D og2ltold-change)E G WT△Psc WT△sB 0510152025051015202505101520250510152025 ays after cohousing Fig. 2. Specific capsular polysaccharides, regulated by ccf, are necessary for single-strain (A)RNAseq gene expression analysis of B. fragilis overexpressing ccfA during laboratory ulture growth, relative to empty vector control(n=3). Green symbols represent PSA genes ed symbols represent PSC genes; blue symbols represent ccfgenes.(B)Heat map of expression levels for all capsular polysaccharide loci in B. fragilis following ccfA overexpression during growth in culture.(C)Relative expression using qRT-PCR (AACt normalized to gyrase)of RNA from colon lumen contents of mice mono-colonized with B fragilis or B fragilis Accf(Sidak 2-way ANOVA, n=4).(D-G)Abundance of foreign strains exchanged between pairs of co-housed mice each mono-colonized with the indicated strains, in colony forming units(CFU) per gram of feces(Sidak repeated measure 2-way ANOVA on log-transformed data, geometric mean and 95%Cl, n=9-12 pairs per plot).(H) Relative expression levels of capsular polysaccharides analyzed by qrT-PCr(AACt normalized to gyrase)of RNA from colon lumen contents of mice mono-colonized with B fragilis or B fragilis APSC (Sidak 2-way ANOVA, n=3, 4).(D) Plating of CFU from ascending colon mucus of mice mono-colonized with B fragilis strains(Tukey ANOVA,n 8)(*p<005,**p<0.01 0001) cience. Author manuscript; available in PMC 2018 November 18Fig. 2. Specific capsular polysaccharides, regulated by ccf, are necessary for single-strain stability (A) RNAseq gene expression analysis of B. fragilis overexpressing ccfA during laboratory culture growth, relative to empty vector control (n = 3). Green symbols represent PSA genes; red symbols represent PSC genes; blue symbols represent ccf genes. (B) Heat map of expression levels for all capsular polysaccharide loci in B. fragilis following ccfA overexpression during growth in culture. (C) Relative expression using qRT-PCR (ΔΔCt normalized to gyrase) of RNA from colon lumen contents of mice mono-colonized with B. fragilis or B. fragilis Δccf (Sidak 2-way ANOVA, n = 4). (D–G) Abundance of foreign strains exchanged between pairs of co-housed mice each mono-colonized with the indicated strains, in colony forming units (CFU) per gram of feces (Sidak repeated measure 2-way ANOVA on log-transformed data, geometric mean and 95% CI, n = 9–12 pairs per plot). (H) Relative expression levels of capsular polysaccharides analyzed by qRT-PCR (ΔΔCt normalized to gyrase) of RNA from colon lumen contents of mice mono-colonized with B. fragilis or B. fragilis ΔPSC (Sidak 2-way ANOVA, n = 3, 4). (I) Plating of CFU from ascending colon mucus of mice mono-colonized with B. fragilis strains (Tukey ANOVA, n = 8) (* p < 0.05, ** p < 0.01, *** p < 0.001). Donaldson et al. Page 11 Science. Author manuscript; available in PMC 2018 November 18. Author Manuscript Author Manuscript Author Manuscript Author Manuscript