Fig. S1. Phylogenetic relationships in Methanobacteriaceae. The tree was determined on the basis of the 16S rRNA gene sequences. The scale bar represents branch length. Reliability of internal branches was assessed using the bootstrap method with 1000 pseudo-replicates. Bootstrap values greater than 50% are indicated at the nodes. Accession numbers are given in parentheses. An unrooted tree was generated using NJplot.

Fig. S2. Circular representation of the CaT2 chromosome. From the outside in: circles 1 and 2 of the chromosome show the positions of protein-coding genes on the positive and negative strands respectively. Circle 3 shows the positions of protein-coding genes (red) that have no ortholog in M. thermautotrophicus ΔH, and clustered regularly interspaced short palindromic repeats (CRISPRs) regions (blue). Circle 4 shows the positions of tRNA genes (purple) and rRNA genes (brown). Circle 5 shows a plot of GC skew [(G − C)/(G + C); khaki indicates values > 0; purple indicates values < 0]. Circle 6 shows a plot of G + C content (outward: higher values than the average).

Fig. S3. Comparison of the CaT2, ΔH, and Marburg genomes.

A. Venn diagram comparing the gene inventories of the three genomes. The numbers of shared and unique genes are shown.

B. Comparison of the genomic location of formate dehydrogenase genes (fdhABC) in CaT2 and ΔH. Genes and their orientations are depicted with arrows. The formate utilizing genes (fdh) are shown as black. Grey lines indicate orthologous regions.

Fig. S4. PCR analyses of strain isolated from the aggregated coculture. PCR analyses of strain isolated from the aggregated coculture. Genomic DNA was isolated from the strain (CaT2) isolated from the aggregated coculture, the strain stocked in our laboratory, and ΔH by FastDNA® SPIN kit. Used primers are described in Table S9. Primer sets for each lane were as follows: A–F, pFZ1 specific primers; G–L, cobalt transporter gene-specific primers; N–S, formate dehydrogenase A gene-specific primers; U–Y, the mcrA gene-specific primers. The template DNA contents of each lane are as follows: A, G, N, 1 ng DNA of the strain stocked in our laboratory; B, H, O, 0.1 ng DNA of the strain stocked in our laboratory; C, I, P, V, 1 ng DNA of the strain isolated from the aggregated coculture; D, J, Q, W, 0.1 ng DNA of the strain isolated from the aggregated coculture; E, K, R, X, 1 ng DNA of ΔH; F, L, S, Y, 0.1 ng DNA of ΔH; M, T, U, φX174/Hae III digest. 1.5% agarose gel.

Fig. S5. Cells aggregation of hydrogenotrophic methanogens.

A. Alteration of AIs during the growth of hydrogenotrophic methanogens. The AI calculation was performed by the difference of the cells density before and after the centrifugation of samples.

B. Growth of hydrogenotrophic methanogens indicated by methane productions. These methanogens were cultivated in 120 ml serum vials containing a 50 ml W medium under an atmosphere of ca. 150 kPa H2–CO2 (20:80, v/v). Error bars indicate standard deviations of the means (n = 3).

Fig. S6. Synteny analysis among Methanothermobacter strains. Each plot point represents reciprocal best matches by BLASTP comparisons between orthologs.

Fig. S7. Phylogenetic tree of formate transporters in methanogens. The amino acid sequence was used for this analysis. The tree was depicted by the UPGMA method. The bar indicates generation distance and the numbers on the branches are bootstrap values. Accession numbers are given in parentheses.

Fig. S8. Methanogenesis pathway map in CaT2. The pathway map was made based on the scheme of Thauer et al. (Thauer et al., 2008). Abbreviations are as follows: F420 (ox), oxidized coenzyme F420; F420 (red), reduced coenzyme F420; Fd (ox) oxidized ferredoxin; Fd (red), reduced ferredoxin; H4MPT, tetrahydrosarcinapterin; MFR, methanofuran; HS-CoB, coenzyme B; HS-CoM, coenzyme M. The numbers in this figure show the reaction numbers listed in Table S5.

Table S1. General genome features of CaT2 and other thermophilic hydrogenotrophic methanogens.

Table S2. Monosaccharide concentrations among CaT2, ΔH and Z-245.

Table S3. The 102 genes present in CaT2 but absent in ΔH.

Table S4. Presence of formate transporter genes in sequenced methanogens.

Table S5. The predicted genes involved in methanogenesis of CaT2.

Table S6. Predicted genes involved in pseudomurein biosynthesis pathway.

Table S7. Classification of putative glycosyltransferase of CaT2 based on the family classification from CAZy database.

Table S8. Annotated functional domains in glycosyltransferases.

Table S9. Primer sets for PCR and real-time RT-PCR.

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