A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl₄) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl₄ dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spe I fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt⁻) phenotype. One cosmid (pT31) complemented the Pdt⁻ phenotype of CTN1 and conferred CCl₄ transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.     

Antimicrobial properties of pyridine-2,6-dithiocarboxylic acid, a metal chelator produced by Pseudomonas spp

Pyridine-2,6-dithiocarboxylic acid (pdtc) is a metal chelator produced by Pseudomonas spp. It has been shown to be involved in the biodegradation of carbon tetrachloride; however, little is known about its biological function. In this study, we examined the antimicrobial properties of pdtc and the mechanism of its antibiotic activity. The growth of Pseudomonas stutzeri strain KC, a pdtc-producing strain, was significantly enhanced by 32 microM pdtc. All nonpseudomonads and two strains of P. stutzeri were sensitive to 16 to 32 microM pdtc. In general, fluorescent pseudomonads were resistant to all concentrations tested. In competition experiments, strain KC demonstrated antagonism toward Escherichia coli. This effect was partially alleviated by 100 microM FeCl3. Less antagonism was observed in mutant derivatives of strain KC (CTN1 and KC657) which lack the ability to produce pdtc. A competitive advantage was restored to strain CTN1 by cosmid pT31, which restores pdtc production. pT31 also enhanced the pdtc resistance of all pdtc-sensitive strains, indicating that this plasmid contains elements responsible for resistance to pdtc. The antimicrobial effect of pdtc was reduced by the addition of Fe(III), Co(III), and Cu(II) and enhanced by Zn(II). Analyses by mass spectrometry determined that Cu(I):pdtc and Co(III):pdtc2 form immediately under our experimental conditions. Our results suggest that pdtc is an antagonist and that metal sequestration is the primary mechanism of its antimicrobial activity. It is also possible that Zn(II), if present, may play a role in pdtc toxicity.     

Identification of siderophores of Pseudomonas stutzeri

We have identified two types of siderophores produced by Pseudomonas, one of which has never before been found in the genus. Twelve strains of Pseudomonas stutzeri belonging to genomovars 1, 2, 3, 4, 5, and 9 produced proferrioxamines, the hydroxamate-type siderophores. Pseudomonas stutzeri JM 300 (genomovar 7) and DSM 50238 (genomovar 8) and Pseudomonas balearica DSM 6082 produced amonabactins, catecholate-type siderophores. The major proferrioxamines detected were the cyclic proferrioxamines E and D2. Pseudomonas stutzeri KC also produced cyclic (X1 and X2) and linear (G1 and G2a-c) proferrioxamines. Our data indicate that the catecholate-type siderophores belong to amonabactins P 750, P 693, T 789, and T 732. A mutant of P. stutzeri KC (strain CTN1) that no longer produced the secondary siderophore pyridine-2,6-dithiocarboxylic acid continued to produce all other siderophores in its normal spectrum. Siderophore profiles suggest that strain KC (genomovar 9) belongs to the proferrioxamine-producing P. stuzeri. Moreover, a putative ferrioxamine outer membrane receptor gene foxA was identified in strain KC, and colony hybridization showed the presence of homologous receptor genes in all P. stutzeri and P. balearica strains tested.     

Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride

Under iron-limiting conditions, Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CT) to CO₂ and nonvolatile products when activated by reduction at cell membranes. Pseudomonas fluorescens and other cell types activate the factor. Triparental mating was used to generate kanamycin-resistant lux::Tn5 recombinants of strain KC. Recombinants were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous ¹⁴C-carbon tetrachloride. CT⁺ recombinants generated nonvolatile ¹⁴C-labeled products, but four CT^– recombinants did not generate significant nonvolatile ¹⁴C-labeled products and had lost the ability to degrade carbon tetrachloride. When colonies of P. fluorescens were grown next to colonies of CT⁺ recombinants and were exposed to gaseous ¹⁴C-carbon tetrachloride, ¹⁴C-labeled products accumulated around the P. fluorescens colonies, indicating that the factor secreted by CT⁺ colonies had diffused through the agar and become activated. When P. fluorescens was grown next to CT^– colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor. Expression of lux reporter genes in three of the CT^– mutants was regulated by added iron and was induced under the same iron-limiting conditions that induce carbon tetrachloride transformation in the wild-type. Received: 23 November 1998 / Accepted: 15 March 1999     

Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis(thiocarboxylate)

Pseudomonas stutzeri strain KC was originally characterized as having, under iron-limiting conditions, novel carbon tetrachloride (CCl(4)) dehalogenation activity, specifically, a net conversion of CCl(4) to CO(2). The exact pathway and reaction mechanisms are unknown, but chloroform is not an intermediate and thiophosgene and phosgene have been identified as intermediates in trapping experiments. Previous work by others using cell-free preparations has shown that cell-free culture supernatants that have been passed through a low-molecular-weight cutoff membrane can confer rapid CCl(4) transformation ability upon cultures of bacteria which otherwise show little or no reactivity toward CCl(4). We used a cell-free assay system to monitor the complete purification of compounds showing CCl(4) degradation activity elaborated by iron-limited cultures of strain KC. Electrospray tandem mass spectroscopy, NMR spectroscopy, and comparisons with synthetic material have identified pyridine-2,6-bis(thiocarboxylate) as a metabolite of strain KC which has CCl(4) transformation activity in the presence of chemical reductants, e.g., titanium[III] citrate or dithiothreitiol, or actively growing bacterial cultures.     

Transformation of carbon tetrachloride via sulfur and oxygen substitution by Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC transforms carbon tetrachloride into carbon dioxide and nonvolatile products, without chloroform as an intermediate. To define the pathway for hydrolysis, nonvolatile products were analyzed. Condensation products containing the carbon atom of carbon tetrachloride as carbonyl and thioxo moieties were identified, indicating the intermediacy of phosgene and thiophosgene in the pathway.     

Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities.     

Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions.

A denitrifying Pseudomonas sp. (strain KC) capable of transforming carbon tetrachloride (CT) was isolated from groundwater aquifer solids. Major products of the transformation of 14C-labeled CT by Pseudomonas strain KC under denitrification conditions were 14CO2 and an unidentified water-soluble fraction. Little or no chloroform was produced. Addition of dissolved trace metals, notably, ferrous iron and cobalt, to the growth medium appeared to enhance growth of Pseudomonas strain KC while inhibiting transformation of CT. It is hypothesized that transformation of CT by this organism is associated with the mechanism of trace-metal scavenging.     

Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions

A denitrifying Pseudomonas sp. (strain KC) capable of transforming carbon tetrachloride (CT) was isolated from groundwater aquifer solids. Major products of the transformation of 14C-labeled CT by Pseudomonas strain KC under denitrification conditions were 14CO2 and an unidentified water-soluble fraction. Little or no chloroform was produced. Addition of dissolved trace metals, notably, ferrous iron and cobalt, to the growth medium appeared to enhance growth of Pseudomonas strain KC while inhibiting transformation of CT. It is hypothesized that transformation of CT by this organism is associated with the mechanism of trace-metal scavenging.     

Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities.     

Plasmid-encoded mercury resistance in a Pseudomonas stutzeri strain that degrades o-xylene

Abstract A Pseudomonas stutzeri strain, previously isolated for its ability to utilize o -xylene, bears a plasmid, pPB, of about 80 kbp. pPB was found to encode resistance to mercuric chloride and organomercury compounds. Loss of the plasmid resulted in a simultaneous loss of the metal resistance, but not of the ability to degrade o -xylene. Transfer of the Hg^r phenotype to an Hg^s strain was achieved by mobilizing pPB with RP4. Mercury reductase activity was induced by mercuric chloride and by phenylmercuric acetate and Thimerosal. pPB may be considered a broad spectrum resistance plasmid.     

Complete nucleotide sequence and evolutionary significance of a chromosomally encoded naphthalene-degradation lower pathway from Pseudomonas stutzeri AN10

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced the entire naphthalene-degradation lower pathway of P. stutzeri AN10, this being, together with the upper-pathway reported previously (Bosch R. et al., 1999a. Gene 236, 149-157) the first complete DNA sequence for an entire naphthalene-catabolic pathway. Eleven open reading frames were identified. The nahGTHINLOMKJ genes encode enzymes for the metabolism of salicylate to pyruvate and acetyl-CoA, and nahR encodes the NahR regulatory protein. Our findings suggest that catabolic modules were recruited through transposition events and recombination among tnpA-like genes, and subsequent rearrangements and deletions of non-essential DNA fragments allowed the formation of the actual catabolic pathway. Our results also suggest that the genes encoding the xylene/toluene-degradation enzymes of P. putida mt-2 (pWW0) have coexisted with the nah genes of the P. stutzeri AN10 ancestral genome. This could allow the selection, via recombination events among homologous genes, for a combination of genes enabling the metabolism of a given aromatic compound in the ancestral host strain. Such events accelerate the evolution of modern catabolic pathways and provide new genetic material to the environment, ultimately resulting in improved, natural, bioremediation potential.     

Molecular characterization of a novel ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215

Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.     

Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

We have worked out a system to obtain mutations that map in the promoter region of the Escherichia coli galactose operon. In order to easily detect small changes in gal promoter activity, we constructed a plasmid containing an operon fusion in which the lactose operon structural genes were controlled by the galactose operon promoter region. In cells harbouring this plasmid, even modest variations in the expression of the lac genes could be detected on MacConkey lactose indicator plates.Enrichment for mutations that map in the promoter segment of the galactose operon was achieved by mutagenesis in vitro of a small fragment of DNA covering the promoter region. After insertion of the mutagenized gal promoter fragment into the gal-lac fusion plasmid, lac^?1 cells were transformed and screened for an altered Lac⁺ phenotype on indicator plates. Several mutants were isolated due to lesions mapping in the small fragment covering the galactose promoter. In these mutants, the level of β-galactosidase was between 15 and 50% of the wild-type level.The mutant promoters were subsequently reinserted into a plasmid containing the intact galactose operon. Cells harbouring such plasmids, reconstituted with mutant galactose promoters, contained decreased levels of galactokinase that paralleled the decreases in β-galactosidase. The biochemical properties of these mutants are reported in the accompanying paper (Busby et al., 1982).     

Complete genome sequence of the type strain Pseudomonas stutzeri CGMCC 1.1803

Here we report the complete genome sequence of Pseudomonas stutzeri strain CGMCC 1.1803 (equivalent to ATCC 17588), the type strain of P. stutzeri, which encodes 4,138 open reading frames on a 4,547,930-bp circular chromosome. The CGMCC 1.1803 genome contains genes involved in denitrification, benzoate/catechol degradation, chemotaxis, and other functions.