Article Figures & Data
Figures
- 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).
Tables
- TABLE 1.
Sample origin and environmental data
Location and sample type Sample Core depth (cm) Water depth (m) Coordinates (°N, °W) Description Eel River T201 4-7 550 40.48, 124.36 Clam patch, high CH4 fosmid library GZfos Monterey Canyon Seep F17.1 0-25 955 36.77, 122.08 Clam patch, high CH4 Nonseep C4.1 AMIS microcosma Blake Ridge PC26 76 2,707 31.53, 75.45 Hemipelagic sediment ↵a AMIS microcosm cultured from nonseep reference core collected at least 25 m away from any known seep site.
- TABLE 2.
Archaeal 16s rDNA and mcrA clone recovery from MOA-containing sediments and fosmid DNA librarya
DNA Group Recovery (no. of clones) Total no. of clones Eel River Monterey Canyon F17.1 AMIS microcosm C4.1 Blake Ridge PC26 T201 Gzfos Archaeal 16s rDNA ANME-1 62 8 1 3 74 ANME-2 ANME-2a 1 41 42 ANME-2b 9 9 ANME-2c 20 1 27 43 91 Methanogen-like 1 1 22 24 Other 6 49 31 5 91 mcrA group a 13 4 9 25 51 b + 6 + ND 6 c 3 4 3 14 5 29 d 1 3 4 e 3 3 ↵a T201 environmental sequences and the Gzfos library were both derived from the same purified cell preparation of MOA from Eel River T201 sediment (see text). Environmental samples and fosmid library were screened with universal mcrA primers ME-1 and ME-2 and ANME-1 type b-specific mcrA primers AOM39 and AOM40 (see text). +, positive amplification with AOM39 and AOM40 primer set. mcrA group represents conceptual translation product. ND, not determined.
