Microbiota conventionalization influences MAFF-dependent B. theta phenotype in the colon. (A) Representative confocal images showing FISH probes targeting Bacteroidales (red) and DAPI (blue) in colon sections. Antibiotics-treated (SPF-Abx) or GF mice (monocolonized) were orally inoculated with PBS or colonized with the indicated B. theta strains. Scale bars represent 20 µm or 2 µm on inset images. OM, outer mucus; IM, inner mucus; Epi, epithelium. (B) Quantification of bacterial length in FISH images, as in A. Data are (A) representative or (B) pooled from three monocolonized or six SPF-Abx mice. Each point represents a single bacteria (n = 353–396); 7–19 images were analyzed from each animal. Statistical analysis was performed with a Kruskal-Wallis test with Dunn’s multiple comparison test. ****P < 0.0001. (C) Experimental scheme. To generate monocolonized animals (upper scheme), GF mice were orally inoculated with either BTWT or BTΔMAFF. For cocolonization (lower scheme), GF mice were orally inoculated with SFB 2 wk before BTWT or BTΔMAFF colonization, and mixtures of culturable strains of Clostridium species (Clost.spp: mixture of C. clostridioforme, C. sphenoides, C. oroticum, and a laboratory-isolated strain C. laboratory), Lactobacillus species (Lacto.spp: mixture of L. acidophilus, L. fermentum, and a laboratory-isolated strain L. laboratory), and the intestinal contents of SPF mice 4 wk after recovery from antibiotic treatment (Li-Cont (Abx→PBS)) were added to some groups at 2-wk intervals. (D) Principal component analysis of BTWT and BTΔMAFF RNA-Seq transcripts from cecal content from individual gnotobiotic mice mono- or cocolonized with increasing microbiota diversity, as depicted in C. Dashed circles indicate BTWT and BTΔMAFF clusters separated on the first two principal components and paired by colonization condition. Each dot represents an individual animal (n = 2–4 for each group).