Genomic and metabolic adaptations of Methanobrevibacter smithii to the human gut

Abstract

The human gut is home to trillions of microbes, thousands of bacterial phylotypes, as well as hydrogen-consuming methanogenic archaea. Studies in gnotobiotic mice indicate that Methanobrevibacter smithii, the dominant archaeon in the human gut ecosystem, affects the specificity and efficiency of bacterial digestion of dietary polysaccharides, thereby influencing host calorie harvest and adiposity. Metagenomic studies of the gut microbial communities of genetically obese mice and their lean littermates have shown that the former contain an enhanced representation of genes involved in polysaccharide degradation, possess more archaea, and exhibit a greater capacity to promote adiposity when transplanted into germ-free recipients. These findings have led to the hypothesis that M. smithii may be a therapeutic target for reducing energy harvest in obese humans. To explore this possibility, we have sequenced its 1,853,160-bp genome and compared it to other human gut-associated M. smithii strains and other Archaea. We have also examined M. smithii's transcriptome and metabolome in gnotobiotic mice that do or do not harbor Bacteroides thetaiotaomicron, a prominent saccharolytic bacterial member of our gut microbiota. Our results indicate that M. smithii is well equipped to persist in the distal intestine through (i) production of surface glycans resembling those found in the gut mucosa, (ii) regulated expression of adhesin-like proteins, (iii) consumption of a variety of fermentation products produced by saccharolytic bacteria, and (iv) effective competition for nitrogenous nutrient pools. These findings provide a framework for designing strategies to change the representation and/or properties of M. smithii in the human gut microbiota.

Fig. 1.

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).

Fig. 2.

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.

Fig. 3.

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.