ISOLATION AND IDENTIFICATION OF METHANOGEN-SPECIFIC DNA FROM BLANKET BOG FEAT BY PCR AMPLIFICATION AND SEQUENCE-ANALYSIS

The presence of methanogenic bacteria was assessed in peat and soil co res taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected t o PCR amplification with primers 1Af and 1100Ar, which specifically am plify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which wer e designed to amplify a 0.75-kb region of the alpha-subunit gene for m ethyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that an aerobiosis is required for activity and survival of the methanogen pop ulation. PCR products from both amplifications were cloned, and the re sulting transformants were screened with specific oligonucleotide prob es internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA w as extracted from probe-positive clones of both types and the insert w as sequenced. The DNA sequences of 8 MCR clones were identical, as wer e those of 16 of the 17 16S rRNA clones. One clone showed marked varia tion from the remainder in specific regions of the sequence. From a co mparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe wa s used to screen primary populations of PCR clones, and all of those t hat were probe negative were checked for the presence of inserts, whic h were then sequenced. By using this strategy, further novel methanoge n 16S rRNA variants were identified and analyzed. The sequences recove red from the peat formed two clusters on the end of long branches with in the methanogen radiation that are distinct from each other. These c annot be placed directly with sequences from any cultured taxa for whi ch sequence information is available.