PROJECT SUMMARY Bacteroides is one of the most abundant genera of the human gut microbiota, representing nearly one-third of the total composition. Bacteroides spp. participate in gut homeostasis and the development of the immune system, being able to promote both health and disease states. Bacteroides spp. unlike other bacteria, produce large quantities of uniformly-sized outer membrane vesicles (OMVs) that present distinct protein composition when compared to the outer membrane (OM). These OMVs are composed mainly of lipoproteins with protease and glycosidase activity. Bacteroides OMVs have been proposed to play pivotal roles in immune modulation, nutrient degradation, and interbacterial mutualistic interactions. Despite their physiological relevance, no mechanism for OMVs biogenesis has been yet established. Recently, we have developed molecular tools that allow the differentiation between bona fide vesicles from lysis byproducts in live Bacteroides strains expressing differentially labeled OMVs- and OM-specific proteins. Moreover, based on specific OMVs markers fused to nano-luciferase, we developed the first high-throughput screen to identify genes involved in OMV biogenesis. The screening for mutants displaying hyper- or hypo- vesiculation resulted in the identification of genes involved in OMV biogenesis. Mutagenes of the gene encoding the protein BT_3341 completely abrogated OMV formation. Preliminary experiments suggest that this strain is not able to surface display the respective glycosylhydrolases required for degradation of glycans. Additionally, we identified two anti-sigma factors with unique structural features. These two proteins appear to span across the bacterial outer and inner membranes, connecting the extracellular space with the bacterial cytoplasm. Thus, we named these proteins Dual Membrane-spanning Anti-sigma factors (DMA) family. Mutations of DMA1 and DMA2 dramatically increase vesiculation, suggesting that they regulate OMV biogenesis. The goal of this proposal is to further understand the mechanism of OMV biogenesis in Bacteroides. In Aim 1, we propose to characterize the novel DMA family and their role in OMV biogenesis. In Aim 2 we will determine the role of BT_3341 and identify additional components of this machinery in OMV biogenesis. In Aim 3 will quantify and visualize OMV formation and evaluate the importance of OMV for fitness in vivo. Our work will generate novel basic knowledge on this poorly understood process in bacteria, and lay the foundation for in- depth investigations into the role of Bacteroides OMVs within the gut. Due to their suspected role in healthy and disease states, this research may lead in the future to the production of novel OMV-based therapies applicable to diseases involving gut dysbiosis, such as inflammatory bowel diseases (IBD).