The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and the aqueous compartment termed the periplasm that is located between the membranes. The long term goal of this grant has always been to understand the mechanisms of envelope biogenesis in molecular terms using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins (LP), and ?-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified most, if not al, of the essential proteins required to transport LPS and OMPs across the periplasm and assemble these molecules in the OM. Work in the previous funding period has provided functional insights into the OM components of these two assembly machines, LptDE and BamABCDE respectively, and their associated periplasmic chaperones. Models derived from this work provide experimentally testable predictions that will guide our efforts. To address the important question of PL transport to the OM, we will develop a technique, using fluorescence microscopy, to follow PL movement between membranes in living cells. This will facilitate the investigation of conditions and mutations that affect this poorly understood, essential process. We have also identified OM lipoproteins in E. coli that are surface exposed, and we will develop methods to probe the mechanism that catalyzes this process. Our interest in envelope stress responses has led to the discovery of a novel role for PldA in the biogenesis of outer membrane vesicles and we will characterize this function. We have also discovered that the Cpx response can reduce the reactive oxygen species produced by certain antibiotics, and we will test the hypothesis that it does so by increasing the synthesis and/or the assembly of cytochrome bo3. LacZ can fold in the periplasm if DsbA is removed, and we will identify the Cpx regulon member(s) that catalyzes this folding reaction, and elucidate its normal function in periplasmic physiology. These studies will provide important information regarding the structure, function, and the interactions within, and between, the cellular components that catalyze envelope biogenesis. Multi-drug resistant Gram- negative bacteria are a growing concern. Insights obtained from this work should facilitate the design of novel antibacterial agents.

Public Health Relevance

The outer membrane of Gram-negative bacteria serves as a barrier to protect these organisms from toxic compounds, including antibiotics. We have identified important cellular components that function to assemble and maintain this essential cellular organelle, and we wish to understand how these components work in molecular detail. Such knowledge would facilitate the development of new antibiotics that would combat the growing problem of antibiotic resistance in this important class of pathogens.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Ainsztein, Alexandra M
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Princeton University
Schools of Arts and Sciences
United States
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Konovalova, Anna; Perlman, David H; Cowles, Charles E et al. (2014) Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of ?-barrel proteins. Proc Natl Acad Sci U S A 111:E4350-8
Maloj?i?, Goran; Andres, Dorothee; Grabowicz, Marcin et al. (2014) LptE binds to and alters the physical state of LPS to catalyze its assembly at the cell surface. Proc Natl Acad Sci U S A 111:9467-72
Sutterlin, Holly A; Zhang, Sisi; Silhavy, Thomas J (2014) Accumulation of phosphatidic acid increases vancomycin resistance in Escherichia coli. J Bacteriol 196:3214-20
Dwyer, Robert S; Malinverni, Juliana C; Boyd, Dana et al. (2014) Folding LacZ in the periplasm of Escherichia coli. J Bacteriol 196:3343-50
Schwalm, Jaclyn; Mahoney, Tara F; Soltes, Garner R et al. (2013) Role for Skp in LptD assembly in Escherichia coli. J Bacteriol 195:3734-42
Grabowicz, Marcin; Yeh, Jennifer; Silhavy, Thomas J (2013) Dominant negative lptE mutation that supports a role for LptE as a plug in the LptD barrel. J Bacteriol 195:1327-34
Ricci, Dante P; Schwalm, Jaclyn; Gonzales-Cope, Michelle et al. (2013) The activity and specificity of the outer membrane protein chaperone SurA are modulated by a proline isomerase domain. MBio 4:
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
Dwyer, Robert S; Ricci, Dante P; Colwell, Lucy J et al. (2013) Predicting functionally informative mutations in Escherichia coli BamA using evolutionary covariance analysis. Genetics 195:443-55
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

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