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BIOLOGICAL SCIENCES / MICROBIOLOGY
Genomic and metabolic adaptations of Methanobrevibacter smithii to the human gut
*Center for Genome Sciences and Genome Sequencing Center, Washington University School of Medicine, St. Louis, MO 63108; and Unité Mixte de Recherche 6098, Centre National de la Recherche Scientifique, Universités Aix-Marseille I & II, 13288 Marseille Cedex 9, France
Contributed by Jeffrey I. Gordon, May 4, 2007 (received for review April 10, 2007)
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Abstract |
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archaeal-bacterial mutualism | comparative microbial genomics | functional genomics and metabolomics | gnotobiotic mice | human gut microbiota
Complex dietary polysaccharides (fiber) and proteins are digested by enzymes encoded by genes in the microbial community's collective genome (microbiome), but not in our human genome (3, 4). Bacterial fermentation of polysaccharides yields short chain fatty acids (SCFAs) (principally acetate, propionate, and butyrate), other organic acids (e.g., formate), alcohols (e.g., methanol and ethanol), and gases [e.g., hydrogen (H2) and carbon dioxide (CO2)]. Host absorption of SCFAs provides up to 10% of daily caloric intake, although this value varies depending on the glycan content of the diet (5). Archaeal methanogenesis improves the efficiency of polysaccharide fermentation in animal gut "bioreactors" by preventing the buildup of H2 and other reaction end products (6).
Several recent observations underscore the importance of delineating the genomic and metabolic underpinnings of M. smithii's contributions to energy balance and, if it is to be a therapeutic target for manipulation, the nature of its adaptations to the gut ecosystem. First, comparative metagenomic analysis of the gut microbiomes of genetically obese ob/ob mice and their lean ob/+ or +/+ littermates revealed that the obese community exhibits increased representation of archaea, division-wide increase in the proportion of Firmicutes relative to Bacteroidetes, and an accompanying enrichment of microbial genes involved in polysaccharide degradation (7). Moreover, transplantation of the gut microbiota from ob/ob or +/+ donors into lean +/+ germ-free (GF) mouse recipients disclosed that this increased energy harvesting capacity is transmissible: i.e., recipients of an obese donor's gut microbiota gained more body fat than did recipients of a lean donor's microbiota (7). Second, colonization of adult GF mice with M. smithii and/or Bacteroides thetaiotaomicron, a prominent sequenced human gut symbiont equipped with a large arsenal of glycoside hydrolases (GHs) not represented in our human proteome (8), revealed that the methanogen increased the efficiency, and changed the specificity of bacterial digestion of dietary glycans (6). Moreover, cocolonized mice exhibited a significantly greater increase in adiposity compared with mice colonized with either organism alone (6). Third, metagenomic studies of the colonic microbiomes of two healthy adults confirmed that M. smithii is a prominent component of this community and that enzymes involved in methanogenesis are well represented (3).
With these observations in mind, we have sequenced the genome of the M. smithii type strain PS, compared it to other M. smithii strains isolated from the human gut and to other archaeons, and have performed functional genomic and biochemical analyses of its properties in gnotobiotic mice that do or do not harbor B. thetaiotaomicron. The results provide insights about M. smithii's niche (profession), its evolved adaptations to its gut habitat, and strategies for identifying targets for development of antiarchaeal agents.
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Compared with nongut methanogens and/or all archaea, M. smithii and M. stadtmanae are significantly enriched (binomial test, P < 0.01) for genes assigned to gene ontology (GO) categories involved in surface variation (e.g., cell wall organization and biogenesis; see below), defense (e.g., multidrug efflux/transport), and processing of bacteria-derived metabolites (SI Tables 6 and 7).
The M. smithii and M. stadtmanae genomes exhibit limited global synteny (SI Fig. 4) but share 968 proteins with mutual best BLAST hit E values 
10–20 (46% of all M. smithii proteins; SI Table 8). A predicted interaction network of M. smithii clusters of orthologous groups (COGs) constructed based on STRING (9) shows that it contains more COGs for persistence, improved metabolic versatility, and machinery for genomic evolution compared with M. stadtmanae (SI Fig. 5 and SI Table 9).
Cell Surface Variation. The ability to vary capsular polysaccharide surface structures in vivo by altering expression of glycosyltransferases (GTs) is a feature shared among sequenced bacterial species that are prominent in the distal human gut microbiota (4, 10–12). Transmission EM studies of M. smithii harvested from gnotobiotic mice after a 14-day colonization revealed that it too has a prominent capsule (Fig. 1A). The proteomes of both human gut methanogens also contain an arsenal of GTs [26 in M. smithii and 31 in M. stadtmanae; see SI Table 10 for a complete list organized on the basis of the Carbohydrate Active enZYme (CAZy) classification scheme (www.cazy.org); ref. 13]. Unlike the sequenced Bacteroidetes, which possess large repertoires of GH and carbohydrate esterases (CE) not represented in the human "glycobiome," neither gut methanogen has any detectable GH or CE family members (Fig. 1B). Both M. smithii and M. stadtmanae dedicate a significantly larger proportion of their "glycobiome" to GT2 family glycosyltransferases than any of the sequenced nongut associated methanogens (binomial test; P < 0.00005; Fig. 1B). These GT2 family enzymes have diverse predicted activities, including synthesis of hyaluronan, a component of human glycosaminoglycans in the mucosal layer.
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The genomes of both human gut methanogens also encode a class of predicted surface proteins that have features similar to bacterial adhesins (48 members in M. smithii and 37 members in M. stadtmanae). A phylogenetic analysis (see Materials and Methods in SI Text) indicated that each methanogen has a specific clade of these adhesin-like proteins (ALPs) (SI Fig. 7A). A subset of M. smithii ALPs has homology to pectin esterases (GO 0030599): this GO family, which is significantly enriched in this compared with other Archaea on the basis of the binomial test (P < 0.0005; SI Table 6) is associated with binding of chondroitin, a major component of mucosal glycosaminoglycans. Several other M. smithii ALPs have domains predicted to bind other sugar moieties (e.g., galactose-containing glycans; SI Fig. 7A). Both methanogens also have ALPs with peptidase-like domains (see SI Table 12 for a complete list of InterPro domains).
We conducted qRT-PCR assays of cecal RNAs from the mono- and cocolonized gnotobiotic mice described above. The results revealed one "sugar-binding" ALP (MSM1305) that was significantly up-regulated in the presence of B. thetaiotaomicron, four that were suppressed (including one with a GAG binding domain), and two that exhibited no statistically significant alterations (SI Fig. 7B). Regulated expression of distinct subsets of ALPs may direct this methanogen to specific intestinal microhabitats where close association with saccharolytic bacterial partners could promote establishment and maintenance of syntrophic relationships.
Methanogenic and Nonmethanogenic Removal of Bacterial End Products of Fermentation. Compared with other sequenced non-gut-associated methanogens, M. smithii has significant enrichment of genes involved in utilization of CO2, H2 and formate for methanogenesis (GO 0015948; SI Table 6). They include genes that encode proteins involved in synthesis of vitamin cofactors used by enzymes in the methanogenesis pathway [methyl group carriers (F430 and corrinoids), riboflavin (precursor for F430 biosynthesis), and coenzyme M synthase (involved in the terminal step of methanogenesis)] (see SI Table 7 for a gene list and Fig. 2A for the metabolic pathways). M. smithii also has an intact pathway for molybdopterin biosynthesis to allow for CO2 utilization (SI Fig. 8). qRT-PCR assays demonstrated that although key central methanogenesis enzymes are constitutively expressed in the presence or absence of B. thetaiotaomicron [Fwd (tungsten formylmethanofuran dehydrogenase), Hmd (methylene-H4MPT dehydrogenase), and Mcr (methyl-CoM reductase)], ribofuranosylaminobenzene 5'-phosphate (RFA-P)-synthase (RfaS, MSM0848), an essential gene involved in methanopterin biosynthesis is significantly up-regulated with cocolonization (see Fig. 2A and SI Table 11 for qRT-PCR results). M. smithii also up-regulates a formate utilization gene cluster (FdhCAB, MSM1403–5) for methanogenic consumption of this B. thetaiotaomicron-produced metabolite (6).
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M. smithii also possesses enzymes that in other methanogens facilitate utilization of two other products of bacterial fermentation, methanol and ethanol (16, 17). qRT-PCR assays showed that cocolonization significantly increased expression of a methanol:cobalamin methyltransferase (MtaB, MSM0515), an NADP-dependent alcohol dehydrogenase (Adh, MSM1381), and an F420-dependent NADP oxidoreductase (Fno, MSM0049) (2.4 ± 0.3, 2.3 ± 0.4, and 3.7 ± 0.4 fold vs. monoassociated controls, respectively; P < 0.01; see Fig. 2A for pathway information, and Fig. 2C for qRT-PCR results]. Follow-up biochemical studies confirmed a significant decrease in ethanol levels in the ceca of cocolonized mice (11 ± 2 µmol/g total protein in cecal contents vs. 35 ± 6 µmol/g in mice with B. thetaiotaomicron alone; n = 5–7 animals per group; P < 0.05; Fig. 2D). Moreover, levels of ethanol in M. smithii monoassociated controls were not significantly different from background levels defined in germ-free controls (n = 5–7 animals per group; P > 0.05; data not shown). Expression of B. thetaiotaomicron's alcohol dehydrogenases (BT4512 and BT0535) was not altered by cocolonization (6), indicating that the reduction in cecal ethanol levels observed in cocolonized mice is not due to diminished bacterial production but rather to increased archaeal consumption.
Collectively, these findings indicate that M. smithii supports methanogenic and nonmethanogenic removal of diverse bacterial end products of fermentation: this capacity may endow it with greater flexibility to form syntrophic relationships with a broad range of bacterial members of the distal human gut microbiota.
M. smithii Utilization of Ammonium as a Primary Nitrogen Source. Host metabolism of amino acids by glutaminases associated with the intestinal mucosa (18) or deamination of amino acids during bacterial degradation of dietary proteins yields ammonium (19). The M. smithii proteome contains a transporter for ammonium (AmtB; MSM0234) plus two routes for its assimilation: (i) the ATP-dependent glutamine synthetase–glutamate synthase pathway, which has a high affinity for ammonium and thus is advantageous under nitrogen-limited conditions, and (ii) the ATP-independent glutamate dehydrogenase pathway, which has a lower affinity for ammonium (20).
Microanalytic biochemical assays revealed that the ratio of glutamine to 2-oxoglutarate was 32-fold lower in the ceca of cocolonized gnotobiotic mice compared with animals colonized with M. smithii alone and that was 5-fold lower compared with B. thetaiotaomicron monoassociated hosts (P < 0.0001; Fig. 2E; n = 5 mice per group). In addition, levels of several polar amino acids were also significantly reduced in mice containing the saccharolytic bacterium and methanogen (Fig. 2F; n = 5 mice per group), providing additional evidence for a more nitrogen-limited gut environment. qRT-PCR analysis established that many of the key M. smithii genes involved in ammonium assimilation are up-regulated with cocolonization, particularly those in the high-affinity glutamine synthetase–glutamate synthase pathway [GlnA (glutamine synthetase, MSM1418); GltA/GltB (two subunits of glutamate synthase, MSM0027, MSM0368); Fig. 2 A and G]. GeneChip analysis of the transcriptional responses of B. thetaiotaomicron to cocolonization with M. smithii indicated that it also up-regulates a high-affinity glutamine synthetase (BT4339; 2.4-fold vs. B. thetaiotaomicron monoassociated mice; n = 4–5 mice per group; P < 0.001; ref. 6). This prioritization of ammonium assimilation by B. thetaiotaomicron and M. smithii is accompanied by a decrease in cecal ammonium levels in cocolonized hosts (11.1 ± 1.3 µmol/g dry weight of cecal contents vs. 14.4 ± 0.6 and 14.3 ± 0.9 in M. smithii- and B. thetaiotaomicron-monoassociated animals, respectively; n = 5–15 per group; P < 0.05; Fig. 2H). Together, these studies indicate that ammonium provides a key source of nitrogen for M. smithii when it exists in isolation in the gut of gnotobiotic mice, and that it must compete with B. thetaiotaomicron for this nutrient resource.
Considering Targets for Development of Anti-M. smithii Agents. As noted in the Introduction, manipulation of the representation of M. smithii in our gut microbiota could provide a means for treating obesity. When considering how to manipulate the representation of M. smithii, several obvious questions arise: (i) is the targeted M. smithii gene/pathway expressed in vivo and is its expression affected by the presence of actively fermenting bacteria, and (ii) are the therapeutic targets being considered conserved among different M. smithii strains? We addressed these questions in a final set of experiments.
Functional genomics studies in gnotobiotic mice illustrate one way to approach issue i. For example, inhibitors exist for several M. smithii enzymes. A class of N-substituted derivatives of para-aminobenzoic acid (pABA) interfere with methanogenesis by competitively inhibiting ribofuranosylaminobenzene 5'-phosphate synthase (RfaS; MSM0848; ref. 21). As noted above, this enzyme, which participates in the first committed step in synthesis of methanopterin, is up-regulated with cocolonization (4.6 ± 0.9 fold vs. monoassociated controls; P < 0.01; Fig. 2A).
Archaeal membrane lipids, unlike bacterial lipids, contain ether linkages. A key enzyme in the biosynthesis of archaeal lipids is hydroxymethylglutaryl (HMG)-CoA reductase (MSM0227), which catalyzes the formation of mevalonate, a precursor for membrane (isoprenoid) biosynthesis (22). Some HMG-CoA reductase inhibitors (statins) have been reported to inhibit growth of Methanobrevibacter species in vitro (22). qRT-PCR revealed that MSM0227 is expressed at high levels in vivo in the presence or absence of B. thetaiotaomicron (P > 0.05; SI Table 11). Although statins are commonly used for treating hypercholesterolemia in humans, we are not aware of any studies that report a causal association between their consumption and weight loss. However, the efficacy of statins as antiarchaeal agents will depend on factors such as their activity against the M. smithii enzyme, their capacity to enter the archaeal cell, and their concentration and stability within the distal human gut.
To address issue ii, we designed a custom GeneChip containing probe sets directed against 99.1% of M. smithii's 1,795 predicted protein-coding genes (see SI Table 13 for details). This GeneChip was used to perform whole-genome genotyping of M. smithii PS (control) plus three other strains recovered from the feces of healthy humans: F1 (DSMZ 2374), ALI (DSMZ 2375), and B181 (DSMZ 11975). Replicate hybridizations indicated that 100% of the ORFs represented on the GeneChip were detected in M. smithii PS, whereas 90–94% were detected in the other strains, including the potential drug targets mentioned above (SI Table 2 and Fig. 3). Approximately 50% of the undetectable ORFs in each strain encode hypothetical proteins. The other undetectable genes are involved in genome evolution [e.g., recombinases, transposases, insertion sequence (IS) elements, and type II restriction modification (R-M) systems], are components of a putative archaeal prophage in strain PS (SI Table 14) or are related to surface variation, including several adhesin-like proteins (ALPs) (e.g., MSM0057 and MSM1585–7, MSM1590; SI Fig. 7). Strains F1 and ALI also appear to lack redundant gene clusters encoding subunits of formate dehydrogenase (MSM1462–3) and methyl-CoM reductase (MSM0902–4) that are found in the PS strain (the latter cluster is also undetectable in strain B181). In addition, the only methanol utilization cluster present in the PS strain (MSM0515–8) was not detectable in strain F1 (SI Table 2).
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Prospectus. Ongoing comparisons of the genomes of multiple strains of a bacterial "species" have revealed remarkable variations in gene content. This has led to the concept that understanding the nature of a microbial "species" requires that we consider the sum of all genes present in all strains (i.e., its pan-genome; ref. 23). Our results provide an initial glimpse of the genetic diversity of a gut archaeon and the operations of one strain's transcriptome and metabolome in vivo. Although there is overall conservation of gene content among different strains of M. smithii, there are differences in metabolic capacities and surface properties represented in the organism's pan-genome. These differences may influence its partitioning within and adaptations to its gut habitat, as well as its relationship with members of the bacterial community.
Tractable genetic systems for manipulating the M. smithii genome are not available at present. However, further characterization of the M. smithii pan-genome by sequencing isolates obtained from related and unrelated individuals represents an opportunity to evaluate genome conservation and evolution in an archaeon that coexists with bacteria in an incredibly dense microbial community. In addition, gnotobiotic mice colonized with different strains of M. smithii together with sequenced representatives of human gut-derived Bacteroidetes and Firmicutes, should help guide drug discovery programs aimed at identifying antiarchaeal therapeutic agents with broad efficacy.
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Materials and Methods |
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TopAbstractResults and DiscussionMaterials and MethodsAcknowledgementsReferences |
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40. ORFs were identified and annotated as described in Materials and Methods in SI Text.
qRT-PCR Analyses.
All mouse experiments were performed using protocols approved by the animal studies committee of Washington University. Gnotobiotic male mice belonging to the NMRI inbred strain (n = 5–6 mice per group per experiment) were colonized with either M. smithii (14 d) or B. thetaiotaomicron (28 d) alone or first with B. thetaiotaomicron for 14 d followed by cocolonization with M. smithii for 14 d. All mice were killed at 12 weeks of age. Cecal contents from each mouse were flash frozen and stored at –80°C. RNA was extracted from an aliquot of the harvested cecal contents (100–300 mg) and used to generate cDNA for qRT-PCR assays (see Materials and Methods in SI Text). qRT-PCR data were normalized to 16S rRNA (
CT method) before comparing treatment groups. PCR primers are listed in SI Table 15. All amplicons were 100–150 bp.
Biochemical Assays. Perchloric and hydrochloric acid extracts and alkali extracts of freeze-dried cecal contents were prepared, and established pyridine nucleotide-linked microanalytic assays (26) were used to measure metabolites (see Materials and Methods in SI Text for details).
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Acknowledgements |
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TopAbstractResults and DiscussionMaterials and MethodsAcknowledgementsReferences |
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Footnotes |
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Abbreviations: ALP, adhesin-like protein; CE, carbohydrate esterase; GH, glycoside hydrolase; GO, gene ontology; GT, glycosyltransferase; qRT-PCR, quantitative RT-PCR.
To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu
Freely available online through the PNAS open access option.
Author contributions: B.S.S., E.E.H., and J.I.G. designed research; B.S.S., E.E.H., J.K.M., R.F., P.L., and K.K. performed research; B.S.S., E.E.H., and J.K.M. contributed new reagents/analytic tools; B.S.S., E.E.H., J.K.M., P.M.C., B.H., R.F., P.L., K.K., R.K.W., and J.I.G. analyzed data; and B.S.S., E.E.H., and J.I.G. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequence reported in this paper for the M. smithii PS genome has been deposited in the GenBank database (accession no. CP000678).
This article contains supporting information online at www.pnas.org/cgi/content/full/0704189104/DC1.
© 2007 by The National Academy of Sciences of the USA
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, P < 0.00005 on the basis of binomial test for enrichment vs. non-gut-associated methanogens).
