The outer membrane (OM) is an essential organelle of Gram-negative bacteria which acts as a selective permeability barrier to protect the cell from toxic compounds like antibiotics. One of the key steps in OM biogenesis is assembly of integral beta-barrel proteins (OMPs) that span the OM. The machinery required for OMP assembly has recently been discovered in Escherichia coli. Called the BAM complex, this multi- subunit complex contains one molecule each of an essential OMP BamA, and four associated OM lipoproteins BamB, BamC, BamD, and BamE. BamA and BamD are both essential for growth;in contrast, bamB, bamC, and bamE mutants are all viable, although each mutant has defects in OMP assembly. BamCDE is now known to form a stable subcomplex that interacts with BamA, although the function of this subcomplex is unclear. The goal of this proposal is to determine the role of the BamCDE subcomplex during OMP assembly in E. coli.
In Specific Aims 1 and 2, I propose several genetic approaches to probe the interactions among the BamCDE subcomplex members and between BamCDE and BamA. I will generate a collection of BamD mutants to identify residues of this protein that are required for normal OMP assembly.
In Specific Aim 3, I will characterize the mutants I generate using suppressor analysis. Each mutant will also be characterized biochemically to identify novel OMP assembly intermediates.
Assembly of proteins in the outer membrane is an essential process that is conserved from bacteria to man. Understanding how this process works in bacteria could lead to identification of novel antibiotic targets. Results from these studies could also illuminate the mechanisms of protein trafficking and protein assembly pathways in higher organisms including plants and humans.
|Rigel, Nathan W; Ricci, Dante P; Silhavy, Thomas J (2013) Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for ?-barrel assembly in Escherichia coli. Proc Natl Acad Sci U S A 110:5151-6|
|Rigel, Nathan W; Schwalm, Jaclyn; Ricci, Dante P et al. (2012) BamE modulates the Escherichia coli beta-barrel assembly machine component BamA. J Bacteriol 194:1002-8|
|Rigel, Nathan W; Silhavy, Thomas J (2012) Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Curr Opin Microbiol 15:189-93|