Determination of Gene‐Level Fitness Contributions Across the Mucin‐Utilization Network of Bacteroides thetaiotaomicro n

Host mucus is a major nutrient reservoir for gut Bacteroidota, yet the gene-level determinants of mucin utilization remain poorly defined. In this study, we used a genome-scale CRISPR interference library in Bacteroides thetaiotaomicron to systematically map fitness contributions across the mucin-utilization network, spanning surface accessibility, glycan degradation, and intracellular sugar assimilation. Our screen identified 1,256 genes with condition-dependent fitness divergence, which revealed a layered organization of mucin-dependent growth. A prominent mucin-specific fitness signature was enrichment of strains with repressed capsular polysaccharide loci. Subsequent competition and deletion experiments validated that specific capsules impede growth on mucin, supporting a surface-accessibility trade-off model. By contrast, most mucin-induced carbohydrate-binding and glycoside hydrolase genes showed negligible fitness contributions, consistent with functional redundancy in extracellular glycan capture and processing. Conversely, downstream sugar catabolism showed pronounced condition-specific constraints, with the galactose-utilization gene BT_0623, causing measurable fitness defects upon repression in both mucin- and galactose-containing media. Finally, comparison with murine colon CRISPRi data revealed broad concordance with the in vitro mucin fitness landscape, while also highlighting environment-specific differences, in CPS- and polysaccharide degradation-associated functions. Together, these results define mucin utilization as a layered fitness architecture governed by surface-accessibility costs, redundant extracellular glycan processing, and downstream metabolic constraints.