Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea

Abstract

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.

FIG. 1.

Distance comparison of SSU rRNA and mcrA-based phylogenetic trees of environmental clones and primary methanogenic lineages. Bootstrap values are based on 1,000 replicates each (neighbor joining on top and parsimony on bottom) and are shown for branches with greater than 50% support. Methanocaldococcus spp. were used as the out group reference. ER, Eel River; MC, Monterey Canyon; AMIS, microcosm; BR, Blake Ridge. Boldface identifies clones identified and sequenced in this study. Red highlights ANME-2 group members, and blue highlights ANME-1 group members. Scale bars represent 0.05 nucleotide or amino acid substitution per site.

FIG.2.

(A) Schematic depiction of mcrA and mrt operon structure for MOA-associated fosmids and major methanogenic lineages. The mcrA operon typically consists of mcrBDCGA. The mrt operon structure varies between lineages. Scale bar, 500 bp. (B) Gene trees for mcrA and mrt subunits depicted in A. Abbreviations for methanogenic species harboring these genes are Mj, Methanocaldococcus jannaschii; Mv, Methanococcus vannielii; Mt, Methanothermobacter thermautotrophicus; and Mm, Methanosarcina mazei. Accession numbers for sequences used in the analyses are shown in parentheses for mcrB-Mj (NP_247836.1), mcrB-Mv (P07956), mcrB-Mt (NP_276296.1), mcrB-Mm (NP_633268.1), mcrC-Mj (NP_247838.1), mcrC-Mv (P07960), mcrC-Mt (NP_276294.1), mcrC-Mm (NP_633266.1), mcrD-Mj (NP_247837.1), mcrD-Mv (P07957), mcrD-Mt (NP_276295.1), mcrD-Mm (NP_633267.1), mcrG-Mj (NP_247839.1), mcrG-Mv (P07963), mcrG-Mt (NP_276293.1), mcrG-Mm (NP_633265.1), mcrA-Mj (NP_247840.1), mcrA-Mv (E27793), mcrA-Mt (NP_276292.1), mcrA-Mm (NP_633264.1), mrtB-Mj (NP_247045.1), mrtB-Mt (NP_276260.1), mrtC-Mj (NP_247058.1), mrtD-Mj (NP_247083.1), mrtD-Mt (NP_276259.1), mrtG-Mj (NP_247046.1), mrtG-Mt (NP_276258.1), mrtA-Mj (NP_247047.1), and mrtA-Mt (NP_276257.1). Bootstrap values are based on 1,000 replicates each (neighbor joining on top and parsimony on bottom) and are shown for branches with greater than 50% support. Trees are unrooted. Scale bars represent 50 amino acid substitutions.

FIG. 3.

Amino acid alignment of representative environmental mcrA types and primary methanogenic lineages. Methanothermobacter thermoautotrophicus was used as the reference sequence (GenBank U10036). Position numbers correspond to the reference sequence. Amino acid identity at a given position is denoted by dots, and gaps are marked by dashes. Conserved amino acids are coded by color according to predicted CH₃ modification (green), F₄₃₀ binding (red), or coenzyme B interaction (blue).