Functional Specialization of Cellular and Extracellular Fractions of Bacteroides thetaiotaomicron Contributes to Glycan Utilization

Bacteroides thetaiotaomicron (Bt) degrades dietary glycans via carbohydrate-active enzymes (CAZymes), but their spatial distribution and functional coordination remain unclear. In this study, we characterized the “labor division” during glycan degradation across four bacterial fractions: cytoplasm, outer membrane, extracellular vesicles, and supernatant. All fractions displayed hydrolytic activity. Proteomic analysis identified abundant glycoside hydrolases, mainly GH32 enzymes encoded by PUL22, and their components varied greatly. Untargeted metabolomics identified 45 shared differential metabolites (e.g., carbohydrates and short-chain fatty acids), indicating distinct hydrolytic capacities among fractions. Notably, cross-feeding experiments confirmed that Bt EVs-mediated degradation can support the growth of recipient bacteria. Collectively, our results reveal a synergistic multicomponent strategy for glycan degradation in Bacteroides , offering new insights into glycan utilization and laying a foundation for developing postbiotics and functional microbial products.