Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem.

A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.     

Selection of clc, cba, and fcb chlorobenzoate-catabolic genotypes from groundwater and surface waters adjacent to the Hyde park, Niagara Falls, chemical landfill

The frequency of isolation of three nonhomologous chlorobenzoate catabolic genotypes (clc, cba, and fcb) was determined for 464 isolates from freshwater sediments and groundwater in the vicinity of the Hyde Park industrial landfill site in the Niagara watershed. Samples were collected from both contaminated and noncontaminated sites during spring, summer, and fall and enriched at 4, 22, or 32 degrees C with micromolar to millimolar concentrations of chlorobenzoates and 3-chlorobiphenyl (M. C. Peel and R. C. Wyndham, Microb. Ecol: 33:59-68, 1997). Hybridization at moderate stringency to restriction-digested genomic DNA with DNA probes revealed the chlorocatechol 1,2-dioxygenase operon (clcABD), the 3-chlorobenzoate 3,4-(4,5)-dioxygenase operon (cbaABC), and the 4-chlorobenzoate dehalogenase (fcbB) gene in isolates enriched from all contaminated sites in the vicinity of the industrial landfill. Nevertheless, the known genes were found in less than 10% of the isolates from the contaminated sites, indicating a high level of genetic diversity in the microbial community. The known genotypes were not enriched from the noncontaminated control sites nearby. The clc, cba, and fcb isolates were distributed across five phenotypically distinct groups based on Biolog carbon source utilization, with the breadth of the host range decreasing in the order clc > cba > fcb. Restriction fragment length polymorphism (RFLP) patterns showed that the cba genes were conserved in all isolates whereas the clc and fcb genes exhibited variation in RFLP patterns. These observations are consistent with the recent spread of the cba genes by horizontal transfer as part of transposon Tn5271 in response to contaminant exposure at Hyde Park. Consistent with this hypothesis, IS1071, the flanking element in Tn5271, was found in all isolates that carried the cba genes. Interestingly, IS1071 was also found in a high proportion of isolates from Hyde Park carrying the clc and fcb genes, as well as in type strains carrying the clcABD operon and the biphenyl (bph) catabolic genes.     

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate.

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.     

Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Distribution of the catabolic transposon Tn5271 in a groundwater bioremediation system.

The distribution of Tn5271-related DNA sequences in samples of groundwater and a groundwater bioremediation system at the Hyde Park (Niagara Falls, N.Y.) chemical landfill site was investigated. PCR amplification of target sequences within the cha genes of Tn5271 revealed similar sequences in the groundwater community and in samples from the sequencing batch reactors treating that groundwater. Cell dilution combined with PCR amplification indicated that cha sequences were carried in about 1 of 10 culturable bacteria from the treatment system. Characterization of isolates involved in chlorobenzoate and toluene biodegradation in the treatment system indicated that two phenotypic clusters, Alcaligenes faecalis type 2 and CDC group IVC-2, contained all of the Tn5271 probe-positive isolates from the community. These two groups differed phenotypically from recipient groups isolated following horizontal transfer of pBRC60 (Tn5271) in pristine freshwater microcosms. A genetic rearrangement in Tn5271 attributable to the intramolecular transposition of the flanking element IS1071R was detected in an isolate from the treatment system. Comparison of the structure of the intramolecular transposition derivative from groundwater isolate OCC13(pBRC13) with a laboratory-derived intramolecular transposition derivative of pBRC60 revealed similarities. The rearrangement was shown to increase the stability of the plasmid under starvation conditions.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols.

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl.

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50.

The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate. Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2. This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp. strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate. The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70.     

Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50.

The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate. Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2. This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp. strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate. The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70.     

Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to beta-carboxy-cis,cis-muconate.

An Escherichia coli system for generating a commercially unavailable catabolite in vivo was developed and was used to facilitate molecular genetic studies of phenolic catabolism. Introduction of the plasmid-borne Acinetobacter pcaHG genes, encoding the 3,4-dioxygenase which acts on protocatechuate, into E. coli resulted in bioconversion of exogenously supplied protocatechuate into beta-carboxy-cis,cis-muconate. This compound has been shown to be an inducer of the protocatechuate (pca) genes required for catabolism of protocatechuate to tricarboxylic acid cycle intermediates in Rhizobium leguminosarum biovar trifolii. The E. coli bioconversion system was used to explore regulation of the pca genes in a related bacterium, Agrobacterium tumefaciens. The pcaD gene, which encodes beta-ketoadipate enol-lactone hydrolase, from A. tumefaciens A348 was cloned and was shown to be adjacent to a regulatory region which responds strongly to beta-carboxy-cis,cis-muconate in E. coli. Site-specific insertional mutagenesis of the regulatory region eliminated expression of the pcaD gene in E. coli. When the mutation was incorporated into the A. tumefaciens chromosome, it eliminated expression of the pcaD gene and at least three other pca genes as well. The regulatory region was shown to activate gene expression in trans. The novel regulatory gene was termed pcaQ to differentiate it from pca regulatory genes identified in other microbes, which bind different metabolites.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4.

Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate. By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.     

Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate.

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.