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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Taylor, Christopher E,
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Doerner, Pamela Arden; Sousa, Marcelo C (2017) Extreme Dynamics in the BamA ?-Barrel Seam. Biochemistry 56:3142-3149
Walder, Robert; LeBlanc, Marc-André; Van Patten, William J et al. (2017) Rapid Characterization of a Mechanically Labile ?-Helical Protein Enabled by Efficient Site-Specific Bioconjugation. J Am Chem Soc 139:9867-9875
Warner, Lisa R; Gatzeva-Topalova, Petia Z; Doerner, Pamela A et al. (2017) Flexibility in the Periplasmic Domain of BamA Is Important for Function. Structure 25:94-106
Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L et al. (2016) BamA POTRA Domain Interacts with a Native Lipid Membrane Surface. Biophys J 110:2698-709
Bergal, Hans Thor; Hopkins, Alex Hunt; Metzner, Sandra Ines et al. (2016) The Structure of a BamA-BamD Fusion Illuminates the Architecture of the ?-Barrel Assembly Machine Core. Structure 24:243-51
Edwards, Devin T; Faulk, Jaevyn K; Sanders, Aric W et al. (2015) Optimizing 1-?s-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope. Nano Lett 15:7091-8
Doerner, Pamela Arden; Sousa, Marcelo Carlos (2015) Small Angle X-ray Scattering (SAXS) Characterization of the POTRA Domains of BamA. Methods Mol Biol 1329:149-55
Jansen, Katarina Bartoš; Baker, Susan Lynn; Sousa, Marcelo Carlos (2015) Crystal structure of BamB bound to a periplasmic domain fragment of BamA, the central component of the ?-barrel assembly machine. J Biol Chem 290:2126-36
Jansen, Katarina Bartoš; Baker, Susan Lynn; Sousa, Marcelo Carlos (2012) Crystal structure of BamB from Pseudomonas aeruginosa and functional evaluation of its conserved structural features. PLoS One 7:e49749
Warner, Lisa R; Varga, Krisztina; Lange, Oliver F et al. (2011) Structure of the BamC two-domain protein obtained by Rosetta with a limited NMR data set. J Mol Biol 411:83-95

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