PMC5011839_01
Transmission electron micrograph of negatively stained thin section of the methanogenic archaeon ISO4-H5. The sample was prepared as previously described. Images were captured using a Philips CM10 Transmission Electron Microscope, using an Olympus SIS Morada camera and SIS iTEM software (Germany)
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Fig1
The organism has only a thin bi-layer cell membrane, and no S-layer or cell wall was observed in electron micrographs of thin sections of ISO4-H5 cells (Fig. 1).
Phylogenetic analysis of Methanomassiliicoccales 16S rRNA gene sequences. Phylogenetic tree showing the relationships of methanogenic archaeon ISO4-H5 (shown in bold print) relative to other type and non-type strains within the order Methanomassiliicoccales. The phylogeny was inferred from 16S rRNA gene nucleotide sequences (1474 bp internal region) aligned using the Maximum Likelihood method based on the Kimura 2-parameter model. Evolutionary analyses were conducted in MEGA6. The bootstrap consensus tree from 1000 replicates was used to infer the evolutionary history of the taxa analysed. Bar: 0.05 substitutions per nucleotide position. The GenBank accession numbers of environmental sequences are displayed with the source habitat given in brackets. Strains whose genomes have been sequenced are marked with an asterisk. The initial tree for the heuristic search was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. All positions containing gaps or missing data were eliminated, giving a total of 455 positions in the final dataset. The 16S rRNA gene sequence from Thermoplasma acidophilum 122-1B2 was used as an outgroup
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Fig2
The 16S ribosomal RNA gene of ISO4-H5 is 96 % identical to "Candidatus Methanomethylophilus alvus" Mx1201 enriched from human feces, and 95 % identical to Thermoplasmatales archaeon BRNA1 enriched from bovine rumen (Fig. 2).
Circular representation of ISO4-H5 genome. Circles are referred to as 1 (outermost) to 4 (innermost). Circle 1: predicted ORFs on the positive (+) and negative (-) strands, respectively. ORFs are colored based on the Clusters of Orthologous (COG) categories. Circle 2: location of the rRNA genes. Circle 3: %G + C content. Circle 4: GC bias [(G - C)/(G + C)], khaki indicates values >1, purple <1. This image was generated with DNA plotter
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Fig3
Therefore, Cdc6.1 is predicted to be the origin of replication for ISO4-H5 (Fig. 3).
Gene synteny plots for genomes of members of the order Methanomassiliicoccales. PROmer alignments of the ISO4-H5 genome against completed genomes from members of Methanomassiliicocaccales are shown. The alignments were plotted using MUMmer with forward matches shown in red and reverse matches in blue. The units displayed on both axes are in million base pairs
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Fig4
The organization of genes within the ISO4-H5 genome shows best synteny with "Candidatus Methanomethylophilus alvus" Mx1201 and Thermoplasmatales archaeon BRNA1 (Fig. 4), its two closest genome-sequenced relatives.
The proposed methanogenesis pathway in ISO4-H5 growing with hydrogen and methanol, mono-, di-, tri-methylamine, or methyl-3-methylthiopropionate. Two methyl groups are needed from the methyl donors for every two methane formed. Methyl groups from methanol (MeOH), monomethylamine (MMA), dimethylamine (DMA), trimethylamine (TMA) and methyl-3-methylthiopropionate (M3MTP) are transferred onto methyl-binding corrinoid proteins CH3-MtaC, CH3-MtmC, CH3-MtbC, CH3-MttC and CH3-MtsB by specific corrinoid methyl transferases MtaB, MtmB, MtbB, MttB and MtsA respectively. The methyl groups are then transferred from the corrinoid proteins to CoM by CoM methyltransferase MtaA, MtmA, and bifunctional MtsA. Methyl-CoM is reduced to methane by the methyl coenzyme M reductase Mrt complex with cofactor HS-CoB. Heterodisulfide reductase complex Hdr and hydrogenase complex Mvh couple electron bifurcation to cofactor regeneration, and are coupled to the Fpo-like complex to generate a membrane potential for ATP production. The H+ (or Na+) ratio to ATP is not known, and the reconstruction of the pathway is based on the schemes proposed by Lang et al.. The presence or absence of each gene or the complete pathway for coenzyme M synthesis, in members of Methanomassiliicoccales is highlighted by colored circles; a white circle indicates absence in a genome. The fpoF and fpoO genes that are not found in members of Methanomassiliicoccales but exist in M. barkeri are represented by dotted red circles in the Fpo-like complex
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Fig5
Analysis of pyrrolysine biosynthesis gene cluster in Methanomassiliicoccales. Gene organization of the pyrrolysine gene clusters for the six Methanomassiliicoccales genomes are displayed. The operon includes a pyrrolysine corresponding tRNA(CUA), pylT; pyrrolysine-tRNA ligase, pylS; methylornithine synthase, pylB; (2R,3R)-3-methylornithyl-N
6-lysine synthase, pylC; and pyrrolysine synthase, pylD. The strain names are given on the left-hand side of each scheme. Strain B10 has two clusters, (1) and (2), as indicated next to the strain name
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Fig6
ISO4-H5 possesses a complete operon predicted to encode the genes required for the biosynthesis of pyrrolysine and for aminoacylation of a transfer RNA (tRNA) to pyrrolysine (Fig. 6), enabling read-through of the amber stop codon, UAG. The operon organization is conserved across the Methanomassiliicoccales (Fig. 6), suggesting pyrrolysine use is important for members of this order.