The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill

Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK 1 Author for correspondence: Paul W. Riley. Tel: +44 1980 612315. Fax: +44 1980 611320. e-mail: paul.riley{at}camr.org.uk Inferred amino acid sequences of the methyl coenzyme-M reductase ( mcrA ) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea , generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously. Keywords: methanogenic Archaea , methyl coenzyme-M reductase, detection, identification Abbreviations: MCR, methyl coenzyme-M reductase; OTU, operational taxonomic unit b The GenBank accession numbers for the mcrA sequences reported in this paper are AF414034 – AF414051 (see Fig. 2) and AF414007–AF414033 (environmental isolates in Fig. 3).