Cocolonization with M. smithii (Ms) and B. thetaiotaomicron (Bt) enhances the representation of both species in the distal intestines of gnotobiotic mice. The density of colonization was defined by using quantitative PCR of DNA isolated from the cecal contents of monoassociated and biassociated mice (n = 5 per group per experiment; three independent experiments; each sample assayed in triplicate; mean values ± SEM plotted); ∗, P < 0.05 vs. monoassociated controls. Bt and Bt + Ms represent the number of B. thetaiotaomicron cells present in mice colonized with Bt alone or with Bt + Ms, respectively; Ms and Ms + Bt represent the number of M. smithii cells present in mice colonized with Ms alone or with Ms + Bt, respectively.

M. smithii enhances B. thetaiotaomicron polyfructose-containing glycan degradation in the distal gut. Results of GeneChip analysis of RNA isolated from cecal contents of individual mice colonized with B. thetaiotaomicron ± M. smithii (n = 4–5 per group) are shown. (A) Unsupervised hierarchical clustering (dchip) of B. thetaiotaomicron (Bt) glycoside hydrolases (GH) and polysaccharide lysases (PL) that are up-regulated (13 genes) or down-regulated (57 genes) in the presence of M. smithii (Ms). Some GH/PL families represented in this data set are highlighted by using the classification scheme in CAZy (http://afmb.cnrs-mrs.fr/CAZY/acc.html; see Table 2 for a complete list). Each column represents data obtained from a cecal sample harvested from an individual mouse, whereas each row represents a B. thetaiotaomicron GH or PL gene. (B) B. thetaiotaomicron polyfructose degradation cluster induced in the presence of M. smithii (see Table 1 for fold changes defined by GeneChip). (C) Biochemical analysis of fructan and glucan levels in cecal contents (n = 5 mice per group; each sample assayed in duplicate; mean values ± SEM plotted). ∗, P < 0.05.

Biassociation with B. thetaiotaomicron and M. smithii increases B. thetaiotaomicron production of acetate and formate. (A) qRT-PCR analysis (boxed numbers) of the effects of M. smithii on expression of selected B. thetaiotaomicron genes encoding enzymes involved in fermentation of polyfructose-containing glycans: fructofuranosidases, BT1765/BT1759; fructokinase, BT1757; phosphofructokinase, BT0307; pyruvate:formate lyase, BT4738; acetate kinase, BT3963, methylmalonyl-CoA decarboxylase, BT1688; butyrate kinase, BT2552. Enzyme classification (EC) numbers are provided in parentheses. Dotted lines indicate multistep pathways. (Expression of fructofuranosidases, acetate kinase, puruvate:formate lyase, and butyrate kinase is constant if the colonization period is extended from 14 to 28 d; see Table 3.) (B) GC-MS analyses of cecal SCFAs (n = 5 per group; each sample assayed in duplicate; mean values ± SEM plotted; ∗, P < 0.05). (C) qRT-PCR study of the in vivo expression of M. smithii genes in a cluster (Lower) containing a formate transporter and dehydrogenase (fdhCAB) plus tungsten-containing formylmethanofuran dehydrogenase subunits (fwdEFDBAC) (n = 5 per group; each sample assayed in triplicate; mean values ± SEM plotted; ∗, P < 0.05).

Cocolonization of mice with M. smithii and B. thetaiotaomicron enhances host energy storage. (A) GC-MS analyses of acetate in sera obtained by retro-orbital phlebotomy from fasted (4 h) 12-week-old male GF, B. thetaiotaomicron monoassociated, and biassociated [B. thetaiotaomicron/M. smithii or B. thetaiotaomicron/D. piger (Dp)] gnotobiotic mice (n = 5 per group per experiment; two independent experiments; mean ± SEM are plotted). (B) Liver triglyceride levels (n = 5 per group; each assayed in duplicate; mean ± SEM plotted). (C) Epididymal fat pad weights (n = 5 per group per experiment; two independent experiments; mean ± SEM plotted). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.005.