Abstract
ABSTRACT
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.