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