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Showing papers on "Pseudomonas putida published in 1998"


Journal ArticleDOI
TL;DR: By sequentially adding community members, induction of Pm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida, and this combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.
Abstract: Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of an Acinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and the meta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in P. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the Pu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pm promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of Pm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

338 citations


Journal ArticleDOI
TL;DR: The data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmids establishment in the biofilm community.
Abstract: Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacIq gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.

295 citations


Journal ArticleDOI
TL;DR: Heterologous expression of phaG in Pseudomonas oleovorans enabled PHA synthesis on gluconate as the carbon source, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

278 citations


Journal ArticleDOI
TL;DR: The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins known to be involved in proton-dependent multidrug efflux systems, opening up the possibilities of using the solvent efflux system to construct bacterial strains capable of performing biocatalytic transformations of insoluble substrates in two-phase aqueous:organic medium.

262 citations


Journal ArticleDOI
TL;DR: The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes and the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase.
Abstract: The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-′phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.

246 citations


Journal ArticleDOI
TL;DR: Using the nucleotide sequences of the genes for 16S RNA, DNA gyrase B subunit (gyrB) and RNA polymerase delta 70 factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments, phylogenetic analysis of 20 Pseudomonas strains showed the necessity for the reclassification of P. putida biovar B strains.
Abstract: Phylogenetic analysis of 20 Pseudomonas strains (Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas chlororaphis) was conducted by using the nucleotide sequences of the genes for 16S RNA, DNA gyrase B subunit (gyrB) and RNA polymerase delta 70 factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments. On the basis of gyrB and rpoD sequences, these strains were split into two major clusters: one including the type strain of P. putida and all biovar A strains and the other including all P. putida biovar B strains, P. fluorescens stains and the P. chlororaphis strain. In the phylogenetic tree reconstructed from the 16S rRNA sequences included variable regions, P. Putida biovar A and B strains were not separated into two independent clusters, whereas in the phylogenetic tree reconstructed from the 16S rRNA sequences excluding the variable region sequences, these strains were separated into P. putida biovar A and biovar B clusters. The pairwise distances estimated from the variable regions of 16S rRNA correlated poorly with the synonymous distances estimated from the gyrB and rpoD genes. On the other hand, a highly significant correlation was observed between the pairwise distances estimated from the non-variable regions of 16S rRNA and the synonymous distances from gyrB and rpoD genes. Consequently, only the 16S rRNA sequences in the non-variable regions should be used for the phylogenetic analysis. The gyrB and rpoD analyses showed the necessity for the reclassification of P. putida biovar B strains.

241 citations


Journal ArticleDOI
TL;DR: This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).
Abstract: Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a β-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

241 citations


Journal ArticleDOI
TL;DR: Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp.
Abstract: Polyhydroxyalkanoates (PHAs) are accumulated in various bacteria as intracellular carbon and energy storage material under nutrient-limited conditions (3, 26, 30) These bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics, since they are biodegradable thermoplastics More than 90 different constituent monomer units have been found (47) The PHA-producing bacteria can be broadly divided into two groups according to the number of carbon atoms in the monomeric units of the PHAs produced (44) One group of bacteria, including Ralstonia eutropha (formerly Alcaligenes eutrophus), produces short chain length PHAs with C3 to C5 monomer units, while the other group, including Pseudomonas oleovorans, produces medium chain length PHAs with C6 to C14 monomer units (3, 43) Although the majority of bacteria accumulate either short chain length PHA or medium chain length PHA, several bacteria have been found to synthesize polyesters containing both short and medium chain length 3-hydroxyalkanoic acids (3HA) The bacteria Rhodospirillum rubrum (4), Rhodocyclus gelatinosus (27), and Rhodococcus sp (13) produced terpolyesters consisting of 3HA units of C4, C5, and C6 from hexanoate Aeromonas caviae produced a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (6, 8, 38) Pseudomonas strain GP4BH1 produced PHA containing 3HB and 3-hydroxyoctanoate (3HO) from octanoate and PHA containing 3HB, 3HO, and 3-hydroxydecanoate (3HD) from gluconate (46) In this bacterium, a polymer blend was suggested to be synthesized rather than a copolymer A recombinant strain of P oleovorans expressing R eutropha poly(3HB) [P(3HB)] biosynthesis genes has been shown to synthesize a blend of a P(3HB) homopolymer and a copolymer of 3HHx and 3HO units when grown on octanoate (49) Both polyesters were stored as separated granules within the cells (32) In addition, Pseudomonas fluorescens and several other Pseudomonas strains were found to produce a poly(3HB-co-3HA) [P(3HB-co-3HA)] copolymer consisting of 3HA units of C4 to C12 from 3HB and 1,3-butanediol (25) Although Thiocapsa pfennigii accumulated only a P(3HB) homopolymer from various carbon sources, a recombinant P putida strain harboring the PHA synthesis genes of T pfennigii produced a P(3HB-co-3HHx-co-3HO) terpolymer from octanoate (28) We have reported that Pseudomonas sp strain 61-3 isolated from soil produces a blend of a P(3HB) homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3HA units of C4 to C12 from sugars and alkanoic acids (1, 19, 20) In addition, two different types of polyester granules were formed in the same cell (9, 21) This suggests that Pseudomonas sp strain 61-3 possesses two types of polyester synthases with different substrate specificities, that is, polyhydroxybutyrate (PHB) synthase and PHA synthase, specific for 3HB and 3HA units ranging from C4 to C12, respectively In this study, we cloned and sequenced the P(3HB) biosynthesis genes, as well as the P(3HB-co-3HA) biosynthesis genes, of Pseudomonas sp strain 61-3 The substrate specificity of each polyester synthase was evaluated by heterologous expression in PHA-negative mutants of P putida and R eutropha In addition, we found that the phbRPs gene product exhibits significant similarity to the AraC/XylS family of transcriptional activators and report that it is a positive regulatory protein that controls the expression of the P(3HB) biosynthesis operon

233 citations


Journal ArticleDOI
TL;DR: A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8, which was used to examine the induction of thetod operon when exposed to benzene, toluene, ethylbenzene, and xylene compounds and aqueous solutions of JP-4 jet fuel constituents.
Abstract: A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8. This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 micrograms/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 micrograms/liter. There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene. The transposon insertion was stable and had no negative effect on cell growth.

208 citations


Journal ArticleDOI
TL;DR: Comparing to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher, and aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.
Abstract: Conjugal plasmid transfer was examined on the phylloplane of bean (Phaseolus vulgaris) and related to the spatial distribution pattern and metabolic activity of the bacteria. The donor (Pseudomonas putida KT2442) harbored a derivative of the TOL plasmid, which conferred kanamycin resistance and had the gfp gene inserted downstream of a lac promoter. A chromosomal insertion of lacIq prevented expression of the gfp gene. The recipient (P. putida KT2440) had a chromosomal tetracycline resistance marker. Thus, transconjugants could be enumerated by plating and visualized in situ as green fluorescent cells. Sterile bean seedlings were inoculated with donors and recipients at densities of approximately 105 cells per cm2. To manipulate the density and metabolic activity (measured by incorporation of [3H]leucine) of the inoculated bacteria, plants were grown at various relative humidities (RH). At 100% RH, the transconjugants reached a density of 3 × 103 CFU/cm2, corresponding to about one-third of the recipient population. At 25% RH, numbers of transconjugants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after 96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.

183 citations


Journal ArticleDOI
TL;DR: The capability of such a bacterial biosensor to accurately measure environmental contaminants is demonstrated and a potential for its inexpensive application in field-ready assays is suggested.
Abstract: A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, Pu. A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and Pu. When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K1/2 values ranging from 39.0 +/- 3.8 microM for 3-xylene to 2,690 +/- 160 microM for 3-methylbenzylalcohol (means +/- standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.

Journal ArticleDOI
TL;DR: Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) and expression of the gfpgene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater.
Abstract: Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385-390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacIq). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 x 10(-6) to 2.2 x 10(-4) transconjugants per recipient were recorded after 3 days of incubation.

Journal ArticleDOI
TL;DR: The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase fromPseudomonas putida, and was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.
Abstract: l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Journal ArticleDOI
TL;DR: In this article, the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of Pseudomonas putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence.
Abstract: Analysis of chlorobenzene-degrading transconjugants of Pseudomonas putida F1 which had acquired the genes for chlorocatechol degradation (clc) from Pseudomonas sp. strain B13 revealed that the clc gene cluster was present on a 105-kb amplifiable genetic element (named the clc element). In one such transconjugant, P. putida RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb clc element occurred in two different loci, and the target sites were located within the 3' end of glycine tRNA structural genes. Tandem amplification of the clc element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the clc element. Two nonadjacent copies of the clc element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate. This result suggests that the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of P. putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene.

Journal ArticleDOI
TL;DR: Analysis of Tn5 mutagenesis and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.
Abstract: To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10−6). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.

Journal ArticleDOI
TL;DR: Although the solvent efflux pump in P. putida S12 is a member of the resistance-nodulation-cell division family of transporters, the srpABC genes were not induced by antibiotics or heavy metals.
Abstract: Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the srpABC genes include aromatic and aliphatic solvents and alcohols. General stress conditions such as pH, temperature, NaCl, or the presence of organic acids did not induce srp transcription. Although the solvent efflux pump in P. putida S12 is a member of the resistance-nodulation-cell division family of transporters, the srpABC genes were not induced by antibiotics or heavy metals. Several Pseudomonas putida strains possess an intrinsic resistance to a wide variety of structurally unrelated hydrophobic solvents (1, 8, 20, 29) that are lethal for most other gramnegative bacteria. The susceptibility of bacteria to hydrophobic solvents is due to the accumulation of these compounds in the membrane (25, 26), causing an adverse effect on its physicochemical properties. Solvent-resistant bacteria are able to counterbalance these effects through a variety of mechanisms mostly affecting the lipid content of the cell membrane: isomerizing cis-unsaturated fatty acids to the more rigid trans-unsaturated fatty acids (4, 5, 7), changing the head group composition of membrane fatty acids (28), increasing the phospholipid content (20), or increasing the basal rate of phospholipid synthesis (19). These adaptations of the membrane are static, acting as a physical but still permeable barrier, and cannot explain the exceptional resistance of some P. putida strains to organic solvents. Therefore, it was anticipated that a dynamic system for exporting a broad range of structurally unrelated organic solvents from the bacterial membrane had to play an essential role in solvent tolerance (28). Such an efflux system was indeed identified in P. putida S12 by means of an assay based on radiolabeled toluene (9). The genes (srpABC) for this solvent efflux system were subsequently cloned, sequenced, and shown to impart the solvent-resistant phenotype to solvent-sensitive P. putida strains (12). This efflux system shows strong homology to those of the resistance-nodulation-cell division family of transporters known to be involved in the extrusion of hydrophobic antibiotics, dyes, detergents, bile salts, heavy metals, and fatty acids from the membrane (17, 18). Recently, several researchers also reported the involvement of active organic solvent efflux in solvent-resistant strains of Pseudomonas (2, 13, 14, 22). Induction in Pseudomonas species of efflux systems involved in either multidrug resistance or solvent tolerance has not been studied in detail.

Journal ArticleDOI
TL;DR: DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoB gene of autotrophic ammonia oxidizers.
Abstract: The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N2O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C2H2, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers.

Journal ArticleDOI
TL;DR: The resultant rpoS mutant of P.putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol.
Abstract: A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strain E. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter, bolAp1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of the nlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele of rpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultant rpoS mutant of P.putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein of P. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

Journal ArticleDOI
TL;DR: Resistance to the toxic compound potassium tellurite (Telr) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release.
Abstract: Resistance to the toxic compound potassium tellurite (Telr) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release. In this study, the activated Telr determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors. In one case, the Telr determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons. In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets. Either in the plasmid or in the transposon vector, the Telr marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes. This allowed construction of antibiotic resistance-free but selectable P. putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway.

Journal ArticleDOI
TL;DR: An antifreeze protein secreted to the growth medium by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was purified to apparent homogeneity and displays a low level of ice-nucleation activity.
Abstract: An antifreeze protein secreted to the growth medium by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was purified to apparent homogeneity. The purified protein has a molecular...

Journal ArticleDOI
TL;DR: Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.
Abstract: Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26.5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical at the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (34.3 kDa), encoded by two genes, vanA and vanB, are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.

Journal ArticleDOI
TL;DR: Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides and could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.
Abstract: Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.

Journal ArticleDOI
TL;DR: Data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ, and suggests that it may have played a role in the adaptation of this microbial community to the coal tar contamination at the study site.
Abstract: The presence of a highly conserved nahAc allele among phylogenetically diverse bacteria carrying naphthalene-catabolic plasmids provided evidence for in situ horizontal gene transfer at a coal tar-contaminated site (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330-2337, 1997). The objective of the present study was to identify and characterize the different-sized naphthalene-catabolic plasmids in order to determine the probable mechanism of horizontal transfer of the nahAc gene in situ. Filter matings between naphthalene-degrading bacterial isolates and their cured progeny revealed that the naphthalene-catabolic plasmids were self-transmissible. Limited interstrain transfer was also found. Analysis of the restriction fragment length polymorphism (RFLP) patterns indicated that catabolic plasmids from 12 site-derived isolates were closely related to each other and to the naphthalene-catabolic plasmid (pDTG1) of Pseudomonas putida NCIB 9816-4, which was isolated decades ago in Bangor, Wales. The similarity among all site-derived naphthalene-catabolic plasmids and pDTG1 was confirmed by using the entire pDTG1 plasmid as a probe in Southern hybridizations. Two distinct but similar naphthalene-catabolic plasmids were retrieved directly from the microbial community indigenous to the contaminated site in a filter mating by using a cured, rifampin-resistant site-derived isolate as the recipient. RFLP patterns and Southern hybridization showed that both of these newly retrieved plasmids, like the isolate-derived plasmids, were closely related to pDTG1. These data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ. The pervasiveness and persistence of this naphthalene-catabolic plasmid suggest that it may have played a role in the adaptation of this microbial community to the coal tar contamination at our study site.

Journal ArticleDOI
TL;DR: The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases, and exhibited great sensitivity to temperatures above 40 degrees C.
Abstract: A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native M-r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 +/- 10 kDa, indicating a homotetrameric enzyme structure (4 x 33.4 kDa). The pi of the enzyme was estimated to be 7.1 +/- 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases, In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40 degrees C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

Journal ArticleDOI
Douglas E. Dennis1, M McCoy1, A Stangl1, Henry E. Valentin1, Z Wu2 
TL;DR: The acetoacetyl-CoA reductase and the polyhydroxyalkanoate synthase from R. eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described.

Journal ArticleDOI
TL;DR: All tested compounds, regardless of their probe size, polarity, or the linker binding them to the siderophore analogue, promote growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the added benefit of signaling microbial activity by fluorescence emission.
Abstract: Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled via piperazine with various fluorescent markers at a site not interfering with iron binding or receptor recognition (compounds 10-12). These iron carriers were built from a tetrahedral carbon symmetrically extended with three strands, each containing an amino acid (G = glycyl, A = alanyl, L = leucyl and P = phenylalanyl) and terminated by a hydroxamic acid, which together define an octahedral iron-binding domain. A fourth exogenous strand provided the site for connecting various fluorescent markers via a short bifunctional linker. Iron(III) titrations, along with fluorescence spectroscopy, generated quenching of fluorescence emission of some of the probes used. The quenching process fits the Perrin model which reinforces the intramolecular quenching process, postulated previously.1 All tested compounds, regardless of their probe size, polarity, or the linker binding them to the siderophore analogue, promote growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the added benefit of signaling microbial activity by fluorescence emission. All G derivatives of compounds 10-12 were found to parallel the behavior of natural ferrichrome, whereas A derivatives mediated only a modest iron(III) uptake by P. putida. Incubation of various Pseudomonas strains with iron(III)-loaded G derivatives resulted in the build-up of the labels' fluorescence in the culture medium to a much larger extent than from the corresponding A derivatives. The fluorescence buildup corresponds to iron utilization by the cells and the release of the fluorescent labeled desferrisiderophore from the cell to the media. The fact that the microbial activity of these compounds is not altered by attachment of various fluorescent markers via a bifunctional linker proposes their application as diagnostic tools for detecting and identifying pathogenic microorganisms.

Journal ArticleDOI
TL;DR: The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes from Pseudomonas strain B13 were investigated in activated-sludge microcosms and the effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.
Abstract: The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (clc) from Pseudomonas sp. strain B13 were investigated in activated-sludge microcosms. When the clc genes were transferred into an appropriate recipient bacterium such as Pseudomonas putida F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10−8 per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB+) transconjugants of strain F1 which had acquired the clc genes were detected but only when strain B13 cell densities of more than 105 CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB+ transconjugants reached final cell densities of between 102 and 103 CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB+ transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the clc genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.

Journal ArticleDOI
TL;DR: The view that binding of σ54‐RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of ρ54‐ RNAP for the −12/−24 site is supported.
Abstract: The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.

Journal ArticleDOI
TL;DR: Glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms and the only example to date in which 3D domain swapping can be regulated by a small organic ligand.
Abstract: 3D domain swapping of proteins involves the interconversion of a monomer containing a single domain−domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderstrom, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386−3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993−999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat ∼500 ± 100 s-1; KM ∼0.4 ± 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat ∼115 ± 40 s-1; KM ∼1.4 ± 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a si...

Journal ArticleDOI
TL;DR: Results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist.
Abstract: Research on the biodegradation of chiral xenobiotics can provide a better understanding of the processes that govern enantioselectivity in the microbial degradation and environmental fate of stereoisomers. We have examined the microbial degradation of the widely used chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by a pure bacterial strain that was isolated from soil. This strain, Sphingomonas herbicidovorans MH, can utilize both enantiomers of mecoprop as sole carbon and energy sources for growth (24). Growth experiments with the pure enantiomers as well as with racemic mecoprop revealed that the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. Based on these results, we concluded that specific catabolic enzymes must be involved in the degradation of each enantiomer of mecoprop (24). This view was confirmed by the finding that two α-ketoglutarate-dependent dioxygenases are involved in the enantioselective degradation of mecoprop in S. herbicidovorans MH, one that is specific for (R)-mecoprop and one that is specific for (S)-mecoprop (17). The (S) enantiomer-specific dioxygenase activity is constitutively expressed, whereas the (R) enantiomer-specific enzyme activity is present only when cells grow on the (R) enantiomer. Both enzymes are present in cells grown on the racemic mixture. Extracts of cells grown on complex medium and extracts of cells grown on (S)-mecoprop contain equally high activities of the (S) enantiomer-specific dioxygenase (17). However, intact cells grown on complex medium show very low oxygen uptake rates with (S)-mecoprop, whereas (S)-mecoprop-grown cells show high oxygen consumption with (S)-mecoprop (24). These findings suggested the existence of specific uptake systems and led us to examine the uptake of phenoxyalkanoic acids by S. herbicidovorans after growth on various substrates. Many aromatic compounds are taken up by bacteria through energy-dependent transport systems (4, 5, 10, 11, 15, 16). The uptake of 4-chlorobenzoate by the coryneform bacterium NTB-1 is inducible and coupled to the proton motive force (4). The uptake of 4-toluene sulfonate by Comamonas testosteroni T-2 is also inducible and shows substrate saturation kinetics (11). Transport of 4-toluene sulfonate is inhibited completely by uncouplers but only marginally by ATPase inhibitors, and the authors proposed that 4-toluene sulfonate is taken up by an inducible secondary proton symport system (11). The uptake of 4-hydroxybenzoate by Pseudomonas putida is driven by the proton motive force, and the gene that encodes a 4-hydroxybenzoate transporter was identified, cloned, and expressed in Escherichia coli. Furthermore, it was found that 4-hydroxybenzoate transport is an integral feature of the β-ketoadipate pathway in P. putida (5, 15, 16). The uptake of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) by Ralstonia eutropha JMP134 is inducible, is sensitive to metabolic inhibitors, and shows substrate saturation kinetics (10). Moreover, the authors identified a gene, designated tfdK, whose translation product shows resemblance to transport proteins of the major facilitator superfamily, and they suggested that tfdK encodes the 2,4-D transporter of R. eutropha JMP134. Our results indicate that S. herbicidovorans MH has two distinct uptake systems for chiral phenoxypropionic acid herbicides, one for the (R) enantiomer and another one for the (S) enantiomer of dichlorprop and mecoprop. Furthermore, evidence was obtained that 2,4-D was taken up by a third transport system.