate . However, the apparent K(m) of TADO for these benzoates was >100 microM, indicating that they do not effectively inhibit the turnover of good substrates.

Biomacromolecules, 2002 Jul-Aug, 3(4), 661 - 7
Heterogeneity in bacterial surface polysaccharides, probed on a single-molecule basis; Camesano TA et al.; The heterogeneity in bacterial surface macromolecules was probed by examining individual macromolecules on the surface of Pseudomonas putida KT2442 via single-molecule force spectroscopy (SMFS) . Using an atomic force microscope (AFM), the silicon nitride tip was brought into contact with biopolymer molecules on bacterial cells and these macromolecules were stretched . Force-extension measurements on different bacterial cells showed a range of adhesion affinities and polymer lengths . However, substantial heterogeneity was also observed in the force-extension curves on a single bacterium . A given bacterium has biopolymers that range in size from tens to hundreds of nanometers, with adhesion affinities for the AFM tip from nearly zero to greater than 1 nN . A distribution of polymer sizes was confirmed by size-exclusion chromatography . The freely jointed chain (FJC) model for polymer elasticity was applied to individual force-extension curves in order to estimate the contour lengths and segment lengths of the polymer chains . A range of segment lengths was obtained using the FJC model, from 0.154-0.45 nm in water, 0.154-0.32 nm in 0.01 M KCl, and 0.154-0.65 nm in 0.1 M KCl . The modeling confirms that the heterogeneity in biopolymers is more than a matter of differences in molecular weights, since a range of stiffnesses (segment lengths) was also observed . The effect of salt concentration on biopolymer conformation and adhesion was also explored . While the biopolymers were flexible in all solvents, they were slightly more extended in water than in either of the salt solutions (0.01 and 0.1 M KCl) . The adhesion of polysaccharides with the AFM tip was not dependent on salt concentration, because the polymers were not highly charged and heterogeneity overwhelmed any trends that could be observed in adhesion with respect to solution ionic strength . These experiments indicate that heterogeneity in biopolymer properties on an individual bacterium and within a population of bacterial cells may be much greater than previously believed and should be incorporated into models of bacterial adhesion.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(6), 1133 - 46
Biodegradation and adsorption of phenol using activated carbon immobilized with Pseudomonas putida; Annadurai G et al.; This paper examined the removal efficiency of phenol from aqueous solution using a suspended culture of Pseudomonas putida (ATCC 3180) or the activated carbon on which the microorganism was immobilized . The kinetics of phenol degradation by immobilized and pure cells was studied . Experiments were performed at various phenol concentrations (0.1-0.4 g/L), pH, temperature (30-36 degrees C), and concentrations of glucose (0.5-0.7 g/L) and (NH4)2SO4 (0.5-0.7 g/L) . The presence of activated carbon markedly enhanced the degradation efficiency, showing its ability of protecting microbes from confronting shock loads of organic pollutants . Degradation rate increased with increasing substrate concentration and decreased after reaching a maximum, indicating substrate-inhibition kinetics . In addition, the degradation rate for immobilized cells was much higher than that of free cells . The inhibition effect for phenol degradation was described by the Andrews model . The kinetic parameters were also determined.

J Bacteriol, 2002 Jul, 184(14), 3785 - 93
Inactivation of cytochrome o ubiquinol oxidase relieves catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway; Dinamarca MA et al.; Expression of the alkane degradation pathway encoded by the OCT plasmid of Pseudomonas putida GPo1 is regulated by two control systems . One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes . The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes . This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway . To identify the factors involved, a screening method was developed to isolate mutants without this control . Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome o ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions . Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source . This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell . Since inactivation of the crc gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome o terminal oxidase . The two mutations had an additive effect in relieving catabolic repression in rich medium . This suggests that crc and cyo belong to different regulation pathways, both contributing to catabolic repression.

J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 127 - 33
Application of metabolic engineering to improve both the production and use of biotech indigo; Berry A et al.; A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli . This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO) . In comparison to a tryptophan-over-producing strain, the first indigo-producing strain made less than half of the expected amount of indigo . Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroGfbr gene product), was observed in cells collected from indigo fermentations . Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo . Indigo production was thereafter improved by increasing the gene dosage of aroGfbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase . By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved . Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.

Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 278 - 85
{Effects of p-nitrophenol and organophosphorous nitroaromatic insecticides on the respiratory activity of free and immobilized cells of strains S-11 and BA-11 of Pseudomonas putida}; Ignatov OV et al.; The possibility of using the respiratory activity (RA) of microbial cells (of strains S-11 and BA-11 of Pseudomonas putida) as an instrument for quantitative determination of organophosphorous nitroaromatic insecticides, metaphors and sumithion, and their hydrolysis product, p-nitrophenol (PNP), has been explored . The dependences of RA on the concentrations of the three compounds were linear within the range 0.5-2.5 microM . The cells of the strain BA-11 exhibited maximum selectivity in the determination of the compounds . The RA of microbial cells differing in the modes of immobilization (adsorption to carrier surfaces vs . incorporation into gels) have been compared . Prospects of development of the microbial cell-based sensor system for determining metaphors, sumithion, and PNP in aqueous media are discussed.

Z Naturforsch {C}, 2002 Mar-Apr, 57(3-4), 356 - 60
Biosurfactant production by a new Pseudomonas putida strain; Tuleva BK et al.; Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN . The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp . Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1) . When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.

Structure (Camb), 2002 Jun, 10(6), 837 - 49
Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5; Chen ZW et al.; The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group . The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase . The PQQ is bound near the axis of the propeller domain about 14 A from the heme . A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.

Biotechnol Prog, 2002 May-Jun, 18(3), 458 - 64
Expanded application of a two-phase partitioning bioreactor through strain development and new feeding strategies; Vrionis HA et al.; This research demonstrated the microbial treatment of concentrated phenol wastes using a two-phase partitioning bioreactor (TPPB) . TPPBs are characterized by a cell-containing aqueous phase and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells on the basis of their metabolic demand and the thermodynamic equilibrium of the system . Process limitations imposed by the capability of wild-type Pseudomonas putida ATCC 11172 to utilize long chain alcohols were addressed by strain modification (transposon mutagenesis) to eliminate this undesirable biochemical characteristic, enabling use of a range of previously bioavailable organics as delivery solvents . Degradation of phenol in a system with the modified strain as catalyst and industrial grade Adol 85 NF (primarily oleyl alcohol) as the solvent was demonstrated, with the system ultimately degrading 36 g of phenol within 38 h . Volumetric phenol consumption rates by wild type P . putida ATCC 11172 and the genetically modified derivative revealed equivalent phenol degrading capabilities (0.49 g/L x h vs 0.47 g/L x h respectively, in paired fermentations), with the latter presenting a more efficient remediation option due to decreased solvent losses arising from the modified strain's forced inability to consume the delivery solvent as a substrate . Two feeding strategies and system configurations were evaluated to expand practical applications of TPPB technology . The ability to operate with a lower solvent ratio over extended periods revealed potential for long-term application of TPPB to the treatment of large masses of phenol while minimizing solvent costs . Repeated recovery of 99% of phenol from concentrated phenol solutions and subsequent treatment within a TPPB scheme demonstrated applicability of the approach to the remediation of highly contaminated "effluents" as well as large masses of bulk phenol . Operation of the TPPB system in a dispersed manner, rather than as two distinct phases, resulted in volumetric consumption rates similar to those previously achieved only in systems operated with enriched air.

Biodegradation, 2001, 12(6), 465 - 75
A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1; Yu H et al.; A two-step model is developed for the aerobic biodegradation of benzene, toluene, and p-xylene (BTX) by Pseudomonas putida F1 . The model contains three unique features . First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomass growth . Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis . Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzene noncompetitively inhibit the mineralization of their catechol intermediate . A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.

Biodegradation, 2001, 12(6), 455 - 63
The roles of intermediates in biodegradation of benzene, toluene, and p-xylene by Pseudomonas putida F1; Yu H et al.; Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1 . Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively . Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis . When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.

Biodegradation, 2001, 12(6), 411 - 8
Metal binding by pyridine-2,6-bis(monothiocarboxylic acid), a biochelator produced by Pseudomonas stutzeri and Pseudomonas putida; Stolworthy JC et al.; Pyridine-2,6-bis(monothiocarboxylic acid) (pdtc), a natural metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida that promotes the degradation of carbon tetrachloride, was synthesized and studied by potentiometric and spectrophotometric techniques . The first two stepwise protonation constants (pK) for successive proton addition to pdtc were found to be 5.48 and 2.58 . The third stepwise protonation constant was estimated to be 1.3 . The stability (affinity) constants for iron(III), nickel(II), and cobalt(III) were determined by potentiometric or spectrophotometric titration . The results show that pdtc has strong affinity for Fe(III) and comparable affinities for various other metals . The stability constants (log K) are 33.93 for Co(pdtc)2(1-); 33.36 for Fe(pdtc)2(1-); and 33.28 for Ni(pdtc)2(2-) . These protonation constants and high affinity constants show that over a physiological pH range the ferric pdtc complex has one of the highest effective stability constants for iron binding among known bacterial chelators.

J Biotechnol, 2002 Jul 3, 96(3), 281 - 9
Membrane-facilitated bioproduction of 3-methylcatechol in an octanol/water two-phase system; Husken LE et al.; Bioproduction of 3-methylcatechol from toluene by Pseudomonas putida MC2 was studied in the presence of an additional 1-octanol phase . This solvent was used to supply the substrate and extract the product, in order to keep the aqueous concentrations low . A hollow-fibre membrane kept the octanol and aqueous phase separated to prevent phase toxicity towards the bacterium . Volumetric production rates increased approximately 40% as compared to two-phase 3-methylcatechol production with direct phase contact . Preliminary investigations on downstream processing of 3-methylcatechol showed that 1 M of sodium hydroxide selectively extracted the disodium salt of 3-methylcatechol into an aqueous phase.

Water Res, 2002 Apr, 36(7), 1794 - 802
Inhibition of p-cresol on aerobic biodegradation of carbazole, and sodium salicylate by Pseudomonas putida; Yu YG et al.; A PAH- and phenol-degrading microorganism, Pseudomonas putida (ATCC 17484), was used to study the substrate interactions during cell growth on carbazole-containing mixtures with p-cresol and sodium salicylate . Both p-cresol and sodium salicylate could be utilised by the bacteria as the sole carbon and energy sources . When cells grew on the mixture of carbazole, p-cresol and sodium salicylate, strong substrate interactions were observed . Carbazole degradation started only after p-cresol was significantly or completely removed, and the removal of carbazole was incomplete when the initial p-cresol concentration was higher than 20 mg/l . No carbazole was removed at all when the initial p-cresol concentration in the system was higher than 120 mg/l . When cells grew on the ternary substrates, the specific growth rate was found to increase with p-cresol concentration up to 50 mg/l (from 0.33 to 0.45 h(-1)) but decreased monotonically with higher concentrations . At 120 mg/l p-cresol, specific growth rate fell to 0.33 h(-1) . The inhibitory effect of p-cresol was demonstrated where carbazole degradation was immediately halted when 50 mg/l p-cresol was spiked to a system containing carbazole and sodium salicylate . Besides, the addition of p-cresol was also found to inhibit the degradation of sodium salicylate . With p-cresol, an increase in lag time was observed and the utilisation of sodium salicylate as carbon source was severely retarded.

Appl Environ Microbiol, 2002 Jun, 68(6), 2651 - 9
Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase; Hasona A et al.; During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced . With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1) . Although the E . coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity . A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change . This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ . Recombinant EstZ esterase was purified to near homogeneity and characterized . It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine . As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate . The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer . By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C . The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol . min(-1) . mg of protein(-1), respectively . Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol . min(-1) . mg of protein(-1)), followed by ethyl acetate (66 micromol . min(-1) . mg of protein(-1)) . Expression of estZ in E . coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).

J Ind Microbiol Biotechnol, 2002 Jun, 28(6), 316 - 24
Substrate-dependent autoaggregation of Pseudomonas putida CP1 during the degradation of mono-chlorophenols and phenol; Farrell A et al.; A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture . The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway . P . putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P . putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol . Growth of P . putida CP1 on higher concentrations of the mono-chlorophenols, >or=1.56 mM 4-chlorophenol and >or=0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium . Increases in cell biomass with no clumping of the cells resulted from growth of P . putida CP1 on phenol or on lower concentrations of mono-chlorophenol . Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol . The results suggested that increased hydrophobicity and autoaggregation of P . putida CP1 were a response to toxicity of the added substrates.

Microb Ecol, 2001 Feb, 41(4), 352 - 359
Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes; Palomares AJ et al.; We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, lPR, fused to luc and lucOR as reporter genes . We have demonstrated that the systems cI857-lPR::luc and cI857-lPR::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E . coli to those Gram-negative backgrounds . Using light for the detection, we have observed plasmid transfer from E . coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms . The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed . When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose . This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.

Microb Ecol, 2001 Feb, 41(4), 310 - 313
The Use of Modified GFP as a Reporter for Metabolic Activity in Pseudomonas putida; Lowder M et al.; Many bacteria are now known to enter into a "viable but nonculturable" (VBNC) state in response to various environmental stresses . In this state, the cells are no longer culturable on routine media, but retain viability and in many cases have been shown to be capable of resuscitating to the metabolically active and culturable state . There have been no simple means of measuring the metabolic activity of cells in the VBNC state . The use of green fluorescent protein (GFP) variants with short half-lives was examined in cells intended for environmental release to examine the potential of GFP as a reporter of metabolic activity . Unlike strains with the native (stable) GFP, Pseudomonas putida strains tagged with unstable GFP rapidly lost GFP fluorescence following exposure to starvation and VBNC-inducing conditions . Our results suggest that tagging cells with the modified GFP provides a method for determining metabolic activity in these cells.

Curr Microbiol, 2002 Jul, 45(1), 30 - 6
Tandem biodegradation of BTEX components by two Pseudomonas sp; Attaway HH et al.; A co-culture of two Pseudomonas putida isolates was enriched from sediment on a mixture of benzene, toluene, ethylbenzene, m-xylene, p-xylene, and o-xylene . The co-culture readily degraded each of the compounds present . Benzene, toluene, and ethylbenzene were used as growth substrates by one isolate, while toluene, m-xylene, and p-xylene were used as growth substrates by the other . Neither isolate could grow on o-xylene, but it was removed in the presence of the other compounds presumably by co-metabolism . The findings presented here support other reports in which constructed communities were effectively used to degrade blends of between two and four of the components of BTEX . However, here the co-culture of two P . putida isolates effectively degraded a complete BTEX stream containing all six of the components.

Microb Ecol, 2001 Aug, 42(2), 168 - 176
The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates; Boenigk J et al.; The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated . Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria) . Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles . In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis . Digestion was found to be particle-specific . Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles . The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content . "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles . The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field . In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 123 - 7
Expression of the iorAB genes from Brevundimonas diminuta 7 encoding the molybdenum hydroxylase isoquinoline 1-oxidoreductase in Pseudomonas putida; Israel I et al.; Isoquinoline 1-oxidoreductase (Ior) from Brevundimonas diminuta 7, encoded by iorAB, is a molybdenum hydroxylase containing a molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD) and two distinct {2Fe2S} clusters . The iorAB genes were inserted into pJB653, generating pIL1 . Pseudomonas putida KT2440, and P . putida 86 which produces a Mo-MCD-containing quinoline 2-oxidoreductase when grown on quinoline, were used as recipients for pIL1 . Upon induction of gene expression, both clones produced Ior protein, but Ior activity was not detectable in P . putida KT2440 pIL1 . In P . putida 86 pIL1, formation of catalytically active Ior required the presence of quinoline, suggesting that accessory gene(s) encoding product(s) essential for the assembly of catalytically competent Ior is (are) part of the quinoline regulon in P . putida 86.

J Environ Biol, 2001 Jul, 22(3), 153 - 62
Inorganic nutrient utilisation by "adapted" Pseudomonas putida strain used in the bioremediation of agricultural soil polluted with crude petroleum; Nwachukwu SC et al.; Garden soil samples polluted with crude petroleum were bioremediated by inorganic nutrient monitoring with appropriate adjustment and inoculation with crude oil-adapted strain of Pseudomonasputida (PP) isolated from oil-impacted soils . Soil samples without PP inoculation served as the control samples to compare the abilities of the native soil microflora with the adapted PP strain in biodegrading crude oil pollutant . In the experimental samples, oil concentration and all the inorganic nutrient sources tested decreased more rapidly with a proportional increase in the population densities of both PP and the native soil microflora than were observed in the control samples . This trend was particularly strong for PO4(3-) and NO3- which eventually became limiting both in all the experimental samples and in some control samples . Inoculation of crude oil-impacted agricultural soils by oil -adapted PP strain with nutrient monitoring and adjustment can be effective as bioremediation methods of agricultural land upon pollution with petroleum or petroleum products.

J Biol Chem, 2002 Jul 12, 277(28), 25831 - 9 Epub 2002 May 13.
Putidaredoxin reductase, a new function for an old protein; Sevrioukova IF et al.; Properties of recombinant wild type (WT) and six-histidine tag-fused (His(6)) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied . Both WT and His(6) Pdr were found to undergo a monomer-dimer association-dissociation and were partially present as an NAD(+)-bound form . Although molecular, spectral, and electron transferring properties of recombinant His(6) Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-His-His-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide . Under anaerobic conditions, NAD(+) induced in His(6) Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD(+) . The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione . In the presence of any of these three reagents, NAD(+) was capable of inducing reduction of the flavin in WT Pdr . Free thiol groups were identified as an internal source of electrons in the enzyme . The results showed that WT and His(6) Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD(+)-dependent oxidation of thiols, and NADH-dependent reduction of disulfides . This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin . Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.

Environ Microbiol, 2002 Apr, 4(4), 225 - 37
The LysR-type regulator SftR is involved in soil survival and sulphate ester metabolism in Pseudomonas putida; Kahnert A et al.; Sulphate esters make up a large proportion of the available sulphur in agricultural soils, and many pseudomonads can desulphurize a range of aryl- and alkylsulphate esters to provide sulphur for growth . After miniTn5 transposon mutagenesis of Pseudomonas putida S-313, we isolated 19 mutants that were defective in cleavage of the chromogenic sulphate ester 5-bromo-4-chloro-3-indoxylsulphate (X-sulphate) . Analysis of these strains revealed that they carried independent insertions in a gene cluster that comprised genes for a sulphate ester/sulphonate transporter (atsRBC) a LysR-type regulator (sftR), an oxygenolytic alkylsulphatase (atsK), an arylsulphotransferase (astA) and a putative TonB-dependent receptor (sftP) . The SftP protein was localized in the outer membrane, and the arylsulfphotransferase was identified as an intracellular enzyme . Expression of sftR was repressed in the presence of inorganic sulphate, and the sftR gene was required for the expression of atsBC, atsRK and sftP-astA . An sftR mutant was unable to grow with aryl- or alkylsulphate esters in laboratory media and showed significantly reduced survival compared with the parent strain during incubation in Danish agricultural and grassland soils . This effect suggests that sulphate esters are an important sulphur source for microbes in aerobic soils and highlights the importance of the microbial population in the soil sulphur cycle.

Biotechnol Bioeng, 2002 Jun 30, 78(7), 715 - 21
Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source; Walker AW et al.; Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy . The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P . putida KT2442 . The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters . Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity . A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P . putida allowing the organism to use 0.5 mM PNP as a carbon and energy source . Transformation of P . putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy . Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable .

Biotechnol Bioeng, 2002 Jun 20, 78(6), 626 - 34
Quantitative analysis of experiments on bacterial chemotaxis to naphthalene; Pedit JA et al.; A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation . The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system . The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation . Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system . For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis . Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated . The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media . Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others .

Appl Biochem Biotechnol, 2001 Spring, 91-93, 219 - 32
Dissemination of catabolic plasmids among desiccation-tolerant bacteria in soil microcosms; Weekers F et al.; The dissemination of catabolic plasmids was compared to bioaugmentation by strain inoculation in microcosm experiments . When Rhodococcus erythropolis strain T902, bearing a plasmid with trichloroethene and isopropylbenzene degradation pathways, was used as the inoculum, no transconjugant was isolated but the strain remained in the soil . This plasmid had a narrow host range . Pseudomonas putida strain C8S3 was used as the inoculum in a second approach . It bore a broad host range conjugative plasmid harboring a natural transposon, RP4::Tn4371, responsible for biphenyl and 4-chlorobiphenyl degradation pathways . The inoculating population slowly decreased from its original level (10(6) colony-forming units {CFU}/g of dry soil) to approx 3 x 10(2) CFU/g of dry soil after 3 wk . Transconjugant populations degrading biphenyl appeared in constant humidity soil (up to 2 x 10(3) CFU/g) and desiccating soil (up to 10(4) CFU/g) . The feasibility of plasmid dissemination as a bioaugmentation technique was demonstrated in desiccating soils . The ecologic significance of desiccation in bioaugmentation was demonstrated: it upset the microbial ecology and the development of transconjugants.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 195 - 204
Enhancement of the conversion of toluene by Pseudomonas putida F-1 using organic cosolvents; Rodriguez M Jr et al.; Pseudomonas putida F-1 (ATCC700007) was used as a model organism in stirred tank reactors to study conversion enhancement of poorly soluble substrates by organic cosolvents . After a literature study, silicone oil was used as a solvent system to enhance the mass transfer rate . To study the benefits of the organic solvent addition, batch experiments were conducted in two side-by-side fermentation vessels (experimental and control) at three different levels of silicone oil (10, 30, and 50%) . Results showed that the presence of silicone oil resulted in a 100% increase in the toluene mass transfer compared to the control . Experiments in continuous stirred-tank reactors showed that improved conversion could be obtained at higher agitation rates.

FEMS Microbiol Lett, 2002 Mar 5, 208(2), 263 - 8
Induction and repression of the sty operon in Pseudomonas putida CA-3 during growth on phenylacetic acid under organic and inorganic nutrient-limiting continuous culture conditions; O'Leary ND et al.; The effects of various nutrient-limiting conditions on expression of the sty operon in Pseudomonas putida CA-3 were investigated . It was observed that limiting concentrations of the carbon source phenylacetic acid, resulted in high levels of phenylacetyl coenzyme A (CoA) ligase activity, this was accompanied also by upper pathway styrene monooxygenase enzyme activity . The introduction of inorganic nutrient limitations, (nitrate, sulfate and phosphate), caused a dramatic reduction in detectable levels of phenylacetyl CoA ligase activity, particularly in the presence of the primary carbon source, succinate . Under these conditions it was no longer possible to detect styrene monooxygenase activity . Reverse transcription PCR analysis of total RNA, isolated under each of the continuous culture conditions examined, revealed that variations in the levels of enzyme activity coincided with altered patterns of corresponding paaK (phenylacetyl CoA ligase) and styA (styrene monooxygenase) gene expression . Transcription of the upper pathway regulatory sensor kinase gene styS was also observed to be growth condition-dependent . These observations suggest that induction/repression of the sty operon in P . putida CA-3, during growth on phenylacetic acid under continuous culture conditions, involves regulatory mechanisms coordinately affecting both the upper and lower pathways and acting at the level of gene transcription.

Appl Microbiol Biotechnol, 2002 Mar, 58(4), 469 - 75 Epub 2002 Jan 16.
Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization; Vrionis HA et al.; Two-phase partitioning bioreactors (TPPBs) comprise an aqueous phase containing all non-carbon nutrients necessary for microbial growth and a solvent phase containing high concentrations of inhibitory or toxic substrates that partition at sub-inhibitory levels to the aqueous phase in response to cellular demand . This work aimed at eliminating the growth of Pseudomonas putida ATCC 11172 on medium-chain-length (C8-C12) aliphatic alcohols, hence enabling their use as xenobiotic delivery solvents within two-phase partitioning bioreactors . Experiments resulted in the isolation of a mini-Tn5 mutant unable to utilize these alcohols . The mutation, which also eliminated growth on glycerol and ethanol, was identified to be within a homologue of the P aeruginosa agmR gene, which encodes a response regulator . Enzyme analysis of the agmR::Tn5Km mutant cell extracts revealed a 10-fold decrease in pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase activity . A knockout in a gene (exaA) encoding a PQQ-linked alcohol dehydrogenase slowed but did not eliminate growth on medium-chain-length alcohols or ethanol, suggesting metabolic redundancy within P . putida ATCC 11172 . Analysis of P . putida KT2440 genome sequence data indicated the presence of two PQQ-linked alcohol dehydrogenase-encoding genes . The successful elimination of alcohol utilization in the agmR mutant indicates control by AgmR on multiple pathways and presents a useful strain for biotechnological applications requiring alcohol non-utilizing microbial catalysts.

J Microbiol Methods, 2002 Jun, 50(1), 75 - 84
Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification; Bakermans C et al.; The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process . In this study, fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria . Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times . Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol . Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts . Only induced cells showed positive hybridization to probe Ac627BR . Results were verified by RNase A or DNase I digestion of samples prior to hybridization . When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (approximately 1%) of the cells present in the contaminated groundwater . This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ-in soil, water, or sediment where they dwell.

J Biochem (Tokyo), 2002 Apr, 131(4), 523 - 31
Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere; Nakajima H et al.; An enzymatic method for measuring the O(2) concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2,3-dioxygenase from Pseudomonas putida . With this system, the amount of O(2) in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction . The product was stable and its anion exhibited strong absorption around 380 nm (molar absorption coefficient of 4.3 x 10(4) M(-1) cm(-1), pK value of 5.4) . A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol . When the O(2) concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O(2) could be successfully measured by the HPLC method . We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon . By applying these devices, the submicromolar O(2) concentration of an anoxic solution and the back diffusion of O(2) from the atmosphere could be directly determined for the first time . The K(m) values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 microM (at pH 6.5), respectively, at 25 degrees C.

Appl Environ Microbiol, 2002 Apr, 68(4), 1988 - 93
Inducible gene expression by nonculturable bacteria in milk after pasteurization; Gunasekera TS et al.; The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion . In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable . Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp . However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units . These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

J Bacteriol, 2002 Apr, 184(8), 2324 - 30
TetR family member psrA directly binds the Pseudomonas rpoS and psrA promoters; Kojic M et al.; We have previously described a Pseudomonas gene, psrA, which enhances transcription of the rpoS sigma factor gene at stationary phase . We present molecular data which demonstrate that in Pseudomonas putida PsrA binds specifically to the rpoS and psrA promoters in DNA regions having similar palindromic sequences, C/GAAAC N(2-4) GTTTG/C, where N is any nucleotide . The position of the initiation of transcription was determined for both promoters, and PsrA binds from positions -59 to -35 in the rpoS promoter and from -18 to +20 in the psrA promoter with respect to the +1 transcription site . Expression studies with a psrA-lacZ transcriptional fusion in wild-type and psrA::Tn5 knockout mutants revealed that psrA was under additional control in response to growth phase . A model for the role of PsrA in the regulation of rpoS and psrA is presented.

Biochemistry, 2002 Mar 26, 41(12), 4048 - 58
Kinetics and thermodynamics of mandelate racemase catalysis; St Maurice M et al.; Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude . The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents . A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions . The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity . However, sucrose did have a slight activating effect on mutant enzyme efficiency . In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed . These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions . The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km . The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction . From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction {Bearne, S . L., and Wolfenden, R . (1997) Biochemistry 36, 1646-1656}, we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/mol, TDeltaS = -4.8 +/- 0.8 kcal/mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/mol, TDeltaS# = +2.0 +/- 0.1 kcal/mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C . Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.

Antimicrob Agents Chemother, 2002 Apr, 46(4), 1053 - 8
bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital; Lee K et al.; We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995 . The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P . aeruginosa isolates from Europe . VIM-2-producing isolates of Pseudomonas putida were also detected . Resistance was transferable from both these species to P . aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid . Serotyping showed that many of the VIM-2-producing P . aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P . putida isolates were diverse . Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons . In contrast to previous VIM-encoding integrons, the integron sequenced from a P . aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4 . bla(VIM) also lay in a class 1 integron in a representative P . putida strain, but the organization of this integron was different from that sequenced from the P . aeruginosa strain . In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread . Strict measures are required in order to control a further spread of resistance.

J Inorg Biochem, 2002 Feb, 88(3-4), 362 - 7
A scanning tunneling microscopy (STM) investigation of complex formation between cytochrome P450(cam) and putidaredoxin; Djuricic D et al.; We have previously reported the scanning tunnelling microscopy (STM) imaging under buffer of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida {Faraday Discuss . 116 (2000) 1} . We describe here the adsorption and STM imaging under buffer of complexes of a mutant of cytochrome P450(cam), K344C, and wild-type putidaredoxin (Pdx) on gold(111) . The images of Pdx on its own on gold(111) are not uniform, presumably due to multiple orientations of protein adsorption because of the presence of five or more cysteines on the protein surface . STM imaging of a 1:1 mixture of P450(cam)-K344C/Pdx showed a regular array of pairs of different-sized proteins 20-25 A apart arranged in rows across the gold(111) surface which we attribute to the P450(cam)/Pdx complex . The images of the pairs are more regular than those of Pdx on its own, probably as a result of complex formation with P450(cam) partly overcoming the heterogeneity of Pdx adsorption . As far as we are aware this is the first report of STM imaging of a protein/protein complex, and the first direct observation of P450(cam)/Pdx complex formation which is a key step in the catalytic cycle of P450(cam) catalysis . The redox centers of the two proteins are ca . 20 A apart, too far for rapid intracomplex electron transfer . Whether the observed complex is competent for electron transfer or physiologically relevant is not known, and further work is in progress to elucidate the protein-protein interaction.

Arch Microbiol, 2002 Apr, 177(4), 345 - 51 Epub 2002 Feb 02.
Ferredoxin-mediated reactivation of the chlorocatechol 2,3-dioxygenase from Pseudomonas putida GJ31; Tropel D et al.; In the chlorobenzene degrader Pseudomonas putida GJ31, chlorocatechol is formed as an intermediate and cleaved by a meta-cleavage extradiol chlorocatechol dioxygenase, which has previously been shown to be exceptionally resistant to inactivation by substituted catechols . The gene encoding this dioxygenase ( cbzE) is preceded by a gene ( cbzT) potentially encoding a ferredoxin, the function of which was studied . The cbzT gene product was overproduced in Escherichia coli and purified in recombinant form . Two homologous proteins, CdoT and AtdS, encoded by genes identified in strains degrading nitrobenzene and aniline, respectively, were also purified and characterized . All three proteins showed spectroscopic properties typical for {2Fe-2S} ferredoxins . The chlorocatechol dioxygenase from strain GJ31 (CbzE) was fully inactivated when 4-methylcatechol was used as substrate . Inactivated CbzE could be rapidly reactivated in vitro in the presence of purified CbzT and a source of reductant . It is inferred that the ability of strain GJ31 to metabolize both chlorobenzene and toluene might depend on the regeneration of the chlorocatechol dioxygenase activity mediated by CbzT . Three CbzT-like ferredoxins, including AtdS, were found to be competent in the reactivation of CbzE, whereas XylT, a protein known to mediate reactivation of the catechol dioxygenase from P . putida mt2 (XylE), was ineffective . Accordingly, CbzT formed a covalent complex with CbzE when cross-linked with a carbodiimide, whereas XylT did not . In the reverse situation, CbzT was found to reactivate XylE as efficiently as XylT and formed an heterologous covalent complex with this enzyme upon cross-linking . We conclude that CbzT, CdoT and AtdS are isofunctional ferredoxins that appear to be involved in the reactivation of their cognate catechol dioxygenases . Based on primary structure comparisons, residues of the ferredoxins possibly involved in the molecular interaction with catechol dioxygenases were identified and their significance is discussed.

J Bacteriol, 2002 Mar, 184(6), 1722 - 32
Rubredoxins involved in alkane oxidation; van Beilen JB et al.; Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems . Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (Alk-Rds) . Complementation tests in an Escherichia coli recombinant containing all Pseudomonas putida GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds . All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in n-octane hydroxylation . Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria . Common to most Rds are the two iron-binding CXXCG motifs . All Alk-Rds possess four negatively charged residues that are not conserved in other Rds . The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif . In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids . Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.

Electrophoresis, 2002 Feb, 23(4), 520 - 7
Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction; Paulino LC et al.; Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium that plays an important role in metal bioleaching processes . Despite the high level of tolerance to heavy metals shown by A . ferrooxidans, the genetic basis of copper resistance in this species remains unknown . We investigated the gene expression in response to copper in A . ferrooxidans LR using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) . One hundred and four differentially expressed genes were identified using eight arbitrary primers . Differential gene expression was confirmed by DNA slot blot hybridization, and approximately 70% of the RAP-PCR products were positive . The RAP-PCR products that presented the highest levels of induction or repression were cloned, sequenced and the sequences were compared with those in databases using the BLAST search algorithm . Seventeen sequences were obtained . The RAP-PCR product with the highest induction ratio showed similarity with the A . ferrooxidans cytochrome c . A high similarity with the thiamin biosynthesis gene thiC from Caulobacter crescentus was observed for another RAP-PCR product induced by copper . An RAP-PCR product repressed by copper showed significant similarity with the carboxysome operon that includes the ribulose-1,5-bisphosphate carboxylase/oxygenase complex from A . ferrooxidans and another copper-repressed product was significantly similar to the XyIN outer membrane protein from Pseudomonas putida . Finally, RAP-PCR products of unknown similarities were also present.

Biosci Biotechnol Biochem, 2002 Jan, 66(1), 85 - 91
Effects of GroESL coexpression on the folding of nicotinoprotein formaldehyde dismutase from Pseudomonas putida F61; Yanase H et al.; The overexpression of fdm, which encodes the formaldehyde dismutase from Pseudomonas putida F61, resulted in the formation of inclusion bodies made up of aggregated enzyme, leaving little activity in the soluble fraction of the transformant cells . On the other hand, coexpression of groESL along with fdm facilitated in vivo solubilization of the enzyme protein in its active form . When coexpressed with groESL, formaldehyde dismutase purified from E . coli had the same crystalline form (i.e., a regular octahedron) as the native enzyme, and like the native enzyme, it bound 1 mol of NAD(H) and 2 mol of zinc in each subunit.

Biometals, 2002 Mar, 15(1), 1 - 13
Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1; Ongena M et al.; Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe3+ acquisition in iron-limited environments . Since it produces a structurally specific pyoverdin, Pseudomonas putida strain BTP1 could represent a valuable tool in an attempt to correlate the structural features of these compounds with some specificity in their two main properties i.e . affinity for iron and recognition rate by other Pseudomonas strains . An uncommonly high affinity for iron of the pyoverdin synthetized by P . putida BTP1 was observed by comparing both the apparent stability constant and the decomplexation kinetic of its ferric complex with those of ferripyoverdins from other strains . On another hand, results from growth stimulation experiments and labeled ferripyoverdin uptake assays highlighted the very low recognition rate of BTP1 isopyoverdins by membrane receptors of foreign strains . By contrast, P . putida BTP1 was able to utilize a broad spectrum of structurally unrelated exogenous pyoverdins by means of multiple receptors that are likely constitutively expressed in its outer membrane . The unusual traits of its pyoverdin-mediated iron acquisition system should contribute to enlarge the ecological competence of Pseudomonas putida BTP1 in terms of colonization and persistence in the rhizosphere.

J Appl Microbiol, 2001 Dec, 91(6), 963 - 71
Evaluation of potential biocontrol rhizobacteria from different host plants of Verticillium dahliae Kleb; Berg G et al.; AIMS: A screening approach was developed to assess the potential of rhizobacterial strains to control Verticillium wilt caused by Verticillium dahliae Kleb . METHODS AND RESULTS: Sixty randomly chosen antagonistic bacterial strains originally isolated from rhizosphere of three different host plants of V . dahliae--strawberry, potato and oilseed rape--were evaluated for biocontrol and plant growth promotion by analysing in vitro antagonism towards V . dahliae and other plant pathogenic fungi, production of fungal cell wall-degrading enzymes and plant growth-promoting effects on strawberry seedlings . To test the plant growth-promoting effect, a microplate assay with strawberry seedlings was developed . Although the rhizobacterial strains were isolated from different plants they showed effects on the growth of strawberry seedlings . According to the in vitro biocontrol and plant growth-promoting activity, the three best candidates Pseudomonas putida B E2 (strawberry rhizosphere), Ps . chlororaphis K15 (potato rhizosphere) and Serratia plymuthica R12 (oilseed rape rhizosphere) were selected for greenhouse experiments to verify the in vitro screening results . Under greenhouse conditions the isolates selected according to this strategy were as effective, or more effective than commercial biocontrol agents and may therefore possibly be valuable as antagonists of V . dahliae . CONCLUSIONS: In this study, the screening strategy resulted in a selection of three interesting biocontrol candidates against Verticillium: Ps . putida B E2 (strawberry rhizosphere), Ps . chlororaphis K15 (potato rhizosphere) and Ser . plymuthica R12 (oilseed rape rhizosphere) . SIGNIFICANCE AND IMPACT OF THE STUDY: A new combination of in vitro screening methods including a microplate assay with strawberry seedlings to test the plant growth promoting effect which allow to more efficiently select potential biological control agents was developed successfully.

Mol Microbiol, 2002 Jan, 43(1), 39 - 49
The parAB gene products of Pseudomonas putida exhibit partition activity in both P . putida and Escherichia coli; Godfrin-Estevenon AM et al.; The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems . We have investigated the role in partition of the parAB locus of a free-living bacterium, Pseudomonas putida, not known to pass through developmental phases . A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponentially growing cultures but much higher frequencies in deceleration phase . This increase was growth medium dependent . Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild-type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies . Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium . The need for the Par proteins in chromosome partition thus appears to be subject to the cell's physiological state . Three sequences similar to cis-acting stabilization sites of Bacillus subtilis are present in the P . putida oriC-parAB region . One was inserted into an unstable mini-F and shown to stabilize it in E . coli in a ParAB-dependent manner.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(2), 149 - 61
Factor optimization for phenol removal using activated carbon immobilized with Pseudomonas putida; Annadurai G et al.; Removal efficiency of phenol from aqueous solutions was measured using a suspended culture of Pseudomonas putida (ATCC 3180) or the activated carbon on which the microorganisms were immobilized . Experiments were performed as a function of pH (7-9), temperature (30-36 degrees C), and concentrations of glucose (0.5-0.7 g/l) and ammonium sulfate (0.5-0.7 g/l) . The Box-Behnken design was applied in a second-degree quadratic, polynomial regression model to identify the significant effects and the interactions among the above four factors . Based on response curve method the conditions for maximizing phenol removal (initially 0.2 g/l) were recognized as pH 7, temperature 30 degrees C, glucose 0.6 g/l, and ammonium sulfate 0.6 g/l . The inhibition effect of carbon and nitrogen sources beyond a concentration of 0.6 g/l on phenol removal was obvious.

J Bacteriol, 2002 Mar, 184(5), 1444 - 8
Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida; Ditty JL et al.; Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P . putida to have a chemotactic response to 4-HBA . An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters.

Microbiology, 2002 Feb, 148(Pt 2), 537 - 48
Chromosome loss from par mutants of Pseudomonas putida depends on growth medium and phase of growth; Lewis RA et al.; The proteins encoded by chromosomal homologues of the parA and parB genes of many bacterial plasmids have been implicated in chromosome partitioning . Unlike their plasmid counterparts, mutant phenotypes produced by deleting these genes have so far been elusive or weakly expressed, except during sporulation . Here the properties of Pseudomonas putida strains with mutations in parA and parB are described . These mutants do not give rise to elevated levels of anucleate bacteria when grown in rich medium under standard conditions . However, in M9-minimal medium different parA and parB mutations gave between 5 and 10% anucleate cells during the transition from exponential phase to stationary phase . Comparison of the DNA content of bacteria at different stages of the growth curve, in batch culture in L-broth and in M9-minimal medium, suggests that the par genes are particularly important for chromosome partitioning when cell division reduces the chromosome copy number per cell from two to one . This transition occurs in P . putida during the entry into stationary phase in M9-minimal medium, but not in L-broth . It is proposed that the partition apparatus is important to ensure proper chromosome segregation primarily when the bacteria are undergoing cell division in the absence of ongoing DNA replication.

Arch Biochem Biophys, 2002 Feb 15, 398(2), 269 - 74
Continuous B-epitope maps of cytochrome P450cam (CYP101) obtained by peptide scanning: correlation to spatial structure; Moshkovskii SA et al.; Protein continuous B-epitopes can be revealed using short synthetic peptides that overlap a known protein sequence . Since the whole protein surface is considered to possess antigenic properties, a question that arises is whether a set of linear B-epitopes determined by peptide scanning correlates with a protein spatial structure . We have chosen cytochrome P450cam (CYP101) of Pseudomonas putida, with known 3D structure, as a template . Sera of two rabbits and antibody egg yolk preparations from three chickens were produced against the P450cam molecule . These polyclonals were analyzed separately in ELISA with 409 overlapping P450cam hexapeptides . The whole set of continuous antigenic sites of P450cam covered about 45% of the P450cam sequence . However, immunodominant sites (those revealed with more than 50% antibody preparations), the so-called "antigenic core," represent only 9% of the protein sequence . While the amount of water-accessible residues in the total antigenic map (42%) was close to that in the whole native P450cam molecule (39%), the amount of water-accessible residues in the antigenic core was significantly higher (64%) . These results led to the conclusion that antigenic core epitopes can be associated to the molecular surface, whereas epitopes with low detection frequency may partly correspond to unfolded regions of the protein molecule.

Biodegradation, 2001, 12(3), 189 - 99
Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate; Wang SJ et al.; A kinetic model to describe the degradation of phenol and cometabolic transformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) has been developed and validated experimentally . The integrated model accounts for cell growth, toxicity of 4-cp, cross-inhibitions among the three substrates, and the different roles of the specific growth substrate (phenol) and the conventional carbon source (SG) in the cometabolism of 4-cp . In this ternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate . Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)(-1)) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is not directly affected by the presence of SG . The competitive inhibition coefficient of 4-cp to phenol degradation (Ki,cp) and that of phenol to 4-cp transformation (Ki,ph) were determined to be 6.49 mg l(-1) and 0.193 mg l(-1), respectively, indicating that phenol imposes much larger competitive inhibition to 4-cp transformation than the converse . The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.

Biodegradation, 2001, 12(3), 179 - 88
Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1; Balashova NV et al.; 1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene . A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1 . The purified protein had a molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors . The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates . The comparison of the Km and Vmax values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate . A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate . The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria . It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate 1-monooxygenase . Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.

Appl Environ Microbiol, 2002 Feb, 68(2), 560 - 8
Characterization and application of xylene monooxygenase for multistep biocatalysis; Buhler B et al.; Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes . Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B . Buhler, A . Schmid, B . Hauer, and B . Witholt, J . Biol . Chem . 275:10085-10092, 2000) . Using E . coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation . We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step . These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type . Growth of E . coli was strongly affected by low concentrations of pseudocumene and its products . Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations . In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol . Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.

Syst Appl Microbiol, 2001 Nov, 24(3), 321 - 30
A siderophore peptide synthetase gene from plant-growth-promoting Pseudomonas putida WCS358; Devescovi G et al.; Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety . Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions . In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene . Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning . This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each . ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans . It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358 . PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P . aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P . aeruginosa . It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.

J Invertebr Pathol, 2001 Oct, 78(3), 135 - 40
Differential infectivity of two Pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera); Schneider M et al.; Pseudomonas aeruginosa and Pseudomonas putida show a profound differential infectivity after inoculation in Oncopeltus fasciatus . Whereas P . putida has no significant impact on nymphs, P . aeruginosa kills all experimental animals within 48 h . Both Pseudomonas species, however, induce the same four hemolymph peptides in O . fasciatus . Also injection of saline solution and injury induced these peptides . In general peptide induction was stronger in nymphs than in adult males . A significantly higher number of nymphs survived a challenge with P . aeruginosa when an immunization with P . putida preceded . The antibacterial properties of the hemolymph were demonstrated in inhibition experiments with P . putida . Two of the four inducible peptides (peptides 1 and 4) could be partially sequenced after Edman degradation and were compared with known antibacterial peptides . Peptide 1, of 15 kDa, showed 47.1% identity with the glycine-rich hemiptericin of Pyrrhocoris apterus . Peptide 4, of 2 kDa, had a 77.8% identity with the proline-rich pyrrhocoricin of P . apterus and a 76.9% identity with metalnikowin 1 of Palomena prasina . Peptides 2 and 3 are also small, with molecular weights of 8 and 5 kDa.

Arch Biochem Biophys, 2002 Feb 1, 398(1), 94 - 100
Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3; Kawanami T et al.; The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied . Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions . The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid . However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid . In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination . Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme . We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.

Biotechnol Bioeng, 2002 Mar 20, 77(6), 717 - 22
Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads; de Roo G et al.; A novel and efficient method for the production of enantiomericaly pure R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters was developed . The described method is based on hydrolysis of poly(hydroxyalkanoate) copolymers synthesized by Pseudomonas putida . The polymer was isolated via solvent recovery and hydrolyzed by acid methanolysis . The obtained 3-hydroxyalkanoic acid methylester mixture was distilled into several fractions with an overall yield of 96.6% (w/w) . Gas chromatography-mass spectrometry analysis of the fractions showed that 3-hydroxyhexanoic-, 3-hydroxyoctanoic-, 3 hydroxydecanoic-, and 3-hydroxydodecanoic acid methylesters were enriched to purities exceeding 96 mol%, with distillation yields of 99.9, 99.8, 88.4, and 56.8% (w/w), respectively . Subsequent saponification of the purified methylester fractions yielded the corresponding 3-hydroxyalkanoic acids, which were recovered up to 92.8% (w/w) . Chiral gas chromatography analysis confirmed that both 3-hydroxyoctanoic acid and 3-hydroxyoctanoic acid methylester are present in the R-form at a very high enantiomeric excess (>99.9%) .

Biosci Biotechnol Biochem, 2001 Nov, 65(11), 2472 - 81
Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes; Shindo K et al.; Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes . E . coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp . strain KP7 converted all of these tricyclic aromatic compounds, while E . coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P . pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates . Surprisingly, E . coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene . The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds . The ability of this actinomycete in their conversion was similar to that of E . coli carrying the corresponding genes . Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses . Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.

J Bacteriol, 2002 Feb, 184(3), 760 - 70
Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms; Sze CC et al.; Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds . The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters . The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator . These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors . In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media . Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation . Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case . The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host . The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties . The mechanistic implications of how each of the host factors exerts its action are discussed.

Appl Environ Microbiol, 2002 Jan, 68(1), 143 - 51
Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling; Pillai BV et al.; Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria . We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches . Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain . In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed . Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry . Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy . Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates . The first step in the pathway involves 3,3'-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate . This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 541 - 7
Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12; Heipieper HJ et al.; The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase . Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids . After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized . Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed . The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h . The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.

Fresenius J Anal Chem, 2001 Oct, 371(4), 467 - 73
Characterization of wastewater toxicity by means of a whole-cell bacterial biosensor, using Pseudomonas putida, in conjunction with chemical analysis; Farre M et al.; A new amperometric biosensor based on inhibition of Pseudomonas putida has been developed to assess the acute toxicity of wastewater . This system uses the biological component immobilized on disposable screen-printed electrodes . The responses for a selected group of polar organic standard substances were studied using Pseudomonas putida as biological component . The results have been compared with responses obtained using the same system and Escherichia coli as biological component and with the bioluminescence inhibition of Vibrio fisheri using ToxAlert 100 . Different properties, e.g . the standard deviation (SD) of the data, the goodness of fit (R2) and the standard deviation (Syx) of the vertical distances of the points from the inhibition curve, the 50% effective concentration (EC50) and the toxicity units (TII50) of the standard substance, were calculated and compared . This biosensor was used to assess the acute toxicity of real wastewater samples collected at different wastewater treatment plants (WWTP) . Finally, a sequential solid-phase extraction (SSPE) procedure followed by liquid chromatography-mass spectrometry (LC-MS) was used to determine the polar organic toxic substances present in the wastewater samples.

Antonie Van Leeuwenhoek, 2001 Oct, 80(2), 163 - 7
Pyrimidine base catabolism in Pseudomonas putida biotype B; West TP; Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B . The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase activities were detected in this pseudomonad . The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor . The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source . The reductive pathway enzyme activities in succinate-grown P . putida biotype B cells were induced when uracil served as the nitrogen source.

Biotechnol Bioeng, 2002 Feb 5, 77(3), 340 - 51
Toluene bioconversion to p-hydroxybenzoate by fed-batch cultures of recombinant Pseudomonas putida; Miller ES Jr et al.; A microbial oxidation process for the production of p-hydroxybenzoate (HBA) from toluene is reported . The oxidation reaction was studied in fed-batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively . An average volumetric HBA productivity of 13.4 mg HBA x L(-1) x h(-1) was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg x L(-1) after 9.8 h of fermentation . This corresponded to an average specific HBA productivity of 7.2 microg HBA (mg total protein)(-1) x h(-1) . In contrast, maximum HBA titers of 35 mg HBA x L(-1) were achieved in 27 h in comparative studies employing glutumate limited fed-batch cultures . A specific productivity of 4.1 microg HBA (mg total protein)(-1) x h(-1) and volumetric productivity of 1.3 mg HBA x L(-1) x h(-1) were calculated for the growth-rate restricted cultures . The differences in HBA production between the two cultures could be correlated to the levels of specific toluene-4-monooxygenase (T4MO) polypeptides . T4MO catalyzes the rate-limiting step in the pathway . Using experimental data, the half-life value of TmoA was calculated to be approximately 28 h . Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 x h(-1) was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co-expression of tmoF, the terminal oxidoreductase subunit of T4MO .

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 165 - 9
Transcription analysis of rpoH in Pseudomonas putida; Aramaki H et al.; We previously determined the complete DNA sequence of the rpoH gene encoding the heat-shock sigma factor (sigmaH) of Pseudomonas putida . In the present study, the transcriptional start sites of rpoH were determined to be 41 nucleotides (T1), 153 nucleotides (T2) and 157 nucleotides (T3) upstream from the translational start codon (AUG) of rpoH by rapid amplification of cDNA 5'-ends . Based on the locations of T2 and T3, a sigma70-type promoter (P2) was determined to be located in the open reading frame region of upstream ftsX in addition to the sigmaE-type promoter (P1; DNA Res . 6 (1999) 241) . In the in vitro transcription assay with reconstituted RNA polymerases (Esigma70, EsigmaE, EsigmaH and EsigmaS) of Pseudomonas aeruginosa, EsigmaE transcribed rpoH from T1 and Esigma(70) transcribed it from T2 and T3 . In both cases, the level of transcription was higher at 42 degrees C than at 30 degrees C . No transcript was detected when EsigmaH or EsigmaS was used . These results indicate that EsigmaE and Esigma70 recognize P1 promoter and P2 promoter, respectively, and also prove that the synthesis of rpoH mRNA is inducible upon heat shock.

Biomacromolecules, 2001 Summer, 2(2), 562 - 7
Microbial synthesis of poly(beta-hydroxyalkanoates) bearing phenyl groups from pseudomonas putida: chemical structure and characterization; Abraham GA et al.; New poly(beta-hydroxyalkanoates) having aromatics groups (so-called PHPhAs) from a microbial origin have been characterized . These polymers were produced and accumulated as reserve materials when a beta-oxidation mutant of Pseudomonas putida U, disrupted in the gene that encodes the 3-ketoacyl-CoA thiolase (fadA), was cultured in a chemically defined medium containing different aromatic fatty acids (6-phenylhexanoic acid, 7-phenylheptanoic acid, a mixture of them, or 8-phenyloctanoic acid) as carbon sources . The polymers were extracted from the bacteria, purified and characterized by using (13)C nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) . Structural studies revealed that when 6-phenylhexanoic acid was added to the cultures, an homopolymer (poly-3-hydroxy-6-phenylhexanoate) was accumulated . The feeding with 8-phenyloctanoic acid and 7-phenylheptanoic acid leads to the formation of copolymers of the corresponding units with the n - 2 carbons formed after deacetylation, copoly(3-hydroxy-8-phenyloctanoate-3-hydroxy-6-phenylhexanoate) and copoly(3-hydroxy-7-phenylheptanoate-3-hydroxy-5-phenylvalerate), respectively . The mixture of 6-phenylhexanoic acid and 7-phenylheptanoic acid gave rise to the corresponding terpolymer, copoly(3-hydroxy-7-phenylheptanoate-3-hydroxy-6-phenylhexanoate-3-hydroxy-5-phenylvalerate) . Studies on the chemical structure of these three polyesters revealed that they were true copolymers but not a mixture of homopolymers and that the different monomeric units were randomly incorporated in the macromolecular chains . Thermal behavior and molecular weight distribution were also discussed . These compounds had a dual attractive interest in function of (i) their broad use as biodegradable polymers and (ii) their possible biomedical applications.

Biomacromolecules, 2001 Spring, 2(1), 45 - 57
Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties; Gorenflo V et al.; A process for the large-scale production of 4-hydroxyvalerate (4HV)-containing biopolyesters with a new monomer composition was developed by means of high-cell-density cultivation applying recombinant strains of Pseudomonas putida and Ralstonia eutropha, harboring the PHA-biosynthesis genes phaC and phaE of Thiocapsa pfennigii . Cell densities of about 20 g/L revealing a PHA content of 52% (w/w) and a molar fraction of 4HV of up to 15.4 mol % were obtained by a two-stage fed-batch cultivation process at a 25-L scale using octanoic acid during the growth phase and levulinic acid for the accumulation of 4HV-containing polyesters . Besides 4HV the polyester contained significant amounts of both 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) and traces of 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO) . With glucose or gluconic acid as the growth substrate, the components of the polyester could be reduced to mainly 3HV and 4HV with only a negligible fraction of 3HB, resulting in a polyester with a new composition . Scale-up of the cultivation process to a 500-L scale was successfully performed, resulting in the production of these polyesters at a pilot plant scale . Short-term shifts in temperature and pH resulted in the formation of cell agglomerates of about 50-100 microm by which the effectiveness of the semicontinuous centrifugation process was drastically increased . Washing of the freeze-dried cells with boiling methanol significantly shortened the extraction process and resulted in a polyester of higher purity . The physical and mechanical properties of these copolyesters were characterized by means of size exclusion chromatography, dynamic mechanical analysis, differential scanning calorimetry, stress-strain measurements, and measurements of the viscosity of the solution . The copolyesters were cast into films, spun to fibers, or processed into test bars by melt spinning and injection molding, respectively . They revealed an almost entirely amorphous structure and consequently were sticky and lacked strength . However they showed high thermal stability and an unusually high elongation at break of about 200%; the molecular weights (M(w)) were between 2.0 x 10(5) and 3.3 x 10(5) g/mol . It was shown that 4HV-containing polyesters belong to the class of thermoplastic elastomeres.

Protein Eng, 2001 Oct, 14(10), 797 - 802
Engineering the CYP101 system for in vivo oxidation of unnatural substrates; Bell SG et al.; The protein engineering of CYP enzymes for structure-activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation . We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system . Wild-type CYP101 expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor without further oxidation to 5-oxo-camphor until >95% of camphor has been consumed . CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L mutant) have been engineered . In particular, the Y96F-V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min . Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h . This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.

Eur J Biochem, 2001 Dec, 268(23), 6011 - 9
Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida; Rother D et al.; Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) {Schwede, T.F., Retey, J., Schulz, G.E . (1999) Biochemistry, 38, 5355-5361} . To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83 . Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity . The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced . Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively . The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A . We propose that the carboxylate group of E414 plays an important role as a base in catalysis . To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator . All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group . The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also . For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.

Appl Environ Microbiol, 2001 Dec, 67(12), 5761 - 70
Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere; Steidle A et al.; Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing N-acyl-L-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species . In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere . For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of Pseudomonas putida IsoF and Serratia liquefaciens MG1, two strains that are capable of colonizing tomato roots . These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants . Furthermore, we integrated into the chromosome of AHL-negative P . putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp . This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules . The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.

Environ Microbiol, 2001 Oct, 3(10), 612 - 8
Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications; Olivera ER et al.; New bioplastics containing aromatic or mixtures of aliphatic and aromatic monomers have been obtained using genetically engineered strains of Pseudomonas putida . The mutation (-) or deletion (Delta) of some of the genes involved in the beta-oxidation pathway (fadA(-), fadB(-) Delta fadA or Delta fad BA mutants) elicits a strong intracellular accumulation of unusual homo- or co-polymers that dramatically alter the morphology of these bacteria, as more than 90% of the cytoplasm is occupied by these macromolecules . The introduction of a blockade in the beta-oxidation pathway, or in other related catabolic routes, has allowed the synthesis of polymers other than those accumulated in the wild type (with regard to both monomer size and relative percentage), the accumulation of certain intermediates that are rapidly catabolized in the wild type and the accumulation in the culture broths of end catabolites that, as in the case of phenylacetic acid, phenylbutyric acid, trans-cinnamic acid or their derivatives, have important medical or pharmaceutical applications (antitumoral, analgesic, radiopotentiators, chemopreventive or antihelmintic) . Furthermore, using one of these polyesters (poly 3-hydroxy-6-phenylhexanoate), we obtained polymeric microspheres that could be used as drug vehicles.

Int J Biol Macromol, 2001 Dec 10, 29(4-5), 243 - 50
Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid; Chung DM et al.; From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique . The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry . The solvent fractionation analysis showed that the PHA synthesized by P . putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P . citronellolis exhibited a rather little compositional separation into the two phases . According to the thermal analysis, the P . putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P . citronellolis PHA exhibited only one glass transition . It was found that the structural heterogeneity of the P . putida PHA was caused by a significant difference in the assimilation rate between PV and OA . The structural heterogeneity present in the P . putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells . The two different first-order degradation rate constants (k(1)), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation . In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h . The k(1) value of 0.083/h, almost the same as for the 3HO-unit in the P . putida PHA, was obtained for the P(3HO) accumulated in P . putida BM01 grown on OA as the only carbon source . In addition, the k(1) value of 0.015/h for the 3HPV-unit in the P . putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P . putida BM01 grown on PV plus butyric acid . On the contrary, the k(1) values for the P . citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P . citronellolis PHA . In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P . putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.

Nucleic Acids Res . 2001 Nov 15;29(22):E110.
General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440; Weinel C et al.; A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project . The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project . A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies . The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage . In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes . Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.

J Biol Chem, 2002 Jan 25, 277(4), 2830 - 4 Epub 2001 Nov 09.
Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida . Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges; Satoh A et al.; The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution . The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone . More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains . Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.

Biochemistry, 2001 Nov 13, 40(45), 13529 - 37
Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B; Nam GH et al.; Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit . The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme . The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively . The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution . Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices . Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type . A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding . Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.

J Biol Chem, 2002 Jan 18, 277(3), 2169 - 75 Epub 2001 Nov 01.
In vivo UV laser footprinting of the Pseudomonas putidasigma 54Pu promoter reveals that integration host factor couples transcriptional activity to growth phase; Valls M et al.; The occupation of the final sigma(54)-dependent Pu promoter of Pseudomonas putida by the integration host factor (IHF) under different growth conditions has been monitored in its native state and stoichiometry (i.e . monocopy) with UV laser footprinting technology . We present evidence that an abrupt change in intracellular IHF concentrations occurs when P . putida cells enter stationary phase . This change results in enhanced binding of the factor to the promoter and in the ensuing bending of the target DNA . Since Pu activity depends rigorously on DNA bending, promoter occupation is in turn translated into a much higher transcriptional output when cells leave exponential growth . Inspection of the residual activity of Pu in an IHF(-) strain reveals that IHF predominantly locks the capacity of the promoter to specific growth stages and also that additional physiological signals are entered in the system through final sigma(54)-RNA polymerase . The results substantiate the notion that final sigma(54) promoters process metabolic co-regulation signals through factor-induced changes in the architecture of the cognate DNA region . Further, they validate UV laser technology as a suitable tool to visualize nondisruptive alterations of DNA shape in vivo.

Curr Microbiol, 2001 Nov, 43(5), 365 - 70
Relationship between antifreeze protein and freezing resistance in Pseudomonas putida GR12-2; Kawahara H et al.; Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that have different freeze-resistant properties were selected . Five of the freeze-sensitive mutants, i.e . FSM-5, -6, -14, -29, and -41, secreted a lower amount of antifreeze protein-(AFP) into the culture broth compared with the wild-type . Among of these five mutants, the three mutants (FSM-6, FSM-14, and FSM-41) that have the lowest level of freezing resistance (4.0-6.0% survival) also produce AFP at low levels (0.5-0.9 microg/mL) compared with the wild-type (4.8 microg/ml) . The antifreeze and ice-nucleating activities of the AFP from these three mutant strains were similar to those of wild-type . Furthermore, the decreased freezing resistance from three mutants could be partially restored by adding purified AFP to mutant cell suspensions . Freezing resistance of three mutants was found to increase in proportion to the addition of AFP up to a concentration of 50 microg/mL . We conclude that accumulation of AFP is one component of the mechanism for freezing resistance in bacteria.

Curr Microbiol, 2001 Nov, 43(5), 316 - 21
Mutational analysis of zinc resistance in Pseudomonas putida strain S4; Choudhury R et al.; The genes for resistance to any essential metal ion are generally tightly regulated . In Pseudomonas putida strain S4, a multiple metal-resistant strain, mutational analysis gave strong evidence to the presence of the same for the expression of Zn resistance . Zn-sensitive mutants showed a lower MTC of Zn and expressed the Zn resistance genes with a lower efficacy . Non-complementation between these mutants suggests that they are possibly involved in the same function . Altered response to Zn of these mutants assisted in predicting the involvement of a repressor protein regulating the expression of Zn resistance genes . Zn hypersensitive mutant, on the other hand, appears to have an unregulated Zn uptake . This seems to provide the sensor component in the regulation . Zn resistance in strain S4 consists of three steps, viz., uptake, efflux, and binding, which are shared by a Zn homeostasis mechanism as well.

Can J Microbiol, 2001 Sep, 47(9), 855 - 60
Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone; Shibata H et al.; The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus . Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor . Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products . This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide . As a consequence of the ubiquinone deficiency, this activity was markedly decreased . In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain . These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone . To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells . When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E . coli in vivo.

Mol Genet Genomics, 2001 Oct, 266(2), 199 - 206
The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation; Petruschka L et al.; A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation . Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified . In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon . The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain . In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate . During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged . The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.

Appl Environ Microbiol, 2001 Nov, 67(11), 5219 - 24
Expression of a Pseudomonas putida aminotransferase involved in lysine catabolism is induced in the rhizosphere; Espinosa-Urgel M et al.; Using a transposon carrying a promoterless lux operon to generate transcriptional fusions by insertional mutagenesis, we have identified a Pseudomonas putida gene with increased expression in the presence of corn root exudates . Expression of the transcriptional fusion, induced by the amino acid lysine, was detected in P . putida in the rhizosphere of plants as well as in response to seed exudates . The mutant was unable to grow on lysine or delta-aminovalerate as carbon sources, which indicates that the affected function is involved in the pathway for lysine catabolism . However, the mutant strain grew with glutaric acid, the product of delta-aminovalerate metabolism via glutaric acid semialdehyde, as a C source . The translated sequence of the interrupted gene showed high levels of similarity with aminotransferases . These sets of data suggest that the product of this gene has delta-aminovalerate aminotransferase activity . This is the first direct genetic evidence correlating a DNA sequence with such activity in Pseudomonadaceae.

Mol Microbiol, 2001 Oct, 42(1), 47 - 59
A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors; Garmendia J et al.; To investigate the activation mechanism of the enhancer-binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled N-terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure . When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g . nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors . Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain . The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding . These results provide a rationale for evolving in vitro regulators a la carte that are responsive to predetermined, natural or xenobiotic chemical species.

J Bacteriol, 2001 Nov, 183(22), 6662 - 6
The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-xylene uptake; Kasai Y et al.; The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives . The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli . To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P . putida harboring the wild-type and xylN mutant TOL plasmids were characterized . The growth of P . putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P . putida harboring the xylN mutant TOL plasmid was not . The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical . We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane . Western blot analysis confirmed the localization of XylN in the outer membrane.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 664 - 9
Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas; Solaiman DK et al.; Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA) . These bacteria are incapable of metabolizing triacylglycerols (TAGs) . We have constructed recombinant P . putida and P . oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis . A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms . The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay . The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l . The recombinant P . putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate . While P . oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l . Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA . The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively . The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.

J Bacteriol, 2001 Nov, 183(21), 6215 - 24
Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53; Tsuda M et al.; It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H . Keil, S . Keil, R . W . Pickup, and P . A . Williams, J . Bacteriol . 164:887-895, 1985) . Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon . This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site . The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653 . Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons . The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.

J Bacteriol, 2001 Nov, 183(21), 6197 - 206
Role of the crc gene in catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway; Yuste L et al.; Expression of the alkane degradation pathway encoded in the OCT plasmid of Pseudomonas putida GPo1 is induced in the presence of alkanes by the AlkS regulator, and it is down-regulated by catabolic repression . The catabolic repression effect reduces the expression of the two AlkS-activated promoters of the pathway, named PalkB and PalkS2 . The P . putida Crc protein participates in catabolic repression of some metabolic pathways for sugars and nitrogenated compounds . Here, we show that Crc has an important role in the catabolic repression exerted on the P . putida GPo1 alkane degradation pathway when cells grow exponentially in a rich medium . Interestingly, Crc plays little or no role on the catabolic repression exerted by some organic acids in a defined medium, which shows that these two types of catabolic repression can be genetically distinguished . Disruption of the crc gene led to a six- to sevenfold increase in the levels of the mRNAs arising from the AlkS-activated PalkB and PalkS2 promoters in cells growing exponentially in rich medium . This was not due to an increase in the half-lives of these mRNAs . Since AlkS activates the expression of its own gene and seems to be present in limiting amounts, the higher mRNA levels observed in the absence of Crc could arise from an increase in either transcription initiation or in the translation efficiency of the alkS mRNA . Both alternatives would lead to increased AlkS levels and hence to elevated expression of PalkB and PalkS2 . High expression of alkS from a heterologous promoter eliminated catabolic repression . Our results indicate that catabolic repression in rich medium is directed to down-regulate the levels of the AlkS activator . Crc would thus modulate, directly or indirectly, the levels of AlkS.

J Appl Microbiol, 2001 Oct, 91(4), 625 - 35
Succession of microbial communities during a biostimulation process as evaluated by DGGE and clone library analyses; Ogino A et al.; AIMS: The objective of this study was to investigate the changes in the indigenous bacterial community structure for assessing the impact of biostimulation on spilled oil . METHODS AND RESULTS: Changes in the bacterial community structure were monitored by denaturing gradient gel electrophoresis (DGGE) and clone library methods based on 16S rRNA gene (rDNA) sequences . The results of DGGE, coupled with the use of the Shannon index and principal component analysis (PCA) and clone library analyses, were consistent . In the treated (fertilized) area, one operational taxonomic unit (OTU) became dominant during the fertilization period, and it was most closely related to Pseudomonas putida . CONCLUSIONS: The bacterial community structure in the treated area was markedly different from that in the control (non-fertilized) area during the fertilization period, but in the two areas it became similar at 14 weeks after the end of fertilization . SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the bacterial community structure was disrupted by the biostimulation treatment, but that it recovered immediately after the end of fertilization.

Appl Environ Microbiol, 2001 Oct, 67(10), 4842 - 9
Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects; Morasch B et al.; Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers . D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography . Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d(8) and nondeuterated toluene . The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve {b} = -1.219), Desulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and Geobacter metallireducens (b = -1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = -2.667) . The D/H isotope fractionation was 3 orders of magnitude greater than the (13)C/(12)C carbon isotope fractionation reported previously . Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D . cetonicum (b = -0.198), respectively . Carbon and hydrogen isotope fractionation during toluene degradation by D . cetonicum remained constant over a growth temperature range of 15 to 37 degrees C but varied slightly during degradation by P . putida mt-2, which showed maximum hydrogen isotope fractionation at 20 degrees C (b = -4.086) and minimum fractionation at 35 degrees C (b = -2.138) . D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study . Use of ring-labeled toluene-d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation . The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D . cetonicum with an initial activity of 3.63 mU (mg of protein)(-1) . The D/H isotope fractionation (b = -1.580) was 30% greater than that in growth experiments with D . cetonicum . Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released . Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation . Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities . Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.

J Biomol Struct Dyn, 2001 Aug, 19(1), 75 - 83
Modeling and analysis of the structure of the thermostable catechol 2,3-dioxygenase from Bacillus Stearothermophilus; Dai L et al.; The three-dimensional structure of thermostable catechol 2,3-dioxygenase(TC230) from Bacillus Stearothermophilus has been modeled basing on the known x-ray structure of catechol 2,3-dioxygenase(metapyrocatechase) from Pseudomonas putida mt-2, using computer graphics energy minimization techniques . The rationality of the resulting model was validated by Ramachandran plot and Profile-3D . The structure-functionally important residues, such as M++ binding residues and the substrate binding residues, were identified from the model . These residues are candidates for further site-directed mutagenesis experiments . The reason that the thermostability of TC230 is greater than metapyrocatechase(MPC) has been found, which may be due to the specific structure of the TC230 in the C-end mainly.

Bioresour Technol, 2001 Nov, 80(2), 137 - 42
Biodegradation of phenolic industrial wastewater in a fluidized bed bioreactor with immobilized cells of Pseudomonas putida; Gonzalez G et al.; The paper presents the main results obtained from the study of the biodegradation of phenolic industrial wastewaters by a pure culture of immobilized cells of Pseudomonas putida ATCC 17484 . The experiments were carried out in batch and continuous mode . The maximum degradation capacity and the influence of the adaptation of the microorganism to the substrate were studied in batch mode . Industrial wastewater with a phenol concentration of 1000 mg/l was degraded when the microorganism was adapted to the toxic chemical . The presence in the wastewater of compounds other than phenol was noted and it was found that Pseudomonas putida was able to degrade these compounds . In continuous mode, a fluidized-bed bioreactor was operated and the influence of the organic loading rate on the removal efficiency of phenol was studied . The bioreactor showed phenol degradation efficiencies higher than 90%, even for a phenol loading rate of 0.5 g phenol/ld (corresponding to 0.54 g TOC/ld).

Cancer Res, 2001 Sep 15, 61(18), 6805 - 10
Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate; Miki K et al.; In this study, we report a novel approach to gene-directed enzyme prodrug therapy for cancer . This gene therapy strategy exploits the toxic pro-oxidant property of methylselenol, which is released from selenomethionine (SeMET) by cancer cells with the adenoviral-delivered methionine alpha,gamma-lyase (MET) gene cloned from Pseudomonas putida . In MET-transduced tumor cells, the cytotoxicity of SeMET is increased up to 1000-fold compared with nontransduced cells . A strong bystander effect occurred because of methylselenol release from MET gene-transduced cells and uptake by surrounding tumor cells . Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and apoptosis . Adenoviral MET-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival . Recombinant adenovirus-encoding MET gene-SeMET treatment thereby offers a new paradigm for cancer gene therapy.

J Biol Chem, 2001 Nov 16, 276(46), 42923 - 31 Epub 2001 Sep 12.
The covalent structure of the small subunit from Pseudomonas putida amine dehydrogenase reveals the presence of three novel types of internal cross-linkages, all involving cysteine in a thioether bond; Vandenberghe I et al.; Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups . Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain . As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage . In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved . Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ . The thioether type bond in all four of these adducts has never been found in other proteins . CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.

Biochemistry, 2001 Sep 18, 40(37), 11007 - 12
Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters; Kakuta Y et al.; Escherichia coli ferredoxin (Fdx) is an adrenodoxin-type {2Fe-2S} ferredoxin . Recent genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins . Fdx probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters . Its crystal structure was determined by the multiple-wavelength anomalous dispersion method using the iron atoms in the {2Fe-2S} cluster of the protein and then refined to R and R(free) values of 0.255 and 0.278, respectively, at 1.7 A resolution . The structure of Fdx is similar to the structures of bovine adrenodoxin (Adx) and Pseudomonas putida putidaredoxin (Pdx) whose respective root-mean-square deviations of the corresponding Calpha atoms are 1.8 and 2.2 A . This analysis also revealed the structure of the C-terminal residues protruding into the solvent, which is missing in Adx and Pdx . The {2Fe-2S} cluster is located at the edge of the molecule and bonds with the Sgamma atoms of Cys42, Cys48, Cys51, and Cys87 . Electrostatic potential analysis showed that the surface of Fdx has two negatively charged areas separated by a hydrophobic lane . One is conserved on the surface of Adx which is an area of interaction with adrenodoxin reductase . Cys46 is located on the molecular surface in the vicinity of the {2Fe-2S} cluster, an indication that it may be involved in Fe-S cluster formation.

Int J Food Microbiol, 2001 Aug 15, 68(1-2), 1 - 9
Combined effect of antimicrobial coating and gamma irradiation on shelf life extension of pre-cooked shrimp (Penaeus spp.); Ouattara B et al.; The present study was conducted to evaluate the combined effect of low-dose gamma irradiation and antimicrobial coating on the shelf life of pre-cooked shrimp (Penaeus spp.) . Antimicrobial coatings were obtained by incorporating various concentrations of thyme oil and trans-cinnamaldehyde in coating formulations prepared from soy or whey protein isolates . Coated shrimps were stored at 4 +/- 1 degrees C under aerobic conditions and were periodically evaluated for aerobic plate counts (APCs) and Pseudomonas putida . Sensory evaluations were performed for appearance, odor, and taste using a hedonic test . Results showed that gamma irradiation and coating treatments had synergistic effects (p < or = 0.05) in reducing the APCs and P . putida with at least a 12-day extension of shelf life . Without irradiation, the inhibitory effects of the coating solutions were closely related to the concentration of thyme oil and trans-cinnamaldehyde . No detrimental effects of gamma irradiation on organoleptic parameters (appearance, odor, and taste) were observed . However, incorporation of thyme oil and trans-cinnamaldehyde reduced the acceptability scores for taste and odor.

J Bacteriol, 2001 Oct, 183(19), 5571 - 9
Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins; Fraile S et al.; We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp . The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes . An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR . One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid) . The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins . The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions . Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules . XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase . Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator . The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage . These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

Plasmid, 2001 Jul, 46(1), 16 - 24
New alkane-responsive expression vectors for Escherichia coli and pseudomonas; Smits TH et al.; We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB . The expression vectors were tested in several E . coli strains, P . putida GPo12 and P . fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE) . Induction factors ranged between 100 and 2700 for pKKPalk in E . coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E . coli . XylE expression levels of more than 10% of total cell protein were obtained for E . coli as well as for Pseudomonas strains .

Ecotoxicol Environ Saf, 2001 Sep, 50(1), 65 - 71
Suitability of the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida NCTC 10936 as an indicator of the acute toxicity of chemicals; Loffhagen N et al.; This study explored the suitability of using the trans/cis ratio of unsaturated fatty acids as an indicator of the acute toxicity of membrane active hazardous chemicals . The conversion of cis into trans fatty acids in Pseudomonas putida NCTC 10936 in response to 4-chlorophenol and temperature changes was compared with the results from another kind of toxicity test using the same organism, based on the sensitivity of its xylose oxidation-driven ATP synthesis to uncoupling . The response of both indicators is believed to be largely due to changes in the fluidity of the cytoplasmic membrane . However, the electron transport phosphorylation reacted faster and more sensitively to the fluidizing effect of 4-chlorophenol than the isomerization of unsaturated fatty acids . Therefore, measuring the trans/cis ratio does not provide as good early warning signals of acute toxicity as monitoring the response of the electron transport phosphorylation . If used as an indicator of chemostress, with Pseudomonas species as test organisms, the ratio should only be used in conjunction with other parameters reflecting the energetic state of the cells .

Appl Environ Microbiol, 2001 Sep, 67(9), 4338 - 41
Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate; Ramos-Gonzalez MI et al.; Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain . We show that it is also able to grow on high concentrations (up to 17 g/liter {123 mM}) of p-hydroxybenzoate (4HBA) . Tolerance to this aromatic carboxylic acid (up to 30 g/liter {217 mM}) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism . The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids . In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.

Appl Environ Microbiol, 2001 Sep, 67(9), 4272 - 8
cumA multicopper oxidase genes from diverse Mn(II)-oxidizing and non-Mn(II)-oxidizing Pseudomonas strains; Francis CA et al.; A multicopper oxidase gene, cumA, required for Mn(II) oxidation was recently identified in Pseudomonas putida strain GB-1 . In the present study, degenerate primers based on the putative copper-binding regions of the cumA gene product were used to PCR amplify cumA gene sequences from a variety of Pseudomonas strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains . The presence of highly conserved cumA gene sequences in several apparently non-Mn(II)-oxidizing Pseudomonas strains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms . Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters . Overall, our results indicate that both the cumA gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse Pseudomonas strains.

Appl Microbiol Biotechnol, 2001 Jun, 55(6), 721 - 6
Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads; Swift RJ et al.; Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene . As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene . In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture . The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them . However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source . Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.

Biochemistry, 2001 Sep 4, 40(35), 10592 - 600
Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction; Sevrioukova IF et al.; The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam . The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system . We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx . Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)) . The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin . The rate of FADH(*) decay was dependent on ionic strength and NAD(+) . In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed . The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively . This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase . Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.

Mol Genet Genomics, 2001 Aug, 265(6), 1004 - 10
The inverted repeats of IS1384, a newly described insertion sequence from Pseudomonas putida strain H, represent the specific target for integration of IS1383; Muller C et al.; Analysis of a region on plasmid pPGH1 from Pseudomonas putida strain H that is flanked by two copies of IS1383 has revealed an additional element with the typical features of a bacterial insertion sequence . This new IS element, designated IS1384, contains a single ORF of 972 bp, and is flanked by 9-bp inverted repeats . Based on sequence homology and structural characteristics of the putative transposase it encodes, IS1384 belongs to the IS5 subgroup of the IS5 family . Two copies of IS1384 are present on plasmid pPGH1, whereas none could be detected on the chromosome of P . putida strain H . Sequence analysis revealed the presence of two truncated copies of IS1384 on the second plasmid in this strain, pPGH2 . The inverted repeats of all IS1384 copies (including the truncated ones) are interrupted by the integration of an IS1383 element . All integrations were found to be site- and orientation-specific . PCR studies and sequence data indicate that IS1383 can form a circular intermediate on excision . In the circular form, the previously described 13-bp inverted repeats of IS1383 are separated by 10 bp that are identical to the 5-bp motif that flanks each side of the element when it is integrated in its target . We provide evidence that these additional nucleotides, although not of inverted symmetry, represent an essential part of the inverted repeats . Furthermore, the data indicate that IS1383 integrated into the inverted repeats of IS1384 by a site-specific recombination rather than a site-specific insertion event.

Syst Appl Microbiol, 2001 Jul, 24(2), 252 - 61
Isolation and characterization of a Pseudomonas putida strain able to grow with trimethyl-1,2-dihydroxy-propyl-ammonium as sole source of carbon, energy and nitrogen; Kaech A et al.; Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care . Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment . However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far . We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen . The strain reached a maximum specific growth rate of 0.4(h-1) when growing with TM as the sole source of carbon, energy and nitrogen . TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium . A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO2 resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures . Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not . The isolated bacterium was identified by 165-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P . putida DSM 291T . Despite their high identity, the reference strain P . putida DSM 291T was not able to grow with TM and the two strains differed even in shape when growing on the same medium . This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion . Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1617 - 26
Azurin involved in alcohol oxidation system in Pseudomonas putida HK5: expression analysis and gene cloning; Toyama H et al.; Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis . Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared . In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB . The azurin gene (azu) was cloned and sequenced . azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found . However, its relative position suggests different transcriptional regulation from that in azu of P . aeruginosa . The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.

J Bacteriol, 2001 Sep, 183(18), 5445 - 8
Involvement of sigma(S) in starvation-induced transposition of Pseudomonas putida transposon Tn4652; Ilves H et al.; Transpositional activity of mobile elements can be induced by different environmental stresses . Here, we present evidence that transposition of Tn4652 is elevated in stationary-phase Pseudomonas putida and suppressed in an isogenic sigma(S)-defective strain . We demonstrate that transcription from the Tn4652 transposase promoter is controlled by the stationary-phase-specific sigma factor sigma(S) . To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase . Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.

J Bacteriol, 2001 Sep, 183(18), 5285 - 92
Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E; Godoy P et al.; Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter . After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock) . Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates . The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA) . Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon . Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.

Biochemistry, 2001 Aug 21, 40(33), 9870 - 8
Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase; Sukumar N et al.; The structure of an active mutant of (S)-mandelate dehydrogenase (MDH-GOX2) from Pseudomonas putida has been determined at 2.15 A resolution . The membrane-associated flavoenzyme (S)-mandelate dehydrogenase (MDH) catalyzes the oxidation of (S)-mandelate to give a flavin hydroquinone intermediate which is subsequently reoxidized by an organic oxidant residing in the membrane . The enzyme was rendered soluble by replacing its 39-residue membrane-binding peptide segment with a corresponding 20-residue segment from its soluble homologue, glycolate oxidase (GOX) . Because of their amphipathic nature and peculiar solubilization properties, membrane proteins are notoriously difficult to crystallize, yet represent a large fraction of the proteins encoded by genomes currently being deciphered . Here we present the first report of such a structure in which an internal membrane-binding segment has been replaced, leading to successful crystallization of the fully active enzyme in the absence of detergents . This approach may have general application to other membrane-bound proteins . The overall fold of the molecule is that of a TIM barrel, and it forms a tight tetramer within the crystal lattice that has circular 4-fold symmetry . The structure of MDH-GOX2 reveals how this molecule can interact with a membrane, although it is limited by the absence of a membrane-binding segment . MDH-GOX2 and GOX adopt similar conformations, yet they retain features characteristic of membrane and globular proteins, respectively . MDH-GOX2 has a distinctly electropositive surface capable of interacting with the membrane, while the opposite surface is largely electronegative . GOX shows no such pattern . MDH appears to form a new class of monotopic integral membrane protein that interacts with the membrane through coplanar electrostatic binding surfaces and hydrophobic interactions, thus combining features of both the prostaglandin synthase/squaline-hopine cyclase and the C-2 coagulation factor domain classes of membrane proteins.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 101 - 7
Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor; Mars AE et al.; Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol . ammonium, and limonene . P . putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene . The p-cymene pathway of P putida GS1 is very similar, if not identical, to the cym pathway of P . putida F1 . The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P . putida Fl, also converted limonene to perillic acid . However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P . putida GS1.

Microbiology, 2001 Aug, 147(Pt 8), 2149 - 56
Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida; Tover A et al.; Expression of the phenol degradation pathway in Pseudomonas putida strain PaW85 requires coordinated transcription of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and phenol monooxygenase, respectively, and the chromosomally encoded catechol degradation catBCA operon . Transcriptional activation from the pheBA and catBCA promoters is regulated by CatR and the catechol degradation pathway intermediate cis,cis-muconate . Here it is shown that physiological control mechanisms are superimposed on this regulatory system . Transcriptional activation from the pheBA and catBCA promoters is growth-phase-regulated in P . putida cells grown on rich medium (LB medium) . CatR-mediated transcription from these promoters is silenced on rich medium until the transition from exponential to stationary phase . A slight positive effect (threefold) of stationary-phase-specific sigma factor sigma(S) on transcription from the pheBA promoter was observed . Expression of the catBCA promoter was not influenced by the activity of this sigma factor . In contrast to rich growth medium, transcription from the pheBA and catBCA promoters in minimal medium containing a mixture of glucose and sodium benzoate was rapidly induced in exponential culture . It was shown that the presence of amino acids in the culture medium causes exponential silencing of the pheBA and catBCA promoters . The possibility that a hypothetical repressor protein could be involved in physiological control of transcription from the pheBA and catBCA promoters is discussed.

J Bacteriol, 2001 Sep, 183(17), 5128 - 33
Role of ptsO in carbon-mediated inhibition of the Pu promoter belonging to the pWW0 Pseudomonas putida plasmid; Cases I et al.; An investigation was made into the role of the ptsO gene in carbon source inhibition of the Pu promoter belonging to the Pseudomonas putida upper TOL (toluene degradation) operon . ptsO is coexpressed with ptsN, the loss of which is known to render Pu unresponsive to glucose . Both ptsN and ptsO, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA(Ntr) and NPr, respectively, have been mapped adjacent to the rpoN gene of P . putida . The roles of these two genes in the responses of Pu to glucose were monitored by lacZ reporter technology with a P . putida strain engineered with all regulatory elements in monocopy gene dosage . In cells lacking ptsO, Pu activity seemed to be inhibited even in the absence of glucose . A functional relationship with ptsN was revealed by the phenotype of a double ptsN ptsO mutant that was equivalent to the phenotype of a mutant with a single ptsN disruption . Moreover, phosphorylation of the product of ptsO seemed to be required for C inhibition of Pu, since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype . A genomic search for proteins able to phosphorylate ptsO revealed the presence of two open reading frames, designated ptsP and mtp, with the potential to encode PTS type I enzymes in P . putida . However, neither an insertion in ptsP nor an insertion in mtp resulted in a detectable change in inhibition of Pu by glucose . These results indicate that some PTS proteins have regulatory functions in P . putida that are independent of their recognized role in sugar transport in other bacteria.

J Bacteriol, 2001 Sep, 183(17), 5074 - 81
Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process; Park HS et al.; The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol . The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes . The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain . It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form . A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster . It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon . The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.

J Environ Biol, 2001 Jan, 22(1), 29 - 36
Impacts of crude oil on the germination and growth of cress seeds (Lepidium sp.) after bioremediation of agricultural soil polluted with crude petroleum using "adapted" Pseudomonas putida; Nwachukwu SC et al.; The impacts of crude oil on the germination, growth and morphology of cress seeds (Lepidium sp.) after bioremediation of agricultural soil polluted with crude petroleum using "adapted" Pseudomonas putida (PP) were examined for 15 days . At day 15 there was 100% germination in the untreated control samples, the mean height of the seedlings was 75.8 +/- 2.6 mm and all appeared to have grown morphologically normal . In the experimental samples treated with oil and PP inoculation, there was 98% germination and the seedlings reached a height of 63.8 +/- 6.9 mm; again, morphologically the seedlings appeared normal . However, in the control samples treated with oil but without PP inoculation, there was 31-38% germination and seedling heights of 34.2 +/- 11.4-42.3 +/- 8.5 mm with abnormal morphology . Treatment of oil-impacted agricultural soil with PP as a bioremediation agent does produce soil which is capable of growing larger and healthier plants than where bioremediation has not taken place.

Appl Environ Microbiol, 2001 Aug, 67(8), 3406 - 12
Effects of iron limitation on the degradation of toluene by Pseudomonas strains carrying the tol (pWWO) plasmid; Dinkla IJ et al.; Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases . In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants . We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P . putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation . The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions . In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased . Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains . Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.

Appl Environ Microbiol, 2001 Aug, 67(8), 3333 - 9
Biotransformation of various substituted aromatic compounds to chiral dihydrodihydroxy derivatives; Raschke H et al.; The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5alpha(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from Pseudomonas sp . strain P51, was examined . CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding cis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring . No higher substituted biphenyls were oxidized . The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined . All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus . With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases . Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene . All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P . putida F39/D . The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol . A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.

Arch Environ Contam Toxicol, 2001 Aug, 41(2), 117 - 22
Toluene mineralization and growth potential of Pseudomonas putida PaW164 under toluene-limiting conditions; Lim TG et al.; Toluene-induced cells of Pseudomonas putida PaW164 (pWWO-164) were monitored for growth potential, maintaining the TOL plasmid, and potential toluene mineralization activity in toluene-amended and nonamended soil . A follow-up study was done in a carbon-free mineral salts solution to obtain further information on physiological changes that occur during starvation . These studies showed that there was a larger decline in colony forming units (CFUs) recovered on a toluate- or benzoate-defined mineral salts medium than on a complex agar medium, a greater percent decrease of CFU than of potential mineralization activity, no decrease in direct counts, and no loss of the TOL plasmid during starvation . Toluene-induced cells also showed an increasing lag time and a decreasing potential for mineralization of (14)C-toluene with starvation . In contrast, the lag time for mineralization of glucose was longest at the onset of starvation and reached a minimum by 3 days; thereafter, the potential for glucose mineralization remained high.

Metab Eng, 2001 Jul, 3(3), 218 - 25
Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene; Zhu MM et al.; Anaerobic glycerol fermentation by Escherichia coli strains expressing genes from the Klebsiella pneumoniae dha regulon showed that cell growth and 1,3-propanediol (1,3-PD) production are significantly inhibited when 5 g/L or higher of glycerol is initially present . One reason for this inhibition may be methylglyoxal (MG) accumulation . Assays of both intracellular and extracellular MG levels indicated an accumulation of MG in anaerobic glycerol fermentation of transgenic E . coli . Pseudomonas putida glyoxalase I was expressed in the transgenic E . coli to enhance MG detoxification . The activity of glyoxalase I in the transgenic E . coli with the P . putida glyoxalase I under anaerobic conditions was 12-fold higher than that in the control cells . Compared to the control cells, the transgenic cells with the P . putida glyoxalase I displayed a reduction of 35-43% in intracellular MG and a decrease of 30% in extracellular MG . These decreases were statistically significant (P>94) . Furthermore, the expression of the P . putida glyoxalase I in the transgenic E . coli markedly improved cell growth and resulted in a 50% increase in 1,3-PD production .

Environ Sci Technol, 2001 Jul 1, 35(13), 2734 - 40
Biodegradation of non-desorbable naphthalene in soils; Park JH et al.; The degradation of naphthalene was studied in soil-slurry systems, and a quantitative model was developed to evaluate the bioavailability of sorbed-phase contaminant . Four soils with different organic matter contents were used as sorbents . Two naphthalene-degrading organisms, Pseudomonas putida G7 and NCIB 9816-4, were also selected . Sorption isotherms and single and series dilution desorption studies were conducted to evaluate distribution coefficients, desorption parameters, and the amount of non-desorbable naphthalene . Biodegradation kinetics were measured in soil extract solutions and rate parameters estimated . Bioavailability assays involved establishing sorption equilibrium, inoculating the systems with organisms, and measuring naphthalene concentrations in both sorbed and dissolved phases over time . For all four soils, the sorption isotherms were linear, and desorption could be described by a model involving three types of sites: equilibrium, nonequilibrium, and non-desorption . Enhanced bioavailability, as evidenced by faster than expected degradation rates based on liquid-phase concentrations, were observed in soils with the higher sorption distribution coefficients . These observations could be described using model formulations that included solid-phase degradation . In all soils studied, degradation of non-desorbable naphthalene was observed.

J Bacteriol, 2001 Aug, 183(15), 4551 - 61
Mechanism of chloride elimination from 3-chloro- and 2,4-dichloro-cis,cis-muconate: new insight obtained from analysis of muconate cycloisomerase variant CatB-K169A; Kaulmann U et al.; Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate to cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin . Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product . The formation of cis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate . To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine . As expected, substrates requiring protonation, such as cis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant . However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate . Interestingly, cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively . Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.

FEMS Microbiol Lett, 2001 May 1, 198(2), 117 - 22
Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes; Kieboom J et al.; Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMod-KmO mutagenesis system . These mutants were unable to grow in the presence of octanol and toluene . By cloning the region flanking the transposon insertion point a partial sequence of the interrupted genes was determined . Comparison of the deduced amino acid sequences with a protein database revealed the following interrupted putative gene products: organic solvent efflux proteins SrpA and SrpB, the flagellar structural proteins FlgK, FlaG, FliI, FliC, and FliH, the transcriptional activator FleQ, the alternative RNA polymerase sigma factor RpoN, and the flagellum-specific RNA polymerase sigma factor FliA (RpoF) . The transposon mutants, except for the organic solvent efflux mutants, were nonmotile as determined by a swarm assay and the formation of the flagellum was totally impaired . Expression studies with a srp promoter probe showed a decreased expression of the SrpABC efflux pump in the nonmotile mutants.

Int J Food Microbiol, 2001 Jun 15, 66(3), 163 - 73
Microstructural factors controlling the survival of food-borne pathogens in porous media; Hills BP et al.; The survival of Salmonella typhimurium LT2, Escherichia coli K-12 and Pseudomonas putida in several model porous media poised at a water activity of 0.94 is shown to depend critically on the microstructure of the particulate matrix and the microscopic water distribution . The porous media were made by randomly dispersing a liquid inoculum containing ca . 10(7) cells/ml throughout the pores and interparticle spaces of packed beds of silica particles and Sephadex microspheres . The purely "microstructural stress" effects were isolated by comparison with a homogeneous liquid growth medium having the same water activity . The possibility of exploiting similar microstructural stress effects in food preservation is discussed.

Biotechnol Bioeng, 2001 Sep 5, 74(5), 355 - 63
Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation; Hermann R et al.; The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen . To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed . The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application . The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction . The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period . The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time . The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode . The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks . The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors .

Appl Environ Microbiol, 2001 Jul, 67(7), 3102 - 9
Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli; Rehm BH et al.; Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P . aeruginosa with respect to biosynthesis of PHAs and rhamnolipids . All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P . aeruginosa showed decreased PHA accumulation and rhamnolipid production . In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased . Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P . aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis . These data suggested that both biosynthesis pathways are competitive . In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P . putida strongly impaired in PHA production from gluconate . All mutants were complemented by the phaG gene from P . putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium . The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E . coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P . putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan . The accumulated PHA contributed to 2 to 3% of cellular dry weight.

Appl Environ Microbiol, 2001 Jul, 67(7), 3010 - 5
Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria; Xu Y et al.; Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml . Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed . Cry11A was detected primarily in insoluble form in recombinant cells . Cry11A was not detected in P . putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P . putida recombinants producing both proteins were toxic at concentrations similar to those for E . coli . A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein . The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact . Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 391 - 399
Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6; Stolz A; Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate . Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816 . In contrast to the naphthalene-degrading pseudomonads, S . xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts . Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads . According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2'-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads . The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40-60% sequence identity to the isofunctional enzymes from the pseudomonads . In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied . S . xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes . Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models.

J Bacteriol, 2001 Jul, 183(14), 4127 - 33
Mutations in genes involved in the flagellar export apparatus of the solvent-tolerant Pseudomonas putida DOT-T1E strain impair motility and lead to hypersensitivity to toluene shocks; Segura A et al.; Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium . Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol) . The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain . The mutation in this strain therefore seemed to specifically affect toluene tolerance . Cloning and sequencing of the mutation revealed that the mini-Tn5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components . FliP is involved in the export of flagellar proteins, and in fact, the P . putida fliP mutant was nonmotile . The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance . An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene . In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain . The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P . putida DOT-T1E to the periplasm.

Appl Microbiol Biotechnol, 2001 May, 55(5), 627 - 31
Efficiency of naphthalene and salicylate degradation by a recombinant Pseudomonas putida mutant strain defective in glucose metabolism; Samanta SK et al.; Metabolically engineered microorganisms may have tremendous potential in removing toxic compounds from nature . In general, microorganisms prefer to utilize simpler carbon sources over toxic compounds when both are present in an environment and, therefore, the presence of simpler carbon sources may greatly reduce the efficiency of a microorganism towards toxic compounds . If a microorganism is prevented from utilizing simpler carbon sources, thereby making it totally dependent upon the toxic compounds, it should increase the specificity for and efficiency of degradation of the toxic compounds in the presence of other, simpler carbon sources . To test this hypothesis, the efficiency of naphthalene and salicylate degradation in the presence of glucose by a recombinant Pseudomonas putida strain mutated in glucose metabolism was determined and compared to the non-mutated strain . Results obtained indicate that the impairment of glucose metabolism leads to better degradation of naphthalene and salicylate in the presence of glucose.

Appl Microbiol Biotechnol, 2001 May, 55(5), 619 - 26
Biodegradation of synthetic and naturally occuring mixtures of mono-cyclic aromatic compounds present in olive mill wastewaters by two aerobic bacteria; Di Gioia D et al.; Two bacterial strains, Ralstonia sp . LD35 and Pseudomonas putida DSM 1868, were assayed for their ability to degrade the monocyclic aromatic compounds commonly found in olive mill wastewaters (OMWs) . The goal was to study the possibility of employing the two strains in the removal of these recalcitrant and toxic compounds from the effluents of anaerobic treatment plants fed with OMWs . At first, the two strains were separately assayed for their ability to degrade a synthetic mixture of nine aromatic acids present in OMWs, both in growing- and resting-cell conditions . Then, due to the complementary activity exhibited by the two strains, a co-culture of the two bacteria was tested under growing-cell conditions for degradation of the same synthetic mixture . Finally, the degradation activity of the co-culture on two fractions was studied . Both fractions one deriving from natural OMWs through reverse osmosis treatment and containing low-molecular weight organic molecules, and the other obtained from an anaerobic lab-scale treatment plant fed with OMWs, were rich in monocyclic aromatic compounds . The co-culture of the two strains was able to biodegrade seven of the nine components of the tested synthetic mix (2, 6-dihydroxybenzoic acid and 3, 4, 5-trimethoxybenzoic acid were the two undegraded compounds) . In addition, an efficient biodegrading activity towards several aromatic molecules present in the two natural fractions was demonstrated.

Appl Microbiol Biotechnol, 2001 May, 55(5), 571 - 7
High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system; Husken LE et al.; The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general . For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained . Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system . A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR . Introducing this construct into wild-type P . putida F1, which degrades toluene via 3-methylcatechol, or into mutant P . putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette . As a result, up to 14 mM (1.74 g l(-1)) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 micromol min(-1) g(-1) cell dry weight, which is four times higher than other catechol production systems . It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process . This is an important step for obtaining designer biocatalysts.

Appl Microbiol Biotechnol, 2001 May, 55(5), 541 - 6
Biotransformation of limonene by Pseudomonas putida; Chatterjee T et al.; From a study of three fungal and 15 bacterial strains, it was observed that Pseudomonas putida MTCC 1072 oxidized limonene with the highest efficiency of . Fermentation of limonene by P . putida MTCC 1072 was conducted for 120 h at 30 degrees C at a fixed pH of 5.0 . Major bioconversion products were isolated and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, and by elemental analysis . The bioconversion products were identified as perillyl alcohol and p-menth-1-ene-6,8-diol, and under optimum conditions the yields were 36% and 44%, respectively (a rate kinetic model indicated corresponding limiting yields of 44% and 56%) . No further degradation of the products was observed using this bacteria.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001, 36(4), 509 - 20
Feasibility of fluidized-bed bioreactor for remediating waste gas containing H2S or NH3; Chung YC et al.; Pseudomonas putida for H2S and Arthrobacter oxydans for NH3 were immobilized with Ca-alginate and packed inside glass columns to form fluidized-bed bioreactors . The feasibility of the lab-scale bioreactor for the treatment of H2S or NH3 was examined . Phosphate salt, being added to the nutrient solution as buffer solution, may chelate with Ca2+ in the Ca-alginate beads, resulting in the disintegration of gel structure . When the buffer capacity of the phosphate solution was over the critical point of 33.5 mM/pH, all calcium ions in the bead were released and beads were broken . Increasing liquid flowrate and inlet gas concentration favored to H2S and NH3 removal . Carbon source addition was essential and facilitated malodorous removal for this system . Removal capacity increased with inlet concentration . However, increasing pattern was dependent of H2S or NH3 . The result clearly indicated that bioreactor was suitable to be applied for the industry of livestock farm for removing wastegas containing H2S or NH3.

EMBO J, 2001 Jun 15, 20(12), 3262 - 71
A broad host range replicon with different requirements for replication initiation in three bacterial species; Caspi R et al.; Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria . The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication . To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors . The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV . Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein . Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation . Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein . Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

J Bacteriol, 2001 Jul, 183(13), 3967 - 73
Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E; Rojas A et al.; In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance . Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons . A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase . In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI . The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter . In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter . By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions . The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out . Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene . The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

Microbiology, 2001 Jun, 147(Pt 6), 1621 - 30
Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes; van Beilen JB et al.; The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA . This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes . In P . putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon . Other insertion sequences flank and interrupt the alk genes in both strains . Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved . Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS:

Microbiology, 2001 Jun, 147(Pt 6), 1611 - 20
Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358; Bertani I et al.; The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied . Protocatechuic acid (PCA) is then degraded via the beta-ketoadipate pathway to form tricarboxylic acid intermediates . In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB . In this study the identification and characterization of the pobC-pobA locus of P . putida WCS358 is presented . The p-hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated POBC: Using the pobA promoter transcriptionally fused to a promoterless lacZ gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA . This PobC-PCA response was genetically demonstrated by making use of pobC::Tn5 and pcaH::Tn5 mutants of strain WCS358 constructed in this study . In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed . Finally, the activation of pobA by PHB varied according to the concentration and it was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced . PcaR is a regulator involved in the regulation of several loci of the beta-ketoadipate pathway, one of which is pcaK . It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.

J Biol Chem, 2001 Aug 10, 276(32), 29833 - 8 Epub 2001 Jun 04.
Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene; Maeda T et al.; Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FD(BphA3)), and a ferredoxin reductase (FDR(BphA4)) . A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K . (1994) J . Bacteriol . 176, 2121-2123) . In this study, ISP, FD, and FDR were purified and characterized . Reconstitution of the dioxygenase components consisting of purified ISP(TodC1BphA2), FD(BphA3), and FDR(BphA4) exhibited oxygenation activities toward biphenyl, toluene, and TCE . Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP(TodC1BphA2) and ISP(BphA1A2) were present as heterohexamers, whereas ISP(TodC1C2) was present as a heterotetramer . The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox . The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox . These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

Biosens Bioelectron, 2001 Jun, 16(4-5), 305 - 12
Monitoring of bacteria growth using a wireless, remote query resonant-circuit sensor: application to environmental sensing; Ong KG et al.; A new technique is presented for in-vivo remote query measurement of the complex permittivity spectra of a biological culture solution . A sensor comprised of a printed inductor-capacitor resonant-circuit is placed within the culture solution of interest, with the impedance spectrum of the sensor measured using a remotely located loop antenna; the complex permittivity spectra of the culture is calculated from the measured impedance spectrum . The remote query nature of the sensor platform enables, for example, the in-vivo real-time monitoring of bacteria or yeast growth from within sealed opaque containers . The wireless monitoring technique does not require a specific alignment between sensor and antenna . Results are presented for studies conducted on laboratory strains of Bacillus subtilis, Escherichia coli JM109, Pseudomonas putida and Saccharomyces cerevisiae.

Biochemistry, 2001 Jun 12, 40(23), 6828 - 35
Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B; Choi G et al.; Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate . Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta in the active site of P . putida KSI . Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca . 26-51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network . To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively . Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations . The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F . In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4beta-proton with a solvent deuterium based on the reaction rate in D2O . Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F.

J Biotechnol, 2001 Jun 1, 88(1), 11 - 9
Integrated bioproduction and extraction of 3-methylcatechol; Husken LE et al.; Pseudomonas putida MC2 is a solvent-tolerant strain that accumulates 3-methylcatechol . In aqueous media, 10 mM of 3-methylcatechol was produced and production was limited by 3-methylcatechol toxicity to the biocatalyst . Production levels increased by introduction of a second, organic phase that provides the substrate toluene and extracts the product from the culture medium . Octanol was shown to be an appropriate second phase with respect to tolerance of the strain for this solvent and with respect to partitioning of both substrate and product . Per unit of overall reactor volume (octanol and water), best results were obtained with 50% (v/v) of octanol: an overall 3-methylcatechol concentration of 25 mM was reached with 96% of the product present in the octanol phase . These product concentrations are much higher than in aqueous media without organic solvent, indicating that biocatalysis in an organic/aqueous two-phase system is an improved set-up for high production levels of 3-methylcatechol.

Appl Environ Microbiol, 2001 Jun, 67(6), 2649 - 56
Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation; Ronchel MC et al.; Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population . These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals . An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit . The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef . In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced . The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted . We reinforced the ABC system by including it in a Deltaasd P . putida background . A P . putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine . We constructed a P . putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome . This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction . In the P . putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8 . The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation) . The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain . However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P . putida CMC4 was still detectable in soils after 100 days.

Appl Environ Microbiol, 2001 Jun, 67(6), 2622 - 6
Complete denitration of nitroglycerin by bacteria isolated from a washwater soakaway; Marshall SJ et al.; Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate {GTN}) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant . The isolates were identified by 16S rRNA gene sequence homology as Pseudomonas putida, an Arthrobacter species, a Klebsiella species, and a Rhodococcus species . Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of the Arthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment . The Klebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity . All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate . Most significant was the finding that the Rhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN . Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source . Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.

Z Naturforsch {C}, 2001 Mar-Apr, 56(3-4), 303 - 7
An isopyoverdin from Pseudomonas putida CFML 90-44; Sultana R et al.; From Pseudomonas putida CFML 90-44 an isopyoverdin was isolated . Its structure could be elucidated by chemical degradation and spectroscopic data.

Biotechniques, 2001 May, 30(5), 988 - 90, 992, 994-6
Screening for ribosomal-based false positives following prokaryotic mRNA differential display; Nagel AC et al.; Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems . However, one of the current drawbacks of both techniques is the high number of false positives generated . In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs . To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.

Extremophiles, 2001 Apr, 5(2), 93 - 9
A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks; Junker F et al.; Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier . To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS . The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis . The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons . It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Protein Sci, 2001 Mar, 10(3), 482 - 90
Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1; Scognamiglio R et al.; A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase . The putative regulatory D subunit was expressed in Escherichia coli and purified . Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence . The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes . The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600 . The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed . The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred . Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2 . Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data . Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Biochim Biophys Acta, 2001 Feb 9, 1545(1-2), 53 - 66
Cloning and biochemical characterization of Co(2+)-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain; Itoh N et al.; The gene encoding Co(2+)-activated bromoperoxidase (BPO)-esterase (EST), catalyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br(-) and quite specific hydrolysis of (R)-acetylthioisobutyric acid methyl ester, was cloned from the chromosomal DNA of the Pseudomonas putida IF-3 strain . The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da . The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step column chromatographies . The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br(-) for the brominating reaction and was activated by Co(2+) ions . It catalyzed the bromination of styrene and indene to give the corresponding racemic bromohydrin . Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzymatic reactions via a peracetic acid intermediate . The results indicated that the enzyme was a novel Co(2+)-activated organic acid-dependent BPO (perhydrolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family.

Biochim Biophys Acta, 2001 Jan 12, 1544(1-2), 151 - 65
Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization; Parschat K et al.; Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity . It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor . In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1) . Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+ . XDH from P . putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs . After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K . Resonances from FeSI and FeSII were detected at 15 K . Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry . The EPR features of the redox-active centers of P . putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes . The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.

Biochemistry, 2001 May 15, 40(19), 5602 - 14
Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin; Pochapsky TC et al.; Putidaredoxin (Pdx), a Cys4Fe2S2 ferredoxin from Pseudomonas putida, exhibits redox-dependent binding to its physiological redox partner, cytochrome P450(cam) (CYP101), with the reduced form of Pdx (Pdx(r)) binding with greater affinity to oxidized camphor-bound CYP101 than the oxidized form, Pdx(o) . It has been previously shown that Pdx(o) is more dynamic than Pdx(r) on all accessible time scales, and it has been proposed that Pdx(r) samples only a fraction of the conformational substates populated by Pdx(o) on a time average . It is postulated that the ensemble subset populated by Pdx(r) is the same subset that binds CYP101, providing a mechanism for coupling the Pdx oxidation state to binding affinity for CYP101 . Evidence from a variety of sources, including redox-dependent shifts of 15N and 13C resonances, indicates that the metal cluster binding loop of Pdx is the primary determinant of redox-dependent conformational selection . Patterns of paramagnetic effects suggest that the metal cluster binding loop contracts around the metal cluster upon reduction, possibly due to the strengthening of hydrogen bonds between the sulfur atoms of the metal cluster and the surrounding polypeptide NH and OH groups . Effects of this perturbation are then transmitted mechanically to other affected regions of the protein . A specific mutation has been introduced into the metal binding loop of Pdx, G40N, that slows conformational exchange sufficiently that the ensemble of conformational substates in Pdx(o) are directly observable as severe broadenings or splittings in affected NMR resonances . Many of the residues most affected by the mutation also show significant exchange contributions to 15N T(2) relaxation in wild-type Pdx(o) . As predicted, G40N Pdx(r) shows a collapse of many of these multiplets and broadened lines to form much sharper resonances that are essentially identical to those observed in wild-type Pdx(r), indicating that Pdx(r) occupies fewer conformational substates than does Pdx(o) . This is the first direct observation of such redox-dependent ensembles at slow exchange on the chemical shift time scale . These results confirm that conformational selection within the Fe2S2 cluster binding loop is the primary source of redox-dependent changes in protein dynamics in Pdx.

Appl Microbiol Biotechnol, 2001 Apr, 55(3), 321 - 5
Biotransformations catalyzed by cloned p-cymene monooxygenase from Pseudomonas putida F1; Nishio T et al.; p-Cymene monooxygenase (CMO) from Pseudomonas putida F1 consists of a hydroxylase (CymA1) and a reductase component (CymA2) which initiate pcymene (p-isopropyltoluene) catabolism by oxidation of the methyl group to p-isopropylbenzyl alcohol (p-cumic alcohol) . To study the possible diverse range of substrates catalyzed by CMO, the cymA1A2 genes were cloned in an Escherichia coli pT7-5 expression system and the cells were used in transformation experiments . The tested substrates include different substituents on the aromatic ring at the 2 (ortho), 3 (meta) or 4 (para) position relative to the methyl moiety . As a result, a distinct preference was observed for substrates containing at least an alkyl or heteroatom substituent at the para-position of toluene . The conversion rate of 4-chlorotoluene or 4-methylthiotoluene to the corresponding benzyl alcohol was found to be as good as the canonical substrate, p-cymene . But 3-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene were relatively poor substrates . CMO is also capable of producing styrene oxide from styrene . However, the oxidation of 4-chlorostyrene to 4-chlorostyrene oxide was by far the fastest among the substrates used in this study . The various biotransformation products were identified by a combined solid phase microextraction/gas chromatographic-mass spectrometric analytical technique.

Water Res, 2001 Jun, 35(8), 1921 - 32
Simultaneous chromium(VI) reduction and phenol degradation in a fixed-film coculture bioreactor: reactor performance; Nkhalambayausi-Chirwa EM et al.; Simultaneous Cr(VI) reduction and phenol degradation was observed in a fixed-film bioreactor consisting of a coculture of phenol-degrading bacteria, Pseudomonas putida DMP-1, and Cr(VI)-reducing bacteria, Escherichia coli ATCC 33456 . Near complete Cr(VI) reduction and phenol degradation was observed during steady-state operation of the reactor under loadings of 5-21 mg Cr(VI) l-1 d-1 and 840-3350 mg phenol l-1 d-1 . 2-hydroxymuconic semialdehyde (2HMSA), succinate, and acetate were the detected steady-state organic acid metabolites which accounted for 13-23% of TOC in the effluent . Optimum Cr(VI) reduction rate was observed under a Cr(VI) loading of approximately 26.5 mg Cr(VI) l-1 d-1 just before system overload . System overload was characterized by the increase in effluent Cr(VI) and phenol concentrations . System resilience to Cr(VI) toxicity was demonstrated by rapid recovery of biological activity and reduced effluent Cr(VI) and phenol concentrations after off-loading the system from overloaded conditions.

J Biotechnol, 2001 May 18, 87(3), 211 - 23
Mathematical model for evaluation of mass transfer limitations in phenol biodegradation by immobilized Pseudomonas putida; Banerjee I et al.; A mathematical model is proposed to analyze the mass transfer limitations in phenol biodegradation using Pseudomonas putida immobilized in calcium alginate . The model takes into account internal and external mass transfer limitations, substrate inhibition kinetics and the dependence of the effective diffusivity of phenol in alginate gel on cell concentration . The model is validated with the experimental data from batch fermentation . The effect of various operating conditions such as initial phenol concentration, initial cell loading, alginate gel loading on the biodegradation of phenol is experimentally demonstrated . Phenol degradation time is found to decrease initially and reach stationary value with increase in cell loading as well as gel loading . The model predicts these trends reasonably well and shows the presence of external mass transfer limitations . A new concept of effectiveness factor is introduced to analyze the overall performance of batch fermentation.

Biochemistry, 2001 Feb 20, 40(7), 2155 - 66
Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase; Efimov I et al.; Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384 . PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde . The Y384F mutant form of PchF (apo-PchF{Y384F}) displayed stoichiometric noncovalent FAD binding . PchF{Y384F}FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH{Y384F}), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)) . Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF{Y384F}, and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C) . The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF{Y384F}FAD associated with PchC . The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein . The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF . Under anaerobiosis, the reduction of PchF{Y384F}FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD . Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH{Y384F} which produced PchF{Y384F}FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme . These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF{Y384F}FAD(rad) x PchC . Steady-state kinetic studies of the reaction of PCMH{Y384F} with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude . The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin . Contributions to this effect likely result from conformational changes.

J Bacteriol, 2001 May, 183(10), 3256 - 60
Effects of mutations in the Pseudomonas putida miaA gene: regulation of the trpE and trpGDC operons in P . putida by attenuation; Olekhnovich I et al.; Tn5 insertion mutants defective in regulation of the Pseudomonas putida trpE and trpGDC operons by tryptophan were found to contain insertions in the P . putida miaA gene, whose product (in Escherichia coli) modifies tRNA(Trp) and is required for attenuation . Nucleotide sequences upstream of trpE and trpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.

Microbiology, 2001 May, 147(Pt 5), 1323 - 30
Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation; Manzanera M et al.; The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa . The unambiguous role of the gene as a sigma factor was confirmed because the cloned P . putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain . Primer extension analysis showed that in P . putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C . Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence . The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e . a sudden heat shock or the addition of aromatic compounds . However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life . An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P . putida growing at temperatures from 18 to 42 degrees C.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 668 - 73
{Characteristic of mutants of Pseudomonas putida IPM-36 recombinant strains, selected by anticipating growth on a media containing an inducer of cry3A gene expression}; Koretskaia NG et al.; Induction of the expression of the delta-endotoxin gene from Bacillus thuringiensis var . tenebrionis in the recombinant strain Pseudomonas putida IPM-36 negatively affected the viability and the growth rate of the culture . In order to optimize the insecticide production by the recombinant strain, mutant clones exhibiting anticipating growth on an inducer-containing medium were selected and studied . These clones differed in such aspects as the localization of mutations (either in plasmid pBTN11, carrying the cry3A gene, or in the chromosome), growth rate, or the level of delta-endotoxin synthesis after induction . Several mutants obtained proved much superior to P . putida IPM-36 in their structural and segregation stability, although they were as efficient as the original strain with respect to the production of the insecticide (protei Cry3A).

Can J Microbiol, 2001 Mar, 47(3), 222 - 8
Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid; Anderson AJ et al.; Peracetic acid is used as a sterilant in several industrial settings . Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A . Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts . Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid . The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture . Amendment of sterilized soils with wild-type P . putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant . The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid . No differential recovery of the wild-type and catalase A mutant of P . putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.

J Biol Chem, 2001 Jun 29, 276(26), 23480 - 5 Epub 2001 Apr 16.
Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage; Goda M et al.; Isonitrile containing an N triple bond C triple bond was degraded by microorganism sp . N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound . The isonitrile-degrading microorganism was identified as Pseudomonas putida . The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide . The enzyme, named isonitrile hydratase, was purified and characterized . The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits . The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected . The apparent K(m) value for cyclohexyl isocyanide was 16.2 mm . Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.

Crit Rev Microbiol, 2001, 27(1), 41 - 55
Allelopathic bacteria and their impact on higher plants; Barazani O et al.; The impact of allelopathic, nonpathogenic bacteria on plant growth in natural and agricultural ecosystems is discussed . In some natural ecosystems, evidence supports the view that in the vicinity of some allelopathically active perennials (e.g., Adenostoma fasciculatum, California), in addition to allelochemicals leached from the shrub's canopy, accumulation of phytotoxic bacteria or other allelopathic microorganisms amplify retardation of annuals . In agricultural ecosystems allelopathic bacteria may evolve in areas where a single crop is grown successively, and the resulting yield decline cannot be restored by application of minerals . Transfer of soils from areas where crop suppression had been recorded into an unaffected area induced crop retardation without readily apparent symptoms of plant disease . Susceptibility of higher plants to deleterious rhizobacteria is often manifested in sandy or so-called skeletal soils . Evaluation of phytotoxic activity under controlled conditions, as well as ways to apply allelopathic bacteria in the field, is approached . The allelopathic effect may occur directly through the release of allelochemicals by a bacterium that affects susceptible plant(s) or indirectly through the suppression of an essential symbiont . The process is affected by nutritional and other environmental conditions, some may control bacterial density and the rate of production of allelochemicals . Allelopathic nonpathogenic bacteria include a wide range of genera and secrete a diverse group of plant growth-mediating allelochemicals . Although a limited number of plant growth-promoting bacterial allelochemicals have been identified, a considerable number of highly diversified growth-inhibiting allelochemicals have been isolated and characterized . Some species may produce more than one allelochemical; for example, three different phyotoxins, geldanamycin, nigericin, and hydanthocidin, were isolated from Streptomyces hygroscopicus . Efforts to introduce naturally produced allelochemicals as plant growth-regulating agents in agriculture have yielded two commercial herbicides, phosphinothricin, a product of Streptomyces viridochromogenes, and bialaphos from S . hygroscopicus . Many species of allelopathic bacteria that affect growth of higher plants are not plant specific, but some do exhibit specificity; for example, dicotyledonous plants were more susceptible to Pseudomonas putida than were monocotyledons . Differential susceptibility of higher plants to allelopathic bacteria was noted also in much lower taxonomical categories, at the subspecies level, in different cultivars of wheat, or of lettuce . Therefore, when test plants are employed to evaluate bacterial allelopathy, final evaluation must include those species that are assumed to be suppressed in nature . The release of allelochemicals from plant residues in plots of 'continuous crop cultivation' or from allelopathic living plants may induce the development of specific allelopathic bacteria . Both the rate by which a bacterium gains from its allelopathic activity through utilizing plant excretions, and the reasons for the developing of allelopathic bacteria in such habitats, are important goals for further research.

Extremophiles, 2001 Feb, 5(1), 11 - 5
Evaluation of the growth and inhibition strength of hydrocarbon solvents against Escherichia coli and Pseudomonas putida grown in a two-liquid phase culture system consisting of a medium and organic solvent; Aono R et al.; The growth of microorganisms is often inhibited in a two-liquid phase culture system consisting of an aqueous medium and a large volume of hydrophobic solvent . Escherichia coli and Pseudomonas putida were cultured in a two-phase system containing a solvent with a log Pow value in a range of 2.1 to 6.0 . The increase in the cell mass was monitored by increase in turbidity of the medium phase . We devised a semiquantitative method to evaluate the growth inhibition strength of solvents based on the relative amount of bacterial growth occurring in the two-phase system . Analyses of growth of the bacteria by this method showed that the growth inhibition strength of a given solvent was usually but not always correlated inversely with its polarity . It is clear that growth inhibition strength is not determined simply by polarity of the solvent.

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 435 - 7
Occurrence of a novel lyase catalyzing beta-elimination reaction toward threo-3-chloro-L-aspartate in Pseudomonas putida TPU 7151; Kato Y et al.; A bacterium, Pseudomonas putida TPU 7151, which degrades threo-3-chloro-L-aspartate, was isolated from soil and the enzyme responsible for the degradation of the amino acid was partially purified from the cell-free extract of the strain . The enzyme, which required PLP for its reaction, catalyzed a stoichiometric beta-elimination reaction of threo-3-chloro-L-aspartate to form oxaloacetate, Cl-, and NH4 . The enzyme was active toward only threo-3-chloro-L-aspartate and L-cysteine, but did not catalyze a beta-replacement reaction . The enzyme can be classified in a new group of PLP-dependent amino acid-lyases {EC 4.2.1.-}.

Eur J Biochem, 2001 Apr, 268(8), 2229 - 38
Nucleotide sequence analysis of 5'-flanking region of salicylate hydroxylase gene, and identification and purification of a LysR-type regulator, SalR; Sato H et al.; The sal gene comprised of 1266 nucleotides encoding salicylate hydroxylase was cloned from the chromosomal DNA of Pseudomonas putida S-1 and sequenced {Suzuki, K., Mizuguchi, M., Ohnishi, K . and Itagaki, E . (1996) Biochim . Biophys . Acta 1275, 154-156} . Here, we describe the nucleotide sequences of the regulatory region of the sal gene and an ORF (salR gene) divergently oriented from the sal gene, which encodes the protein SalR . This gene product positively controls sal gene expression at the transcriptional level . The salR gene consists of 930 base pairs starting from a GTG codon and encodes a protein of 309 amino acids with a molecular mass of 34 542 Da . The amino-acid sequence is homologous to LysR-family regulatory proteins such as CatR of P . putida RB1 and has helix-turn-helix DNA binding motif near its N-terminal . Transcription start sites of sal and salR genes were determined to lie 30- and 24-bp upstream of the respective initiation codons and separated from each other by 78 nucleotides . A Shine-Dalgarno sequence and the putative promoter sequences containing -10 and -35 sequences were seen in the sal and salR genes . Expression of the salR gene on a plasmid in Escherichia coli cells was confirmed by DNA mobility shift assay . For the overexpression of the salR gene, it was cloned to pET28a (pSAHR) which was transferred to E . coli BL21 (E . coli BL21/pSAHR), and expressed by an inducer, isopropyl thio-beta-D-galactoside . SalR was further purified to homogeneity from the cell-free extracts in yields of approximately 3 mg.L-1 culture volume . The molecular mass was determined to be 33 kDa and the N-terminal amino-acid sequence was the same as that deduced from the nucleotide sequence of salR gene . Native SalR was also purified to homogeneity from P . putida S-1 with very low contents . The properties of the protein were similar to those of SalR expressed in E . coli.

FEMS Microbiol Ecol, 2001 Apr, 35(2), 217 - 221
The limits to genomic predictions: role of sigma(N) in environmental stress survival of Pseudomonas putida; Cases I et al.; Based on genomic data and on the phenotypes of an FlhF mutant of Pseudomonas putida, the alternative sigma factor sigma(N) (sigma(54)) has been proposed to play a key role in survival to various nutritional and environmental stresses in this bacterium . Quite in contrast, we show that unlike sigma(S) (sigma(38)) the loss of sigma(N) does not impair to any significant extent the ability of P . putida to survive long-term starvation . rpoN mutants (lacking sigma(N)) are indistinguishable from the wild-type with respect to solvent tolerance, resistance to heat shock or sensitivity to hydrogen peroxide . These data suggest that while sigma(N) is a key component of expression of alternative biodegradative pathways for unusual carbon sources (i.e . m-xylene or dimethylphenols), its loss does not compromise bacterial endurance to gross types of environmental stress . Moreover, these results point out the limitations, if not the deception, of genomic predictions when confronted with experimental data.

J Bacteriol, 2001 May, 183(9), 2910 - 7
Expression of the putative siderophore receptor gene bfrZ is controlled by the extracytoplasmic-function sigma factor BupI in Bordetella bronchiseptica; Pradel E et al.; A new gene from Bordetella bronchiseptica, bfrZ encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay . A bfrZ null mutant was constructed by allelic exchange . The protein profile of this mutant is similar to that of the wild-type parent strain . The BfrZ(-)-BfrZ(+) isogenic pair was tested for utilization of 132 different siderophores as iron sources . None of these iron sources acted as a ligand for BfrZ . Translational bfrZ::phoA and transcriptional bfrZ::lacZ fusions were introduced into the B . bronchiseptica bfrZ locus . No alkaline phosphatase or beta-galactosidase activity was detected . Sequence analysis of the bfrZ upstream region revealed the presence of two tightly linked genes, bupI and bupR . Both of these genes are located downstream from a Fur-binding sequence . BupI is homologous to Escherichia coli FecI and Pseudomonas putida PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions . BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane . Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension . We found that bfrZ is not transcribed when bupI and bupR are expressed at the same level . However, overexpression of bupI from a multicopy plasmid triggers bfrZ transcription, and under these conditions BfrZ was detected in membrane fractions . By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that bfrZ expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope.

Environ Technol, 2001 Jan, 22(1), 39 - 46
Removal of toluene in gas streams by a fibrous-bed trickling filter; Tseng DH et al.; A novel fibrous-bed trickling filter was developed to remove toluene present in contaminated air . Pure culture of Pseudomonas putida F1 was attached on fibrous-bed and utilized toluene as the carbon source . Experimental results indicated the removal efficiency decreased with the increase of inlet concentration . In general, the removal efficiency of toluene was greater than 90% when the inlet loading capacity was below 70 g m-3h-3 . The elimination capacity increased with increasing inlet loading capacity, but the increased rate decreased gradually . When the inlet loading capacity increased to 300 g m-3h-1, the elimination capacity could approach to 130 g m-3h-1 . The first order kinetics model was useful to describe the removal of toluene in this filter and an excellent linear relationship was found between the apparent first order parameter and inlet concentration (ranging from 1.2 g m-3 to 3.5 g m-3) . Also, the performance of fibrous-bed trickling filter was relatively stable during the four-month period of continuous operation . Slight clogging phenomena of filters were observed only under high loading capacity.

Microbiology, 2001 Apr, 147(Pt 4), 973 - 9
Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3; O'Leary ND et al.; The styrene degradative pathway in Pseudmonas putida CA-3 has previously been shown to be divided into an upper pathway involving the conversion of styrene to phenylacetic acid and a lower pathway for the subsequent degradation of phenylacetic acid . It is reported here that expression of the regulatory genes styS and styR is essential for transcription of the upper pathway, but not for degradation of the lower pathway inducer, phenylacetic acid . The presence of phenylacetic acid in the growth medium completely repressed the upper pathway enzymes even in the presence of styrene, the upper pathway inducer . This repression is mediated at the transcription level by preventing expression of the styS and styR regulatory genes . Finally, an examination was made of the various stages of the diauxic growth curve obtained when P . putida CA-3 was grown on styrene together with an additional carbon source and it is reported that catabolite repression may involve a different mechanism to transcriptional repression by an additional carbon source.

Appl Environ Microbiol, 2001 Apr, 67(4), 1613 - 8
Development and application of a most-probable-number-pcr assay to quantify flagellate populations in soil samples; Fredslund L et al.; This paper reports on the first successful molecular detection and quantification of soil protozoa . Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations . Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists . Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences . This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene . The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey . There was satisfactory overall agreement between H . globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

Appl Environ Microbiol, 2001 Apr, 67(4), 1437 - 44
Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator; Lee SW et al.; Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharacterized . A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR . CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli . CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems . A related gene, zntR, regulates zntA in E . coli, but it is not contiguous with zntA in the E . coli genome as cadA and cadR were in P . putida . In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P . aeruginosa genome, the P . putida CadA sequence had a histidine-rich N-terminal extension . CadR and the product of PA3689 of P . aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators . Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance . However, unlike zntA, they did not confer significant levels of lead resistance . The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II) . CadR apparently represses its own expression at the transcriptional level . However, CadR apparently does not repress cadA . Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.

Res Microbiol, 2001 Jan-Feb, 152(1), 83 - 93
Biodegradation of hydroxylated and methoxylated benzoic, phenylacetic and phenylpropenoic acids present in olive mill wastewaters by two bacterial strains; Di Gioia D et al.; Two aerobic bacterial strains, a chlorophenol-degrading bacterium characterized in this work as a Ralstonia sp . LD35 on the basis of the sequence of the gene encoding for 16S ribosomal RNA, and Pseudomonas putida DSM 1868, capable of metabolizing 4-methoxybenzoic acid, were tested for their capacity to degrade monocyclic aromatic acids responsible for the toxicity of olive mill wastewaters (OMWs) . Both strains possess interesting and complementary degradation capabilities in resting cell conditions: Ralstonia sp . LD35 was found to metabolize 4-hydroxybenzoic, 4-hydroxyphenylacetic, 3,4-dihydroxycinnamic and cinnamic acid, whereas DSM 1868 was capable of metabolizing 4-hydroxy-3-methoxybenzoic, 3,4-dimethoxybenzoic and 4-hydroxy-3,5-dimethoxybenzoic acid, as well as 4-hydroxybenzoic and 4-hydroxyphenylacetic acid . The kinetic parameters describing the growth of the two strains on the same compounds were determined in growing-cell batch conditions, and showed that both strains presented high affinity and high specific growth rates towards all assayed substrates . In addition, the two strains were capable of growing on and extensively biodegrading a mixture of monocyclic aromatic acids commonly found at high concentrations in OMWs, and of growing on a 20% dilution of a natural OMW . All these features make the two strains attractive candidates for the development of a biotechnological process for the biodegradation of aromatic compounds found in OMWs.

J Biol Chem, 2001 May 18, 276(20), 16641 - 8 Epub 2001 Feb 13.
The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions; Bartels F et al.; A protein mixture containing two major components able to catalyze a beta-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract . N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant . The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence . The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified . Their activities were examined in vitro in beta-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis . HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins . This was reflected in the maintenance of in vivo activity of the final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN strains . However, hupB/hupN double mutants were not viable . It is suggested that the requirement for protein-facilitated DNA bending is met in P . putida by two independent proteins that ensure an adequate supply of an essential cellular activity.

Protein Sci, 2001 Apr, 10(4), 741 - 52
Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B; Kim DH et al.; Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B . Folding was reversible in secondary and tertiary structures as well as in activity . Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two-state mechanism without thermodynamically stable intermediates . Consistent with the two-state model, one dimensional (1D) NMR spectra and gel-filtration chromatography analysis did not show any evidence for a folded monomeric intermediate . Interestingly enough, Cys 81 located at the dimeric interface was modified by DTNB before unfolding . This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation . The refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step . Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step . During the refolding process, negative ellipticity at 225 nm increased very fast within 80 msec to account for >80% of the total amplitude . This result suggests that the protein folds into a monomer containing most of the alpha-helical structures before dimerization . Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active . Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.

Biosci Biotechnol Biochem, 2001 Jan, 65(1), 190 - 3
Isolation of the rpoD gene encoding the principal sigma factor of the deep-sea piezophilic bacterium Shewanella violacea strain DSS12 and its overexpression in Escherichia coli; Nakasone K et al.; The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced . The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively . Comparison with E . coli sigma70 and P . putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding . Using an expression plasmid harboring the rpoD gene, the S . violacea sigma70 factor was overexpressed in E . coli and successfully purified to near homogeneity.

Cancer Detect Prev, 2001, 25(1), 102 - 7
Biodegradation of the antineoplastics vindesine, vincristine, and vinblastine and their toxicity against bacteria in the aquatic environment; Al-Ahmad A et al.; Antineoplastics are excreted into sewage, because patients often poorly metabolize them after administration or they are metabolized into more biologically reactive metabolites . There is little information on their biodegradation and toxicity in aquatic environments . Therefore, the biodegradability of the vinca alkaloids, and their toxicity towards wastewater bacteria were investigated in this study . The biodegradability of vindesine, vincristine, and vinblastine was examined in the closed bottle test (CBT) . Additionally, the biodegradability of vinblastine as a model compound of the vinca alkaloids was tested in the Zahn-Wellens test (ZWT) . The growth inhibition test with Pseudomonas putida was conducted, and a toxicity control in the CBT and the ZWT was used . The colony-forming units were monitored in the CBT; the test results for the biodegradability after 28 days were: 30% for vincristine, 20% for vindesine, and 10% for vinblastine . Therefore, none of the test compounds met the criteria for being readily biodegradable (> or = 60%) . Vinblastine was biodegraded up to 18% in the ZWT after 40 days, and therefore, not inherently . Toxicity towards wastewater bacteria was not found.

Water Res, 2001 Apr, 35(5), 1201 - 8
Stability analysis of the biodegradation of mixed wastes in a continuous bioreactor with cell recycle; Ajbar A; The stability characteristics of a continuous bioreactor with cell recycle for biodegradation of mixed wastes are investigated . The system involves a pure culture of Pseudomonas putida and media containing phenol and glucose as carbon and energy sources . The model growth kinetics for the two substitutable substrates were experimentally validated in a previous study . The stability analysis carried out using elementary principles of bifurcation theory shows rich dynamics characteristics of the reactor model, including steady-state multiplicity and hysteresis . The effect of the bioreactor operating parameters on the stability behavior of the model is discussed . Practical criteria are also derived for the safe operation of the unit and to prevent the occurrence of wash-out conditions.

Bioresour Technol, 2001 May, 78(1), 47 - 54
Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil; Milcic-Terzic J et al.; Bioremediation is often used for in situ remediation of petroleum-contaminated sites . The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation . Diesel . toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates . The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA DNA colony hybridization . The diesel DNA-extract possessed both the xy/E catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation . The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene . Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973 . A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene . 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene . These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site . Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present.

Biochemistry, 2001 Mar 6, 40(9), 2669 - 77
Structural characterization of n-butyl-isocyanide complexes of cytochromes P450nor and P450cam; Lee DS et al.; Alkyl-isocyanides are able to bind to both ferric and ferrous iron of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra . While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm . The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme . To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm . Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively . The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined . The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam {d(Fe-C) = 1.86 A, angleFe-C-N = 159 degrees } versus P450nor {d(Fe-C) = 1.85 A, angleFe-C-N = 175 degrees } . Another difference in the structures was the chemical environment of the heme pocket . In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules . On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.

Biotechnol Bioeng, 2001 Apr 5, 73(1), 69 - 73
Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system; Hage A et al.; We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium . The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen . For both P . chrysosporium and P . putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives . Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed . Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P . putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences . Toxicity of aromatic carbonylic compounds in an established biotransformation system with P . chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products . P . putida can be used to test the toxicity of aromatic ketones to the basic diomycete P . chrysosporium .

Mol Microbiol, 2001 Feb, 39(4), 1100 - 6
Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida; Duque E et al.; Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase . Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family . The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene . The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA . The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence . In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter . In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene . In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type . Analysis of a P . putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene . The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon . The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.

Mol Microbiol, 2001 Feb, 39(4), 863 - 74
Two different pathways are involved in the beta-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications; Olivera ER et al.; In Pseudomonas putida U, the degradation of n-alkanoic and n-phenylalkanoic acids is carried out by two sets of beta-oxidation enzymes (betaI and betaII) . Whereas the first one (called betaI) is constitutive and catalyses the degradation of n-alkanoic and n-phenylalkanoic acids very efficiently, the other one (betaII), which is only expressed when some of the genes encoding betaI enzymes are mutated, catabolizes n-phenylalkanoates (n > 4) much more slowly . Genetic studies revealed that disruption or deletion of some of the betaI genes handicaps the growth of P . putida U in media containing n-alkanoic or n-phenylalkanoic acids with an acyl moiety longer than C4 . However, all these mutants regained their ability to grow in media containing n-alkanoates as a result of the induction of betaII, but they were still unable to catabolize n-phenylalkanoates completely, as the betaI-FadBA enzymes are essential for the beta-oxidation of certain n-phenylalkanoyl-CoA derivatives when they reach a critical size . Owing to the existence of the betaII system, mutants lacking betaIfadB/A are able to synthesize new poly 3-OH-n-alkanoates (PHAs) and poly 3-OH-n-phenylalkanoates (PHPhAs) efficiently . However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants . The genetic and biochemical importance of these results is reported and discussed.

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