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

Abstract

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.

Rumen microbial (meta)genomics and its application to ruminant production

“…The smallsubunit ribosomal RNA gene (rrs) is the most commonly used target for characterising this diversity. For methanogens, the methyl coenzyme-M reductase (mcrA) gene of the methanogenesis pathway is also a phylogenetic marker (Luton et al, 2002) that has been used in rumen studies (Denman et al, 2007). Recently, the diversity of rumen methanogens was investigated using the gene encoding type II chaperonins (Chaban and Hill, 2012).…”

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

“…Functional genes of methanogenesis and methane oxidation Within the methanogenic and methane-oxidizing archaea, the mcrA gene has similar phylogenetic resolution as the 16S rRNA gene (see Luton et al, 2002 for one of many case studies), and therefore permits an independent analysis of the methane-cycling archaeal community. In the hot and cool cores, mcrA genes formed a new Guaymas-specific monophyletic lineage (Figure 3), a sister lineage to the previously defined mcrA groups a and b that constitute the mcrA equivalent to the 16S rRNA-defined ANME-1 group (Hallam et al, 2003).…”

Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a deep-sea whale-fall in Monterey Canyon, California

“…PCR mixtures (25 ml) contained 0.4 mM of either Archaea-specific 16S rRNA primers, 20F and 958R (Delong, 1992), or mcrA-F and mcrA-R for the mcrA analysis (Luton et al, 2002). Reactions also contained 2.5 ml of 10 Â PCR buffer (containing 2 mM MgCl 2 ), 0.2 mM each of deoxynucleotide triphosphates and 0.025 U of Eppendorf HotMaster Taq (Westbury, NY, USA).…”

“…Primers, modified from Luton et al (2002), were used to amplify an B470-bp fragment from the archaeal mcrA gene (mcrA-f, 5 0 GGTGGTGTMGGATTCA-CA CAR-3 0 , T m 56.5 1C, and mcrA-r, 5 0 -TTCATTGCR TAGTTWGGRTAG-3 0 , T m 50.0 1C). Triplicate realtime PCR reactions (20 ml) contained 0.5 mM of each primer and 1 Â PCR buffer containing MgCl 2 , dNTPs and Amplitaq Gold in the SYBR green master mix (Applied Biosystems, Foster City, CA, USA).…”