Abstract The human intestine is colonized with ~1014 microorganisms that make up the gut microbiota. Approximately 40% of the bacteria inhabiting the human gut belong to the phylum Bacteroidetes, which can promote both healthy and diseased states. Long-term colonization of Bacteroidetes in the human gut is largely due to their ability to utilize dietary polysaccharides that are indigestible by the host and endogenous host glycans. Polysaccharide degradation in Bacteroidetes is controlled by a diverse array of multi-gene polysaccharide utilization loci (PUL), which encode the receptors, glycosidases (GH), and transporters needed to sense and digest various glycans. Through proteomic analyses, we determined that numerous GH are preferentially packaged into outer membrane vesicles (OMV). OMV are spherical, membranous structures generated by blebbing of the outer membrane (OM) of Gram-negative bacteria. Bacteroidetes OMV have been proposed to play important roles in immune modulation, maintenance of intestinal homeostasis, and promotion of interbacterial mutualistic interactions. Despite their increasing importance, no definitive mechanism for OMV biogenesis has been established, and OMV biology remains one of the least studied fundamental processes in microbiology. We have previously demonstrated that Bacteroides fragilis and B. thetaiotaomicron produce significant amounts OMV that are homogenous in size and shape. The OMV-specific proteome showed a high prevalence of GH and proteases. The GHs specifically packed into OMV are usually encoded within PUL, and their cognate transporters are not detected in OMV, which suggests that PUL systems partition between OM and OMV. We constructed fusions of OMV- and OM-specific proteins with fluorescent proteins and employed them as markers for live fluorescence microscopy. For the first time, we visualized the formation of OMV. The use of different fluorescent markers allowed us to differentiate between bona fide OMV and lysis by products. Our preliminary data demonstrate that, in Bacteroidetes, OMV are the result of a highly orchestrated physiological process. In aim 1 of this proposal we will employ biochemistry and mass spectrometry to investigate how OMV cargo is regulated to maximize the digestion of dietary and endogenous host glycans. In aim 2, we will employ fusions between OMV proteins and luciferase to investigate the molecular machinery required for OMV biogenesis. Understanding these processes may lead, in the future, to manipulation of bacterial vesiculation for innovative interventions to treat pathologies involving gut dysbiosis, such as Crohn’s disease and IBD.