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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI080709-04
Application #
8417662
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Taylor, Christopher E,
Project Start
2009-05-22
Project End
2017-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$348,367
Indirect Cost
$113,367
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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
Sandoval, Cristina M; Baker, Susan L; Jansen, Katarina et al. (2011) Crystal structure of BamD: an essential component of the ýý-Barrel assembly machinery of gram-negative bacteria. J Mol Biol 409:348-57
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
Gatzeva-Topalova, Petia Zvezdanova; Warner, Lisa Rosa; Pardi, Arthur et al. (2010) Structure and flexibility of the complete periplasmic domain of BamA: the protein insertion machine of the outer membrane. Structure 18:1492-501
Gatzeva-Topalova, Petia Z; Walton, Troy A; Sousa, Marcelo C (2008) Crystal structure of YaeT: conformational flexibility and substrate recognition. Structure 16:1873-81