Functional genomic and biochemical assays of M. smithii metabolism in the ceca of gnotobiotic mice. (A) In silico metabolic reconstruction of M. smithii pathways involved in (i) methanogenesis from formate, H₂/CO₂, and alcohols, (ii) carbon assimilation from acetate and bicarbonate, and (iii) nitrogen assimilation from ammonium. Acs, acetyl-CoA synthase; Adh, alcohol dehydrogenase; AmtB, ammonium transporter; BtcA/B, bicarbonate (HCO₃) ABC transporter; Cab, carbonic anhydrase; CH₃, methyl; CoB, coenzyme B; CoM, coenzyme M; COR, corrinoid; F₄₂₀, cofactor F₄₂₀; F₄₃₀, cofactor F₄₃₀; Fd, ferredoxin (ox, oxidized; red, reduced); FdhAB, formate dehydrogenase subunits; FdhC, formate transporter; Fno, F₄₂₀-dependent NADP oxidoreductase; Ftr, formylmethanofuran: tetrahydromethanopterin (H₄MPT) formyltransferase; Fum, fumarate hydratase; Fwd, tungsten formylmethanofuran dehydrogenase; GdhA, glutamate dehydrogenase; GlnA, glutamine synthetase; GltA/B, glutamate synthase subunits A and B; Hmd, H₂-forming methylene-H₄MPT dehydrogenase; Kor, 2-oxoglutarate synthase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-CoM reductase; Mdh, malate dehydrogenase; MeOH, methanol; Mer, methylene-H₄MPT reductase; MFN, methanofuran; MtaB, methanol:cobalamin methyltransferase; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT:CoM methyltransferase; NH₄, ammonium; OA, oxaloacetate; PEP, phosphoenol pyruvate; Por, pyruvate:ferredoxin oxidoreductase; Pps, phosphoenolpyruvate synthase; PRPP, 5-phospho-a-d-ribosyl-1-pyrophosphate; Pyc, pyruvate carboxylase; RfaS, ribofuranosylaminobenzene 5′-phosphate (RFA-P) synthase; Sdh, succinate dehydrogenase; Suc, succinyl-CoA synthetase. Potential drug targets are denoted by “Rx.” (B, C, and G) qRT-PCR assays of the expression of key M. smithii (Ms) genes in gnotobiotic mice that do or do not harbor B. thetaiotaomicron (Bt) (n = 5–6 animals per group; each sample assayed in triplicate; mean values ± SEM plotted; see SI Table 11 for full list of analyses). Results are summarized in A by using the following color codes: red, up-regulated; green, down-regulated; gray, assayed but no significant change; black arrows, transcript not assayed. (D) Ethanol (EtOH) levels in the ceca of mice colonized with B. thetaiotaomicron ± M. smithii (n = 5–7 animals per group representing two independent experiments; each sample assayed in duplicate; mean values ± SEM plotted). (E) Ratio of cecal concentrations of glutamine (Gln) and 2-oxoglutarate (2-OG) (n = 5 animals per group; samples assayed in duplicate; mean values ± SEM). (F) Cecal levels of free Gln, Glu (glutamate), and Asn (asparagine) (n = 5 animals per group; samples assayed in duplicate; mean values ± SEM). (H) Cecal ammonium and urea levels (n = 5–15 mice per group; three independent experiments). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.005, according to Student's t test.

M. smithii decorates its cell surface to mimic the host glycan landscape. (A) Transmission electron microscopy (TEM) of M. smithii harvested from the ceca of adult germ-free (GF) mice after a 14-d colonization. (Inset) A comparable study of stationary phase M. smithii recovered from a batch fermentor containing Methanobrevibacter complex medium (MBC). Note that the size of the capsule is greater in cells recovered from the cecum (open vs. closed arrow). (Scale bar: 100 nm.) (B) Comparison of GT, GH, and CE families (defined in CAZy; SI Table 10) represented in the genomes of the following sequenced methanogens (see SI Table 5): M. smithii (Msm); M. stadtmanae (Msp); Methanothermobacter thermoautotrophicus (Mth); Methanosarcina acetivorans (Mac); Methanosarcina barkeri (Mba); Methanosarcina mazei (Mma); Methanococcus maripaludis (Mmr); Methanococcus jannaschii (Mja); Methanospirillum hungatei (Mhu); Methanococcoides burtonii (Mbu); and Methanopyrus kandleri (Mka). Gut methanogens (highlighted in orange) have no GH or CE family members but have a larger proportion of family 2 GTs (Ψ, P < 0.00005 on the basis of binomial test for enrichment vs. non-gut-associated methanogens).

Analysis of the M. smithii pan-genome. Schematic depiction of the conservation of M. smithii PS genes [depicted in the outermost circle where the color code is orange for forward strand ORFs (F) and blue for reverse strand ORFs (R)] in (i) other M. smithii strains (GeneChip-based genotyping of strains F1, ALI, and B181; circles in increasingly lighter shades of green, respectively; see SI Table 2 for details), (ii) the fecal microbiomes of two healthy individuals [human gut microbiome (HGM), shown as the red plot in the fifth innermost circle with nucleotide identity plotted from 80% (closest to the purple circle) to 100% (closest to lightest green ring); see also SI Fig. 9 for details], and (iii) two other members of the Methanobacteriales division [M. stadtmanae (Msp) (purple circle), another human gut methanogen, and M. thermoautotrophicus (Mth) (yellow circle), an environmental thermophile; mutual best BLASTP hits (E value <10⁻²⁰)]. Tick marks indicate nucleotide number in kilobases. Asterisks denote the positions of ribosomal rRNA operons. Letters highlight distinguishing features among M. smithii genomes: the table below the figure summarizes differences in M. smithii gene content between strains F1, ALI, and B181, as well as the two human fecal metagenomic data sets.