FEMS Microbiol Ecol, 2001 Mar, 35(1), 57 - 65 The role of motility in the in vitro attachment of Pseudomonas putida PaW8 to wheat roots; Turnbull GA et al.; The attachment of motile and non-motile strains of Pseudomonas putida PaW8 to sterile wheat roots was assessed in both non-competitive and intra-specific competitive assays . The motile strain showed significantly greater attachment to wheat roots than non-motile strains in phosphate buffer . Overall, the motile strain attached better than the non-motile strain at 10(6), 10(7) and 10(8) cfu ml(-1) in competitive assays and at 10(6) and 10(7) cfu ml(-1) in non-competitive assays . When attachment was studied in Luria broth no significant difference between motile and non-motile strains was detected . P . putida PaW8 cells marked with the luxAB genes were used to compare direct detection of attached cells by luminometry with indirect detection by dilution plate counts following extraction from root material . Although direct detection permitted a rapid assessment (60 s) of attachment to surfaces, dilution plate counts provided a more sensitive method for quantification of bacteria . The detection limits were approximately 10 cfu root(-1) using dilution plate counts compared with 1000 cfu root(-1) using luminometry . All results highlighted the importance of motility for the attachment of P . putida to plant roots in simple model systems . To take this work further, studies to assess the role of motility using complex non-sterile systems are needed. FEBS Lett, 2001 Mar 2, 491(3), 207 - 11 Interactions of the XylS regulators with the C-terminal domain of the RNA polymerase alpha subunit influence the expression level from the cognate Pm promoter; Ruiz R et al.; The Pseudomonas putida meta-cleavage operon encodes the enzymes for the catabolism of alkylbenzoates . Activation of meta-operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between -70 and -35 with respect to the main transcription initiation point at the Pm promoter . Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C-terminal domain of the alpha subunit of RNA polymerase (alpha-CTD) . For these studies we expressed a derivative of alpha deprived of the entire C-terminal domain (alpha-Delta235) and found that expression from Pm with XylS or XylS1 was significantly decreased . To discern whether alpha-CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within alpha-CTD . Most substitutions that had an effect on XylS and XylS1-dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in alpha-CTD interactions with DNA . Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of alpha-CTD/XylS regulator interactions. Eur J Biochem, 2001 Mar, 268(5), 1460 - 7 Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam)); Jones JP et al.; Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro-organisms for the biodegradation of aromatic compounds . We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono-oxygenase CYP101 (cytochrome P450(cam)) from Pseudomonas putida with the aim of generating novel systems for their biodegradation . Wild-type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes . Increasing the active-site hydrophobicity with the Y96F mutation increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol . Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10-fold increase in activity with most substrates . Introducing the F98W mutation, also at the top of the active site, decreased the NADH-turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W--Y96F--F98W mutant . The V247L mutation generally increased the NADH-turnover rates, and the F87W--Y96F--V247L mutant showed reasonably fast NADH turnover (229 min(-1)) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents . As all chlorophenols are degraded by micro-organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways. Appl Environ Microbiol, 2001 Mar, 67(3), 1388 - 91 Substrate selectivity of a 3-nitrophenol-induced metabolic system in Pseudomonas putida 2NP8 transforming nitroaromatic compounds into ammonia under aerobic conditions; Zhao JS et al.; The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production . All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed . Ammonia production from most nitroaromatic substrates appeared to be stoichiometric. Appl Environ Microbiol, 2001 Mar, 67(3), 1107 - 15 Genetic diversity among 3-chloroaniline- and aniline-degrading strains of the Comamonadaceae; Boon N et al.; We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA) . Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1 . Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D . acidovorans . Both Delftia and Comamonas belong to the family Comamonadaceae . All five strains possess a large plasmid of ca . 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen . Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline . Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA . For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe . This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment . By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE) . With DGGE, three different clusters of the tdnQ fragment could be distinguished . Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P . putida UCC22 were only about 83% identical to the other sequences . Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present. Appl Environ Microbiol, 2001 Mar, 67(3), 1052 - 62 Analysis of bacterial community structure in sulfurous-oil-containing soils and detection of species carrying dibenzothiophene desulfurization (dsz) genes; Duarte GF et al.; The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil . Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed . Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used . Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts . However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil . Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp., Arthrobacter sp., and a bacterium of uncertain affiliation . dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis . Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities . The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum . Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment . PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands . Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonas sp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis . Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures . Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively . Most of the strains carrying dszA or dszC were classified as R . erythropolis related, and all revealed the capacity to desulfurize DBT . A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R . erythropolis strain IGTS8) dszA sequence and a large degree of internal conservation . The 37 sequences recovered were grouped in three clusters . One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar . The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil. Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 169 - 70 Arthrobacter siderocapsulatus Dubinina and Zhdanov 1975AL is a later subjective synonym of Pseudomonas putida (Trevisan 1889) Migula 1895AL; Chun J et al.; The taxonomic position of Arthrobacter siderocapsulatus Dubinina and Zhdanov 1975AL was investigated using 16S rDNA, fatty acid and phenotypic analyses . The type strain (NCIMB 11286T) showed 99.85% 16S rDNA similarity to the type strain of Pseudomonas putida . Phenotypic properties of the two strains were compared using API 20NE and BIOLOG kits . Identical reactions were recorded for all tests, except for assimilation of malonic acid . The two strains also showed almost identical cellular fatty acid profiles . On the basis of evidence presented in this and earlier studies, it is proposed that Arthrobacter siderocapsulatus is a later subjective synonym of Pseudomonas putida (Trevisan 1889) Migula 1895AL. Bioresour Technol, 2001 Feb, 76(3), 245 - 51 Biodegradation of phenol in a continuous process: comparative study of stirred tank and fluidized-bed bioreactors; Gonzalez G et al.; The paper presents the main results obtained from the study of the biodegradation process of phenol by a pure culture of Pseudomonas putida ATCC 17484 . The experimental work was carried out in two different systems: a stirred tank where cells grew as a suspended culture and a fluidized bed where cells were immobilized within calcium alginate gel beads . The influence of the hydraulic residence time (HRT) and organic loading rate on the removal efficiency of phenol was determined for both bioreactors . Also, the stability of the fluidized-bed bioreactor (FBB) in terms of its ability to withstand sudden phenol overdoses is also reported . Experimental values indicated that both bioreactors showed high phenol degradation efficiencies, higher than 90%, even for a phenol loading rate in the influent as high as 4 g phenol/l day . The FBB showed better performance than the suspended-culture bioreactor due to its better control and because it could operate with lower HRT. Biotechniques, 2001 Jan, 30(1), 142 - 8 Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria; Felske A et al.; A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA . The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment . The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE) . Gene quantification is provided by a competitively co-amplified DNA standard constructed by point mutation PCR . After computing the denaturation behavior of the target DNA stretch, a single base difference was introduced to achieve maximum migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency . This competitive PCR strategy is a highly specific and sensitive way to detect small sequence differences and to monitor recombinant DNA in effluxes of biotechnological plants. Mikrobiologiia, 2000 Nov-Dec, 69(6), 783 - 9 {Degradation of phenanthrene by mutant strains--naphthalene degraders}; Kosheleva IA et al.; Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy . The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol . The latter products are further metabolized by the meta- and ortho-cleavage pathways . In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-borne genes . The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive. Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2336 - 43 Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida; Inoue H et al.; L-Methionine gamma-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of y-family pyridoxal 5'-phosphate dependent enzymes . A mutant form of L-methionine y-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for alpha,gamma-elimination of L-methionine, while the Km remained the same as the wild type enzyme . The Y114F mutant had the reduced kcat by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for alpha,beta-elimination and deamination substrates . The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma-elimination process for L-methionine is the rate-limiting determination step in alpha,gamma-elimination overall reaction of the Y114F mutant . These results indicate that Tyr114 of L-methionine gamma-lyase is important in y-elimination of the substrate. J Bacteriol, 2001 Feb, 183(3), 1032 - 7 Evidence of multiple regulatory functions for the PtsN (IIA(Ntr)) protein of Pseudomonas putida; Cases I et al.; The ptsN gene of Pseudomonas putida encodes IIA(Ntr), a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the sigma(54)-dependent promoter Pu of the TOL (toluate degradation) plasmid pWW0 . Using two-dimensional gel electrophoresis, we have examined the effect of ptsN disruption on the general expression pattern of P . putida . To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the P . putida proteome was used as a reference for comparisons . Among all gene products whose expression was lowered by this carbon source (247 spots {about 22%}), only 6 behaved as Pu (i.e., were depressed in the ptsN background) . This evidenced only a minor role for IIA(Ntr) in the extensive inhibition of gene expression in P . putida caused by glucose . However, the same experiments revealed a large incidence of glucose-independent effects brought about by the ptsN mutation . As many as 108 spots (ca . 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIA(Ntr) . By matching this pattern with that of an rpoN::OmegaKm strain of P . putida, which lacks the sigma(54) protein, we judge that most proteins whose expression was affected by ptsN were unrelated to the alternative sigma factor . These data suggest a role of IIA(Ntr) as a general regulator, independent of the presence of repressive carbon sources and not limited to sigma(54)-dependent genes. J Bacteriol, 2001 Feb, 183(3), 928 - 33 p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6; O'Connor KE et al.; Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde . Cell extracts of P . putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid . Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde . A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds . 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage. Environ Microbiol, 1999 Dec, 1(6), 479 - 88 Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil; Rodriguez-Herva JJ et al.; To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn5luxAB-Km transposon . A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P . putida LPS . The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced . Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen) . The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays . The cloned gene was able to restore the wild-type phenotype of the P . putida wzy mutant . We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL::xylE cassette . The wzy mutants of P . putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains . We analysed the ability of wzy, oprL and wzy oprL mutants of P . putida to colonize soil . In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures. Environ Microbiol, 1999 Oct, 1(5), 409 - 14 13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation; Meckenstock RU et al.; The influence of microbial degradation on the 13C/12C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound . Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers . The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors alphaC for toluene degradation by Pseudomonas putida (1.0026 +/- 0.00017), Thauera aromatica (1.0017 +/- 0.00015), Geobacter metallireducens (1.0018 +/- 0.00029) and the sulphate-reducing strain TRM1 (1.0017 +/- 0.00016) were in the same range for all four species, although they use at least two different degradation pathways . A similar 13C/12C isotope fractionation factor (alphaC = 1.0015 +/- 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions . No carbon isotope shifts resulting from soil-hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions . The results imply that microbial degradation of toluene can produce a 13C/12C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions. Curr Microbiol, 2001 Apr, 42(4), 231 - 6 Bioremediation of sterile agricultural soils polluted with crude petroleum by application of the soil bacterium, Pseudomonas putida, with inorganic nutrient supplementations; Nwachukwu SU; The effects of bioremediation program of sterile agricultural soils contaminated with crude petroleum were determined with a view to developing a suitable technique for rehabilitation of similar environments upon pollution by oil spillage . Sterile soils inoculated with the soil bacterium, Pseudomonas putida (PP), with inorganic nutrients monitoring and supplementation constituted the experimental set-ups (ESU) . The control set-ups (CSU) contained all the materials present in ESU except that they were not inoculated with PP . In ESU at week 9, the oil pollutant was completely biodegraded, and the inorganic nutrient ions, particularly PO4(-3) and NO3(-1), were significantly utilized . In contrast, there were no significant changes in the concentrations of oil and inorganic nutrients in CSU . Also, the percentage germination and growth profiles of cress seeds (Lepidium sp.) planted as evidence of the recovery of the oil-impacted soils were poor in CSU (27.5%) with pronounced abnormal morphology when compared with the results obtained for ESU (98.8%) . Inoculation of PP with addition of appropriate inorganic nutrients may be a suitable method for a rapid rehabilitation of agricultural land upon pollution with crude petroleum. Biochem, Eng . J. . 2001 Mar, 7(2), 113 - 115 Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates; Duetz WA et al.; Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects . The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates . For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates . Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5cm at 300rpm (24mmolO(2)l(-1)h(-1) at a culture volume of 0.75ml) in comparison to a shaking diameter of 2.5cm . Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow. Enzyme Microb Technol, 2001 Feb 1, 28(2-3), 183 - 188 The use of oxygen uptake rate measurements to control the supply of toxic substrate: toluene hydroxylation by Pseudomonas putida UV4; Carragher JM et al.; During toluene hydroxylation, catalyzed by Pseudomonas putida UV4 one molecule of oxygen is added to the aromatic ring to produce the dihydroxylated (non-aromatic) ring structure, toluene cis-glycol . Toluene, which is toxic to the cells at aqueous phase concentration above ( approximately 2.4 mmol), is fed to the reactor . A feed-back control system based on oxygen uptake rate measurements was used to control the feed rate, and thus maintain the aqueous phase toluene concentration in the desired range for zero order kinetics. J Biotechnol, 2001 Feb 13, 85(2), 103 - 13 Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene; Haro MA et al.; In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene . The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates . A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P . putida mt-2 . The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate . These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator . Such gene cassettes (mini-Tn5 {UPP2} and mini-Tn5 {TOD2}) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P . aeruginosa JB2 . GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene . However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source . These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process . Choices to address this general problem are discussed. Nucleic Acids Res, 2001 Feb 1, 29(3), 759 - 66 Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity; Cebolla A et al.; Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties . Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal . We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and PM: Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate . When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the PM: promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems . By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect . Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals. Microbiology, 2001 Jan, 147(Pt 1), 43 - 51 Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance; Kieboom J et al.; The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12 . Recently, it was shown that organic solvents and not antibiotics induce this operon . In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants . The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin . The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux . These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics . However, ArpABC does not contribute to organic solvent tolerance in P . putida S12 but is solely involved in multidrug resistance. Microbiology, 2001 Jan, 147(Pt 1), 31 - 41 Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P . putida F1 except for a single base difference in the operator-promoter region of the cmt operon; Ohta Y et al.; Psudomonas putida CE2010 can assimilate biphenyl despite its high similarity to P . putida F1 . Biphenyl degradation in strain CE2010 was achieved using a mosaic of pathways consisting of the cmt and tod operons . CmtE hydrolysed 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product of 2,3-dihydroxybiphenyl . This enzyme was expressed differently in strains CE2010 and F1 . A cmtE disruption mutant, a tod operon disruption mutant and a cmt operon disruption mutant were unable to utilize biphenyl . The introduction of the cmtE gene enabled the cmt operon disruption mutant to grow on biphenyl . A single base difference was found in the cmt promoter-operator region in strain CE2010, compared with that of strain F1 . CymR protein was purified from Escherichia coli and binding assays were performed, the results of which suggested that the protein bound less strongly to the CE2010 operator sequence than to the F1 operator sequence . Exchanging the F1 promoter-operator fragment into strain CE2010 resulted in a loss of biphenyl degradation capacity . These results indicate that cmtE is not effectively repressed by CymR in strain CE2010, leading to low constitutive expression and, therefore, low growth on biphenyl. Microbiology, 2001 Feb, 147(Pt 2), 337 - 44 A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector; Holtwick R et al.; In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e . pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (approximately 100 kbp) . Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D) . No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates . Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin . Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion . This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both Escherichia coli and P . putida. J Bacteriol, 2001 Feb, 183(4), 1225 - 32 Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3; Hughes MA et al.; Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate {PCA}) to central metabolites . We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA . In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway . The central pnbR gene is transcribed in the opposite direction to the other four genes . These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P . putida strain TW3. J Bacteriol, 2001 Jan, 183(2), 700 - 8 nag genes of Ralstonia (formerly Pseudomonas) sp . strain U2 encoding enzymes for gentisate catabolism; Zhou NY et al.; Ralstonia sp . strain U2 metabolizes naphthalene via gentisate to central metabolites . We have cloned and sequenced a 21.6-kb region spanning the nag genes . Upstream of the pathway genes are nagY, homologous to chemotaxis proteins, and nagR, a regulatory gene of the LysR family . Divergently transcribed from nagR are the genes for conversion of naphthalene to gentisate (nagAaGHAbAcAdBFCQED) (S . L . Fuenmayor, M . Wild, A . L . Boyes, and P . A . Williams, J . Bacteriol . 180:2522-2530, 1998), which except for the insertion of nagGH, encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of Pseudomonas putida PpG7 . Downstream of nahD is a cluster of genes (nagJIKLMN) which are probably cotranscribed with nagAaGHAbAcAdBFCQED as a single large operon . By cloning into expression vectors and by biochemical assays, three of these genes (nagIKL) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate . NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates . NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate . NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate . The three other genes (nagJMN) have also been cloned and overexpressed, but no biochemical activities have been attributed to them . NagJ is homologous to a glutathione S-transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function . Downstream of the operon is a partial sequence with homology to a transposase. J Bacteriol, 2001 Jan, 183(2), 490 - 9 The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation; Lu CD et al.; The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined . This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions . The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa) . Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits . Comparison of the derived amino acid sequence of NAD-GDH from P . aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus . A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa . Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function . NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate . Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins . NAD-GDH was purified to homogeneity from a recombinant strain of P . aeruginosa and characterized . The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate . NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively . Both effectors act by influencing the affinity of the enzyme for glutamate . NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases. Appl Environ Microbiol, 2001 Jan, 67(1), 453 - 8 Genetic and molecular organization of the alkylbenzene catabolism operon in the psychrotrophic strain Pseudomonas putida 01G3; Chablain PA et al.; The 11-kb sequence encompassing the alkylbenzene upper pathway in Pseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized . Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P . putida RE204 . ebd genes appeared to be preferentially induced by ethylbenzene . Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities . Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster. Appl Microbiol Biotechnol, 2000 Nov, 54(5), 711 - 4 The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance; Isken S et al.; The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically . The mutant P . putida JK1, which lacks the active efflux system, was compared with the wild-type organism . None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type . The amount of {14C}toluene accumulating in P . putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers . The effect of uncouplers confirms the proton dependency of the efflux system in P . putida S12 . Other compounds, potential substrates for the solvent pump, did not affect the accumulation of {14C}toluene . These results show that the efflux system in P . putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps. Appl Microbiol Biotechnol, 2000 Nov, 54(5), 665 - 70 Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway; Hoffmann N et al.; Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6-C14), when grown on simple carbon sources such as, for example, gluconate or acetate . In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG . Southern hybridization experiments with digoxigenin-labeled phaG(Pp) from P . putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads . Although P . oleovorans does not accumulate PHA(MCL) from non-related carbon sources, its genomic DNA reveals a strong hybridization signal . We employed PCR to amplify this phaG homologue . The respective PCR product comprising the coding region of phaG(Po) was cloned into pBBR1MCS-2, resulting in plasmid pBHR84 . DNA sequencing revealed that putative PhaG(Po) from P . oleovorans exhibited about 95% amino acid sequence identity to PhaG(Pp) from P . putida . Reverse transcriptase-PCR analysis demonstrated that phaG(Po) was not transcribed even tinder inducing conditions, i.e . in the presence of gluconate as carbon source, whereas induction of phaG(Pp) transcription was obtained in P . putida . When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P . putida . Plasmid pBHR84 complemented the phaG-negative mutant PhaG(N)-21 from P . putida . Interestingly, reintroduction of phaG(Po) under lac promoter control into the natural host P . oleovorans established PHA(MCL) synthesis from non-related carbon sources in this bacterium . These data indicated that phaG(Po) in P . oleovorans is not functionally expressed and does not exert its original function. Metab Eng, 2000 Oct, 2(4), 339 - 48 Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion; Zhang N et al.; Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone . The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS . To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method . High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format . Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC . Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes . A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis . After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield. J Bacteriol, 2001 Jan, 183(1), 162 - 70 Control of the ferric citrate transport system of Escherichia coli: mutations in region 2.1 of the FecI extracytoplasmic-function sigma factor suppress mutations in the FecR transmembrane regulatory protein; Stiefel A et al.; Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane of Escherichia coli K-12 . Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active . In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of the Pseudomonas aeruginosa, Pseudomonas putida, Bordetella pertussis, Bordetella bronchiseptica, and Caulobacter crescentus genomes . The cytoplasmic portion of the FecR mutant proteins, FecR(1-85), did not interact with wild-type FecI, in contrast to wild-type FecR(1-85), which induced FecI-mediated fecB transport gene transcription . Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of the fecR mutants by ferric citrate . Region 2.1 of sigma(70) is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI . In addition, missense mutations in the fecI promoter region resulted in a twofold increased transcription in fecR wild-type cells and a partial restoration of fec transport gene transcription in the fecR mutants . The mutations reduced binding of the Fe(2+) Fur repressor and as a consequence enhanced fecI transcription . The data reveal properties of the FecI ECF factor distinct from those of sigma(70) and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity. Appl Environ Microbiol, 2000 Dec, 66(12), 5221 - 5 Control of expression of divergent Pseudomonas putida put promoters for proline catabolism; Vilchez S et al.; Pseudomonas putida KT2440 uses proline as the sole C and N source . Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein . Sequence analysis revealed that the putA and putP genes are transcribed divergently . Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline . Expression from the put promoters was induced by proline . The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes . The PutA protein acts as a repressor of put gene expression in P . putida because expression from the put promoters is constitutive in a host background with a knockout putA gene . This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896-->Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity . Expression from the put promoters in the presence of proline in a putA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be sigma(54) dependent because the put genes were not expressed in a sigma(54)-deficient background . Expression of the putA and putP genes was equally high in the presence of proline in sigma(38)- and ihf-deficient P . putida backgrounds. FEMS Microbiol Lett, 2000 Dec 1, 193(1), 123 - 7 Versatile biosensor vectors for detection and quantification of mercury; Hansen LH et al.; Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp . In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively . Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments . The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria . The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil. J Biol Chem, 2001 Feb 23, 276(8), 5700 - 6 Epub 2000 Nov 27. An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress; Wery J et al.; The novel insertion sequence ISS12 plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress . Under normal culturing conditions the P . putida S12 genome contained seven copies of ISS12 . However, a P . putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies . The survival frequency of cells in this variant P . putida S12 population was 1000 times higher than in "normal" P . putida S12 populations . Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene . srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC . SrpABC makes a major contribution to solvent tolerance in P . putida S12 and is induced by toluene . The basal level of srp promoter activity in the P . putida S12 variant was seven times higher than in wild-type P . putida S12 . Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock . These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents . We propose that P . putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions. Acta Crystallogr D Biol Crystallogr, 2000 Dec, 56 Pt 12, 1665 - 7 Crystallization and preliminary crystallographic characterization of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane lyase (methioninase); Sridhar V et al.; L-Methionine-alpha-deamino-gamma-mercaptomethane lyase (rMETase) is involved in the alpha,gamma-elimination of methionine to alpha-ketobutyrate, methanethiol and ammonia . The reaction catalyzed by rMETase reduces the methionine concentration of methionine-dependent tumor cells, arresting their growth . Towards the goal of developing rMETase into an effective antitumor therapeutic and also to understand the catalytic mechanism of this enzyme, rMETase from Pseudomonas putida has been expressed, purified and crystallized . The crystals belong to space group P2(1)2(1)2 and diffract X-rays to at least 2.68 A resolution at 100 K using synchrotron radiation . The unit cell has parameters a = 152.8, b = 154.6, c = 80.8 A and contains four molecules in the asymmetric unit. DNA Seq, 2000, 11(3-4), 193 - 7 Automated sample-preparation technologies in genome sequencing projects; Hilbert H et al.; A robotic workstation system (BioRobot 96OO, QIAGEN) and a 96-well UV spectrophotometer (Spectramax 250, Molecular Devices) were integrated in to the process of high-throughput automated sequencing of double-stranded plasmid DNA templates . An automated 96-well miniprep kit protocol (QIAprep Turbo, QIAGEN) provided high-quality plasmid DNA from shotgun clones . The DNA prepared by this procedure was used to generate more than two mega bases of final sequence data for two genomic projects (Arabidopsis thaliana and Schizosaccharomyces pombe), three thousand expressed sequence tags (ESTs) plus half a mega base of human full-length cDNA clones, and approximately 53,000 single reads for a whole genome shotgun project (Pseudomonas putida). J Mol Biol, 2000 Dec 1, 304(3), 397 - 410 Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase; Senda T et al.; Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others . These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase . Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen . BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp . strain KKS102 . The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria . To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems . In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved . The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family . Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system . Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4 . This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD . Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs . J Bacteriol, 2000 Dec, 182(23), 6707 - 13 Effects of combination of different -10 hexamers and downstream sequences on stationary-phase-specific sigma factor sigma(S)-dependent transcription in Pseudomonas putida; Ojangu EL et al.; The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is sigma(S) . In contrast to other minor sigma factors, RNA polymerase holoenzyme containing sigma(S) (Esigma(S)) recognizes a number of promoters which are also recognized by that containing sigma(70) (Esigma(70)) . We have previously shown that transposon Tn4652 can activate silent genes in starving Pseudomonas putida cells by creating fusion promoters during transposition . The sequence of the fusion promoters is similar to the sigma(70)-specific promoter consensus . The -10 hexameric sequence and the sequence downstream from the -10 element differ among these promoters . We found that transcription from the fusion promoters is stationary phase specific . Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P . putida, the effect of sigma(S) on transcription from the fusion promoters was established only in some of these promoters . The importance of the sequence of the -10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the -10 element can affect sigma(S)-dependent transcription . Combination of the -10 hexameric sequences and downstream sequences of different fusion promoters revealed that sigma(S)-specific transcription from these promoters is not determined by the -10 hexameric sequence only . The results obtained in this study indicate that the sequence of the -10 element influences sigma(S)-specific transcription in concert with the sequence downstream from the -10 box. Mol Microbiol, 2000 Oct, 38(2), 401 - 10 The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida; Garmendia J et al.; In the presence of toluene and other structural analogues, the enhancer binding protein XylR activates the sigma54 promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida . Introduction of amino acid changes Val-219Asp and Ala-220Pro, which enter a proline kink at the interdomain region (B linker) between the A (signal reception) module and the central portion of XylR, originated a protein with unforeseen properties . These included a minor ability to activate Pu in the absence of aromatic effectors, a much higher responsiveness to m-xylene and a significant response to a large collection of aromatic inducers . Such changes could not be attributed to variations in XylR expression levels or to the fortuitous creation of a novel promoter, but to a genuine change in the properties of the activator . Structural predictions suggested that the mutation entirely disrupted an otherwise probable coiled-coil structure . A second directed mutant within the same region consisting of a major replacement of amino acids A220-N221 by the peptide HHHR produced an even more exacerbated phenotype . These data support a model in which the linker B region influences the effector profile by modifying at a distance the operative shape of the effector pocket and fixing the protein in an intermediate step of the activation process. J Mol Biol, 2000 Nov 10, 303(5), 797 - 811 How do substrates enter and products exit the buried active site of cytochrome P450cam? 1 . Random expulsion molecular dynamics investigation of ligand access channels and mechanisms; Ludemann SK et al.; Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics . Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s . However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle . While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations . Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field . The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product . Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data . Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed . The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors . In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations . Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed . J Bacteriol, 2000 Nov, 182(22), 6451 - 5 Novel toluene elimination system in a toluene-tolerant microorganism; Kobayashi H et al.; In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture . These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins . The MVs also contained a higher concentration of toluene molecules (0.172 +/- 0 . 012 mol/mol of lipid) than that found in the cell membrane . In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane . The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000. J Bacteriol, 2000 Nov, 182(22), 6339 - 46 BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida; Cowles CE et al.; Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate . Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded meta pathway operon for benzoate and methylbenzoate degradation . Finally, benzoate represses the ability of P . putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease . Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes . A benR mutant isolated by random transposon mutagenesis was unable to grow on benzoate . The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators . An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences . The benABC genes likely encode benzoate dioxygenase, and benD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase . benK and benF were assigned functions as a benzoate permease and porin, respectively . The possible function of a final gene, benE, is not known . benR activated expression of a benA-lacZ reporter fusion in response to benzoate . It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E . coli chromosome . Third, benR was required for benzoate-mediated repression of pcaK-lacZ fusion expression . The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter . It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate . The action of BenR in repressing 4-HBA uptake is probably indirect. J Med Chem, 2000 Oct 19, 43(21), 3981 - 6 Role of hydrophobic interactions in binding S-(N-aryl/alkyl-N-hydroxycarbamoyl)glutathiones to the active site of the antitumor target enzyme glyoxalase I; Kalsi A et al.; Hydrophobic interactions play an important role in binding S-(N-aryl/alkyl-N-hydroxycarbamoyl)glutathiones to the active sites of human, yeast, and Pseudomonas putida glyoxalase I, as the log K(i) values for these mechanism-based competitive inhibitors decrease linearly with increasing values of the hydrophobicity constants (pi) of the N-aryl/alkyl substituents . Hydrophobic interactions also help to optimize polar interactions between the enzyme and the glutathione derivatives, given that the K(i) value for S-(N-hydroxycarbamoyl)glutathione (pi = 0) with the human enzyme is 35-fold larger than the interpolated value for this compound obtained from the log K(i) versus pi plot . Computational studies, in combination with published X-ray crystallographic measurements, indicate that human glyoxalase I binds the syn-conformer of S-(N-aryl-N-hydroxycarbamoyl)glutathiones in which the N-aryl substituents are in their lowest-energy conformations . These studies provide both an experimental and a conceptual framework for developing better inhibitors of this antitumor target enzyme. FEMS Microbiol Lett, 2000 Sep 15, 190(2), 279 - 85 Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida; Mordukhova EA et al.; Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation . The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied . Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source . No difference was observed in other carbon or nitrogen sources . It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid. Int J Biol Macromol, 2000 Oct 10, 28(1), 23 - 9 Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida; Kim DY et al.; The ability of Pseudomonas putida to synthesize polyhydroxyalkanoate (PHA) from 36 different carboxylic acids containing various functional groups was examined . This bacterium did not utilize short carboxylic acids (C(4)-C(6)) containing bromine, methoxy, ethoxy, cyclohexyl, phenoxy, and olefin groups as the sole carbon substrate . No polymer was isolated from the cells grown with carboxylic acids bearing hydroxyl, amino, para-methoxyphenoxy, and para-ethoxyphenoxy groups regardless of the carbon substrate chain lengths used even when they were cofed with nonanoic acid . Of all the carbon substrates evaluated, only 6-para-methylphenoxyhexanoic acid, 8-para-methylphenoxyoctanoic acid, 8-meta-methylphenoxyoctanoic acid, 10-undecenoic acid, and 10-undecynoic acid supported both growth and the production of PHA containing the corresponding functional groups by P . putida . The present results indicate that the carbon availability of P . putida for growth and PHA production is significantly different from that of P . oleovorans. Ecotoxicol Environ Saf, 2000 Oct, 47(2), 156 - 66 A battery of toxicity tests as indicators of decontamination in composting oily waste; Juvonen R et al.; Heterogeneous oily waste from an old dumping site was composted in three windrows constructed from different proportions of waste, sewage sludge, and bark . The objectives of this pilot study were to examine the usefulness of composting as a treatment method for this particular waste and to study decontamination in the composting process by using a battery of toxicity tests . Five samples from the windrow having intermediate oil concentrations were tested with toxicity tests based on microbes (Pseudomonas putida growth inhibition test, ToxiChromotest, MetPLATE, and three different modifications of a luminescent bacterial test), enzyme inhibition (reverse electron transport), plants (duckweed growth inhibition and red clover seed germination), and soil animals (Folsomia candida, Enchytraeus albidus, and Enchytraeus sp.) . The luminescent bacterial tests were used as prescreening tests . Chemical analyses of samples were carried out simultaneously . Both toxicity and oil concentration, including those of polyaromatic hydrocarbons (PAHs), were reduced during composting and soil quality improved significantly . The total oil hydrocarbon concentration decreased from 90,000 to 19,000 mg/kg, measured with the IR method, in 4 months, and from 86,000 to 1400 mg/kg, measured with GC method . The concentration of PAHs decreased from 135 to 23.5 mg/kg . During the fourth month of composting (stabilization stage), the proportion of the heaviest oil fractions (asphaltenes) became dominant . Toxicity varied between different samples and between different bioassays; however, the first sample was significantly more toxic than the others, and most of the tests revealed a decrease in toxicity during the composting process . Microbiology, 2000 Oct, 146 ( Pt 10), 2565 - 72 Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex; Braun P et al.; The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium . Since expression of the lasB gene, which encodes elastase, in Pseudomonas putida did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secretion machinery of the heterologous host . Here, it is demonstrated that the proenzyme is normally processed in P . putida and that it is indeed not actively secreted by the Xcp machinery . Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium . Co-immunoprecipitations revealed that the extracellular enzyme was associated with the propeptide, which explains the lack of enzymic activity . Since the propeptide-enzyme complex in P . putida apparently does not dissociate spontaneously, it is concluded that a host-specific factor is required to induce this event . Mutants were selected which showed extracellular elastase activity . Two mutations, located within the lasB gene, were further characterized . These mutations, resulting in the substitution of Ala and Thr at positions -15 and -153, respectively, of the propeptide (where position +1 is defined as the first residue of the mature enzyme) destabilized the propeptide-enzyme complex . It is concluded that Ala-15 and Thr-153 are required for the inhibitor function, but not for the chaperone function of the propeptide. Microbiology, 2000 Oct, 146 ( Pt 10), 2555 - 63 Visualization of DNA-protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida; Garmendia J et al.; The ATP-dependent multimerization process undergone by the sigma(54)-dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate PU: promoter was examined by transmission electron microscopy (TEM) . To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylRDeltaA, with or without ATP or its non-hydrolysable analogue ATPgammaS, and the resulting complexes were visualized by TEM . The different types of DNA-protein association were analysed and a statistical study of the frequency of the various forms was made . ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA-protein complexes . The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest . Such complexes often engaged otherwise separated UAS-containing plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans . Whilst ATP caused the different types of XylR-DNA complex to occur at quite balanced frequencies, ATPgammaS appeared to displace the distribution predominantly towards the higher order forms . These data are compatible with the notion that each time ATP is hydrolysed the transcriptional activation complex is disassembled. Biometals, 2000 Jun, 13(2), 147 - 52 A pyoverdine from Pseudomonas putida CFML 90-51 with a Lys epsilon-amino link in the peptide chain; Sultana R et al.; From Pseudomonas putida CFML 90-51--a hospital isolate--a pyoverdine was obtained which is characterized by the unusual linkage by the epsilon--rather than the alpha-amino group of Lys in the peptide chain . The structure elucidation by spectroscopic methods and degradation reactions is reported. Appl Environ Microbiol, 2000 Oct, 66(10), 4575 - 8 Phenylacetyl-coenzyme A is the true inducer of the phenylacetic acid catabolism pathway in Pseudomonas putida U; Garcia B et al.; Aerobic degradation of phenylacetic acid in Pseudomonas putida U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A {CoA} catabolon core) . Induction of this route was analyzed by using different mutants specifically designed for this objective . Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds. Can J Microbiol, 2000 Sep, 46(9), 809 - 16 Development of formulations of biological agents for management of root rot of lettuce and cucumber; Amer GA et al.; The effect of various carrier formulations of Bacillus subtilis and Pseudomonas putida were tested on germination, growth, and yield of lettuce and cucumber crops in the presence of Pythium aphanidermatum and Fusarium oxysporum f.sp . cucurbitacearum, respectively . Survival of B . subtilis and P . putida in various carriers under refrigeration (about 0 degree C) and at room temperature (about 22 degrees C) was also studied . In all carrier formulations, B . subtilis strain BACT-0 survived up to 45 days . After 45 days of storage at room temperature (about 22 degrees C), populations B . subtilis strain BACT-0 were significantly higher in vermiculite, kaolin, and bacterial broth carriers compared with other carriers . Populations of P . putida were significantly higher in vermiculite, peat moss, wheat bran, and bacterial broth than in other carriers when stored either under refrigeration (about 0 degree C) or at room temperature (about 22 degrees C) for 15 or 45 days . Germination of lettuce seed was not affected in vermiculite, talc, kaolin, and peat moss carriers, but germination was significantly reduced in alginate and bacterial broth carriers of B . subtilis compared to the non-treated control . Germination of cucumber seed was not affected by any of the carriers . Significantly higher fresh lettuce and root weights were observed in vermiculite and kaolin carriers of B . subtilis compared with P . aphanidermatum-inoculated control plants . Lettuce treated with vermiculite, and kaolin carriers of B . subtilis, or non-inoculated control lettuce plants had significantly lower root rot ratings than talc, peat moss, bacterial broth, and P . aphanidermatum-inoculated control plants . Growth and yield of cucumber plants were significantly higher in vermiculite-based carrier of P . putida than the other carriers and Fusarium oxysporum f.sp . cucurbitacearum-inoculated plants. Eur J Biochem, 2000 Oct, 267(19), 5926 - 34 Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin; Twilfer H et al.; Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured . These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects . This value favours a concerted oxygenation mechanism of the substrate . Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake . This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent . The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent . With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime . These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates . These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen . Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water . The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate. Genetika, 2000 Jul, 36(7), 915 - 9 {Isolation and comparative study of a group of temperate bacteriophages of rhizospheric pseudomonads Pseudomonas putida}; Shaburova OV et al.; We have isolated several new temperate bacteriophages for rhizosphere pseudomonads Pseudomonas putida . Examination of these phages, along with two previously isolated temperate phages PP56 and PP71 of P . putida PpG1 (biovar A), allowed us to classify them into four species on the basis of DNA cross-homology; relative genomic size; and, to a certain extent, the morphology of phage particles . Two of these species are represented by nonidentical variants . No transposable phages were found among these two new species . Three phage species cause various-types of lysogenic conversion manifested in growth suppression of other phage species . This seems to account for the fact that the temperate phage of rhizosphere pseudomonads are seldom encountered . The new phages described can be used for selection of phage-resistant bacterial forms exhibiting antifungal activity that are commercially produced and used for treatment of seeds of cultivated plants. Biotechnol Bioeng, 2000 Nov 5, 70(3), 291 - 9 Effect of temperature on the inhibition kinetics of phenol biodegradation by Pseudomonas putida Q5; Onysko KA et al.; The temperature-dependent performance of mixed-culture wastewater treatment processes may be strongly influenced by their content of psychrotrophic bacteria . In this work, the effect of temperature on cell growth and phenol biodegradation kinetics of the psychrotrophic bacterium Pseudomonas putida Q5 were determined using both batch and continuous cultures in the range of 10-25 degrees C . The Haldane equation was found to be the most suitable substrate-inhibition model for the specific growth rate . The Haldane parameters mu(max) and K(I) were best modeled by a square-root dependency on temperature . However, the Arrhenius model provided a better prediction of the temperature dependence of K(S) . The variation of the yield constant with temperature also was studied experimentally . Comparisons with results of previous workers are presented . Methods Enzymol, 2000, 324, 329 - 35 Purification of branched-chain keto acid dehydrogenase regulator from Pseudomonas putida; Madhusudhan KT et al.; BkdR can be isolated in nearly pure form as a tetramer by this procedure, which involves hyperexpressing bkdR from a plasmid, purification by chromatography on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, and dialysis to precipitate BkdR . BkdR is relatively insoluble in aqueous buffers but can be kept in solution in buffer with 50% (v/v) glycerol and 0.2 M NaCl . Cultures of E . coli DH5 alpha (pJRS119) should be maintained at 30 degrees to promote plasmid stability . Because BkdR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (v/v) 2-mercaptoethanol. Mol Cells, 2000 Aug 31, 10(4), 475 - 9 Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp . S-47; Noh SJ et al.; Pseudomonas sp . S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage . The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed . The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain . The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids . A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp . CF600 . However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp . KKS102 . The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites . Therefore, Pseudomonas sp . S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains. J Bacteriol, 2000 Oct, 182(19), 5580 - 5 Characterization of three XylT-like {2Fe-2S} ferredoxins associated with catabolism of cresols or naphthalene: evidence for their involvement in catechol dioxygenase reactivation; Hugo N et al.; The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel {2Fe-2S} ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE) . In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE . The three XylT analogues show general spectroscopic characteristics common to plant-type {2Fe-2S} ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins . The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively . Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT . The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide . A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases . The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction . In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P . putida which is unable to grow on p-methylbenzoate . All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE . Our results provide evidence for a subgroup of {2Fe-2S} ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons. J Bacteriol, 2000 Oct, 182(19), 5495 - 504 Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit; Parales RE et al.; The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp . strain NCIB 9816-4 . The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation . Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene . In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates . NDO variants F352W and F352Y were marginally active with all substrates tested . F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation . All active enzymes had altered regioselectivity with biphenyl and phenanthrene . In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol {77 and 60% (-)-(3S,4R), respectively} to that formed by wild-type NDO {>98% (+)-(3R,4S)} . The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36 . A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively. Appl Microbiol Biotechnol, 2000 Aug, 54(2), 186 - 94 Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme; Liebergesell M et al.; The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment . Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions . Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T . pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively . This confirmed that the T . pfennigii PHA synthase was composed of two different subunits . Also, with respect to the molecular organization of phaE and phaC, this region of the T . pfennigii genome resembled very much the corresponding regions of C . vinosum and of Thiocystis violacea . A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T . pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite . The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC . Hybrid PHA synthases composed of PhaE from T . pfennigii and PhaC from C . vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P . putida; and the resulting PHAs were analyzed. Appl Microbiol Biotechnol, 2000 Aug, 54(2), 180 - 5 A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical; Wery J et al.; The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion . For this purpose the genes involved in this conversion, todC1C2BAD from P . putida F1, were introduced into P . putida S12 with high stable expression . Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium-octanol system . The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium-octanol system, irrespective of the selected medium. Appl Environ Microbiol, 2000 Sep, 66(9), 4142 - 4 Anhydrobiotic engineering of gram-negative bacteria; Garcia De Castro A et al.; Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis . This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state. Appl Environ Microbiol, 2000 Sep, 66(9), 4119 - 23 Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions; Hassett DJ et al.; Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs . Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains . More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase . Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity . Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used. J Biochem (Tokyo), 2000 Sep, 128(3), 349 - 54 Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida; Motoshima H et al.; L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine . We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods . The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data . A homotetramer with 222 symmetry is built up by non-crystallographic symmetry . Two monomers associate to build the active dimer . The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli. J Bacteriol, 2000 Sep, 182(18), 5278 - 9 Genetic analysis of an incomplete mutS gene from Pseudomonas putida; Kurusu Y et al.; We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria . This gene is able to function in the mutS mutants of Escherichia coli and Bacillus subtilis . A P . putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain. Biochemistry, 2000 Aug 22, 39(33), 10055 - 65 Role of arginine 277 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate binding and transition state stabilization; Lehoux IE et al.; (S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase . Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group {Lindqvist, Y., Branden, C.-I., Mathews, F . S., and Lederer, F . (1991) J . Biol . Chem . 266, 3198-3207} . The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments . The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms . The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K . The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants . The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value . Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents . Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate . The positive charge has a relatively small contribution toward substrate binding . R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate. J Appl Microbiol, 2000 Jul, 89(1), 40 - 8 Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos; Karpouzas DG et al.; Two strains of Pseudomonas putida (epI and epII), isolated previously from ethoprophos-treated soil, were able to degrade ethoprophos (10 mg 1(-1)) in a mineral salts medium plus nitrogen (MSMN) in less than 50 h with a concurrent population growth . Addition of glucose or succinate to MSMN did not influence the degrading ability of Ps . putida epI, but increased the lag phase before rapid degradation commenced with Ps . putida epII . The degrading ability of the two isolates was lost when the pesticide provided the sole source of phosphorus . Degradation of ethoprophos was most rapid when bacterial cultures were incubated at 25 and 37 degrees C . Pseudomonas putida epI was capable of completely degrading ethoprophos at a slow rate at 5 degrees C, compared with Ps . putida epII which could not completely degrade ethoprophos at the same time . Pseudomonas putida epI was capable of degrading ethoprophos when only 60 cells ml(-1) were used as initial inoculum . In contrast, Ps . putida epII was able to totally degrade ethoprophos when inoculum densities of 600 cells ml(-1) or higher were used . In general, longer lag phases accompanied the lower inoculum levels . Both isolates rapidly degraded ethoprophos in MSMN at pHs ranging from 5.5 to 7.6, but not at pH 5 or below. J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 71 - 8 Marine Bacillus spores as catalysts for oxidative precipitation and sorption of metals; Francis CA et al.; The oxidation of soluble manganese(II) to insoluble Mn(III,IV) oxide precipitates plays an important role in the environment . These Mn oxides are known to oxidize numerous organic and inorganic compounds, scavenge a variety of other metals on their highly charged surfaces, and serve as electron acceptors for anaerobic respiration . Although the oxidation of Mn(II) in most environments is believed to be bacterially-mediated, the underlying mechanisms of catalysis are not well understood . In recent years, however, the application of molecular biological approaches has provided new insights into these mechanisms . Genes involved in Mn oxidation were first identified in our model organism, the marine Bacillus sp . strain SG-1, and subsequently have been identified in two other phylogenetically distinct organisms, Leptothrix discophora and Pseudomonas putida . In all three cases, enzymes related to multicopper oxidases appear to be involved, suggesting that copper may play a universal role in Mn(II) oxidation . In addition to catalyzing an environmentally important process, organisms capable of Mn(II) oxidation are potential candidates for the removal, detoxification, and recovery of metals from the environment . The Mn(II)-oxidizing spores of the marine Bacillus sp . strain SG-1 show particular promise, due to their inherent physically tough nature and unique capacity to bind and oxidatively precipitate metals without having to sustain growth. J Bacteriol, 2000 Sep, 182(17), 4764 - 72 Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability; Llamas MA et al.; The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents . OprL protein of Pseudomonas putida has been shown to be crucial to maintain the stability of this cell component (J . J . Rodriguez-Herva, M.-I . Ramos-Gonzalez, and J . L . Ramos . J . Bacteriol . 178:1699-1706, 1996) . In the present study we cloned and mutagenized the orf1, tolQ, tolR, tolA, and tolB genes from P . putida KT2440, which were located upstream of the oprL gene . Polar and nonpolar mutations of the P . putida tolQ, tolR, tolA, and tolB genes were generated in vitro by using the omega-Km(r) interposon, which carries two transcriptional stop signals, or a promoterless xylE cassette, lacking any transcriptional stop signal, respectively . The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes . The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterized P . putida oprL::xylE mutant . All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility . Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity in P . putida and suggest a possible role of these proteins in assembling outer membrane components. J Bacteriol, 2000 Sep, 182(17), 4711 - 8 In vivo and in vitro effects of (p)ppGpp on the sigma(54) promoter Pu of the TOL plasmid of Pseudomonas putida; Carmona M et al.; The connection between the physiological control of the sigma(54)-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro . To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA . Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter . Overexpression of the relA gene {predicted to increase intracellullar (p)ppGpp levels} did, however, cause a significant gain in Pu activity . Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery . Although we did observe a significant increase in Pu performance through a range of sigma(54)-RNAP concentrations, such an increase never exceeded twofold . The difference between these results and the behavior of the related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of sigma(54)-systems. Metab Eng, 1999 Jan, 1(1), 63 - 74 Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN; Buckland BC et al.; Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp . isolates . Indene metabolism is consistent with monooxygenase and dioxygenase activity . P . putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS . Molecular cloning of P . putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol . Rhodococcus sp . isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity . Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described. Rev Latinoam Microbiol, 1997 Jul-Dec, 39(3-4), 109 - 15 Isolation of Vibrio and Pseudomonas from brown shrimp (Penaeus californiensis Holmes) intestine; Hernandez-Lopez J et al.; Bacteria of the genera Vibrio, Pseudomonas and Aeromonas were isolated from the intestine of apparently healthy brown shrimp (Penaeus californiensis Holmes) cultured in a tidal pond . Species from these genera of bacteria have been reported as shrimp pathogens and have been involved in human gastrointestinal disorders related to seafood consumption . Isolation was done first in Marine broth, then in selective media (TCBS, Cetrimide and MacConkey) . The oxidase negative strains were discarded as insignificant to shrimp culture . The identification of oxidase positive strains was based in morphological and colonial characteristics, biochemical capabilities, and both salinity and temperature tolerance . API 20E system and fatty acid analysis were also included . Three potentially pathogenic bacteria, Vibrio parahaemolyticus, Vibrio furnissii and Pseudomonas putida were isolated and identified from healthy shrimp intestine. Appl Environ Microbiol, 2000 Aug, 66(8), 3357 - 62 Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta; Abadulla E et al.; Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes . Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes . Immobilization of the T . hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives . The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC(50)) of the immobilized enzyme was 85 mM . Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes) . Textile effluents decolorized with T . hirsuta or the laccase were used for dyeing . Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water . However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (DeltaE*) below 1.1 were measured for most dyes. Appl Environ Microbiol, 2000 Aug, 66(8), 3297 - 304 Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons; Dejonghe W et al.; Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation . When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed . These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days . In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil . However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days . With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started . Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils . The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates . This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil . Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon. Appl Environ Microbiol, 2000 Aug, 66(8), 3255 - 61 Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics; Klemba M et al.; The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp . strain P51, has been cloned into a Tn5-based minitransposon . The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance . Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants . The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate . Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy . Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent . Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source . Expression of the tcb chlorocatechol catabolic operon in P . putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates. Appl Microbiol Biotechnol, 2000 Jun, 53(6), 748 - 53 Broad substrate specificity of naphthalene- and biphenyl-utilizing bacteria; Baldwin BR et al.; Although aromatic compounds are most often present in the environment as components of complex mixtures, biodegradation studies commonly focus on the degradation of individual compounds . The present study was performed to investigate the range of aromatic substrates utilized by biphenyl- and naphthalene-degrading environmental isolates and to ascertain the effects of co-occurring substrates during the degradation of mono-aromatic compounds . Bacterial strains were isolated on the basis of their ability to utilize either biphenyl or naphthalene as a sole source of carbon . Growth and transformation assays were conducted on each isolate to determine the range of substrates degraded . One isolate, Pseudomonas putida BP18, was tested for the ability to biodegrade benzene, toluene, ethylbenzene and xylene isomers (BTEX) individually and as components of mixtures . Overall, the results indicate that organisms capable of growth on multi-ring aromatic compounds may be particularly versatile in terms of aromatic hydrocarbon biodegradation . Furthermore, growth and transformation assays performed with strain BP18 suggest that the biodegradation of BTEX and biphenyl by this strain is linked to a catabolic pathway with overlapping specificities . The broad substrate specificity of these environmental isolates has important implications for bioremediation efforts in the field. Rapid Commun Mass Spectrom, 2000, 14(15), 1316 - 20 Isotopic composition of inorganic carbon as an indicator of benzoate degradation by pseudomonas putida: temperature, growth rate and pH effects; Barth JA et al.; Degradation experiments of benzoate by Pseudomonas putida resulted in enzymatic carbon isotope fractionations . However, isotopic temperature effects between experiments at 20 and 30 degrees C were minor . Averages of the last three values of the CO(2) isotopic composition (delta(13)C(CO2(g))) were more