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. 1999 Apr;65(4):1627-35.
doi: 10.1128/AEM.65.4.1627-1635.1999.

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

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

M C Peel et al. Appl Environ Microbiol. 1999 Apr.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Representative metabolic pathways encoded by the nonhomologous CBA-degradative operons clcABD, cbaABC, and fcbBAC. The genes that make up these operons are indicated in open boxes below the metabolic steps they encode. Schematic representations of the locations of the three DNA probes used in this study, with respect to the operons, are shown as black bars. (a) Chlorocatechol ortho-ring-fission pathway. Following initial attack by a nonspecific dioxygenase such as the benzoate (benABC) or toluate (xylXYZ) dioxygenase and dihydrodiol dehydrogenase to form chlorocatechols, the clcABD gene products catalyze ortho-ring fission to open the aromatic ring and continue metabolism. (b) CBA 3,4-(4,5)-dioxygenase pathway. The cbaABC genes encode dioxygenase, reductase, and dehydrogenase enzymes that initiate 3-CBA and 3,4-DCBA (not shown) degradation. Protocatechuic acid is also formed in this pathway. The host’s protocatechuate 4,5-dioxygenase (meta-ring-fission) enzymes then complete the pathway. (c) 4-CBA dehalogenase pathway. The fcbA gene encodes a 4-CBA-CoA ligase; fcbB encodes the dehalogenase; fcbC encodes a 4-hydroxybenzoate-CoA thioesterase. Note that the gene order in the control strain Pseudomonas sp. strain CBS-3 is fcbBAC.
FIG. 2
FIG. 2
Phenotype distance cluster dendrogram of the clcABD, cbaABC, and fcbB isolates from the Hyde Park landfill based on carbon source utilization (Biolog GN). Control strains (clcABD, cbaABC, and fcbBAC) and representative β- and γ-Proteobacteria members from the Biolog GN database are included for reference. Phenotype grouping, an arbitrary division of isolates into five groups at a rescaled distance of 15. Isolate, the numbers following the source designation (Table 3) in each isolate name give the sampling site, the isolate number, and the sampling trip number (35), respectively. The superscript preceding the isolate name refers to the enrichment carbon source: a, 3-CBA; b, 4-CBA; c, 3,4-DCBA; d, 3-CBA (3-CBP). The superscript after the isolate name gives the enrichment temperature (degrees Celsius). Genotype, as described in the legend to Fig. 1. RFLP pattern, as described in footnote d to Table 3. IS1071, detection of the insertion sequence in the genome by hybridization to NheI digests. Rescaled distance cluster combine, dendrogram created from average linkage (between groups) data (SPSS).
FIG. 3
FIG. 3
Hybridization of the IS1071 internal probe H4 to genomic DNA treated with NheI for selected isolates that carried clcABD and fcbB genes for chlorobenzoate degradation. Control genomic DNA digests for C. testosteroni BR60 (cbaABC); P. putida AC866 (clcABD); and the biphenyl-CBP-degrading strains Burkholderia sp. strain LB400 (bph clc), Alcaligenes sp. strain H850 (bph), and Pseudomonas sp. strain B-356 (bph) are also shown. The size of hybridizing DNA fragments was determined relative to the mobility of HindIII-digested λ DNA. Note that the copy number of IS1071 is not apparent in these digests because the NheI recognition sites are located in the inverted repeats of the element. For example, the genomic DNA of strain BR60 contains seven copies of IS1071.

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