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Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

ABSTRACT: It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

SUBMITTER: Erb RW

PROVIDER: S-EPMC195868 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

Erb R W RW Wagner-Döbler I I

Applied and environmental microbiology 19931201 12

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further puri ...[more]

PMID: 8285706

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Project description:We analyzed the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 to biphenyl and polychlorinated biphenyls (PCBs). This species has not been extensively exposed to PCBs, as it was first isolated from a toluene contaminated aquifer, rather than a site contaminated with polychlorinated hydrocarbons. Using a microarray targeting 3524 genes, we assessed gene expression in minimal medium supplemented with various substrates (e.g. PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were up-regulated in the various treatments. In medium and in sediment, PCBs elicited the up-regulation of a common set of 100 genes, including chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were up-regulated in response to PCBs or biphenyl. This study is one of the first which utilizes microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions which more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism. In addition, the identification of numerous genes expressed in contaminated soil specifically may have implications for the development of biosensors. Finally, comparative genomic and transcriptomic analyses suggest that the mere presence of orthologs of the required enzymes may not be sufficient to confer a vigorous biphenyl/PCB metabolism. RNA was isolated from cells incubated in the following: sediment from a PCB-contaminated industrial site, uncontaminated sediment from a comparable site, and defined media supplemented with glucose (3 g/L), glucose and biphenyl (3 g/L, 4.5 Î¼M), or glucose and PCBs (3 g/L, 5 mg/L Aroclor 1254). In all cases, there were 3 biological replicates and 2 technical replicates (repeat hybridizations). A total of 3524 genes are represented on the arrays; of these, 41 and 176 are found on the plasmids pRA2 and pRA3, respectively. On average, there are 3 distinct 24nt probes per gene.

Dataset Information

Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

Publications

Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

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