The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane (IM) is in contact with the cytosol while the outer membrane (OM) contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide (LPS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments such as the gut, where the OM resistance to bile salts allows enteric bacteria to thrive. The components of the OM are the first to come in contact with a host upon infection and strongly modulate the interaction of symbiotic and pathogenic bacteria with their host. A clear understanding of the OM biogenesis proces is esential to understand host- pathogen interactions as well as a fundamental aspect of bacterial physiology. Outer membrane proteins (OMPs) are integral membrane proteins with ?-barrel structures embedded in the OM. Many OMPs are immunogenic and some of them serve as adhesins mediating adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the IM by the Sec translocation machinery . However, how these hydrophobic proteins cross the periplasm and insert specifically into the OM is poorly understood. A number of periplasmic chaperones and the BAM complex in the OM have been implicated in the transport and insertion of OMPs. In this proposal we will establish the mechanisms of OMP transport and assembly focusing on the BAM complex. We will (i) determine the structure of the BAM complex; (ii) test mechanistic hypotheses derived from the structures and (iii) develop an integrated model of OMP transport folding and insertion in the outer membrane. PUBLIC HEALTH RELEVANCE: Transport and assembly of outer membrane proteins is an essential process in bacteria required for viability. Therefore, it represents an attractive targetfor development of antimicrobial. These would be analogous to beta-lactams, which are effective by interfering with cell wall synthesis. This application seeks to understand the molecular mechanisms underlying outer membrane protein transport, folding and insertion.