Analysis of mer Gene Subclasses within Bacterial Communities in Soils and Sediments Resolved by Fluorescent-PCR-Restriction Fragment Length Polymorphism Profiling

Abstract

Bacterial mer (mercury resistance) gene subclasses in mercury-polluted and pristine natural environments have been profiled by Fluorescent-PCR-restriction fragment length polymorphism (FluRFLP). For FluRFLP, PCR products were amplified from individual mer operons in mercury-resistant bacteria and from DNA isolated directly from bacteria in soil and sediment samples. The primers used to amplify DNA were designed from consensus sequences of the major subclasses of archetypal gram-negative mer operons within Tn501, Tn21, pDU1358, and pKLH2. Two independent PCRs were used to amplify two regions of different lengths (merRT(Delta)P [ca. 1 kb] and merR [ca. 0.4 kb]) starting at the same position in merR. The oligonucleotide primer common to both reactions (FluRX) was labelled at the 5(prm1) end with green (TET) fluorescent dye. Analysis of the mer sequences within databases indicated that the major subclasses could be differentiated on the basis of the length from FluRX to the first FokI restriction endonuclease site. The amplified PCR products were digested with FokI restriction endonuclease, with the restriction digest fragments resolved on an automated DNA sequencing machine which detected only those bands labelled with the fluorescent dye. For each of the individual mer operon sources examined, this single peak (in bases) position was observed in separate digests of either amplified region. These peak positions were as predicted on the basis of DNA sequence. mer PCR products amplified from DNA extracted directly from soil and sediment bacteria were studied in order to determine the profiles of the major mer subclasses present in each natural environment. In addition to peaks of the expected sizes, extra peaks were observed which were not predicted on the basis of DNA sequence. Those appearing in the restriction endonuclease digests of both study regions were presumed to be novel mer types. Genetic heterogeneity within and between mercury-polluted and pristine sites has been studied by this technique. Profiles generated were highly similar for samples taken within the same soil type. The profiles, however, changed markedly on crossing from one soil type to another, with gradients of the different groupings of mer genes identified.