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 wight) 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. Thus, for the first time it was possible to directly amplify and detect a chromosomally encoded, single-copy gene from a highly specialized subpopulation of the total microbial community in natural sediments.