ANALYSIS OF MER GENE SUBCLASSES WITHIN BACTERIAL COMMUNITIES IN SOILSAND SEDIMENTS RESOLVED BY FLUORESCENT-PCR-RESTRICTION FRAGMENT LENGTHPOLYMORPHISM PROFILING

Bacterial mer (mercury resistance) gene subclasses in mercury-polluted and pristine natural environments have been profiled by Fluorescent-P CR-restriction fragment length polymorphism (FluRFLP). For FluRFLP, PC R products were amplified from individual mer operons in mercury-resis tant 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-negativ e mer operons within Tn501, Tn21, pDU1358, and pKLH2. Two independent PCRs were used to amplify two regions of different lengths (merRT Delt a 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' 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 w ere 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 b ases) position was observed in separate digests of either amplified re gion. These peak positions were as predicted on the basis of DNA seque nce. 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 ad dition to peaks of the expected sizes, extra peaks were observed which were not predicted on the basis of DNA sequence. Those appearing in t he restriction endonuclease digests of both study regions were presume d to be novel mer types. Genetic heterogeneity within and between merc ury-polluted and pristine sites has been studied by this technique. Pr ofiles generated were highly similar for samples taken within the same soil type. The profiles, however, changed markedly on crossing from o ne soil type to another, with gradients of the different groupings of mer genes identified.