Transport of molecules across biological membranes is a fundamental process shared by all living cells. In Gram-negative bacteria, such as Escherichia coil, transport occurs across two membranes, of which the outer membrane represents the first permeability barrier. Outer membrane protein-mediated transport is needed to bring nutrients inside and expel unwanted metabolites and inhibitors. Ironically, toxic biomolecules and infectious agents, present in the hostile environment in which bacteria live, frequently exploit these outer membrane proteins to gain entry into the cell. The focus of this research is a unique outer membrane protein, TolC, which mediates the diffusion of a diverse group of molecules including the export of alpha-hemolysin and the efflux of antibiotics. Despite the resolution of TolC's three-dimensional structure, the role of individual residues and the mechanism by which it accomplishes the transport of a diverse group of molecules remains poorly understood. Moreover, it is unknown how TolC interacts with other proteins of the inner membrane to form the complexes that facilitate the two-way transport activity. The present study is being undertaken to examine the role of TolC in export (antibiotic) and import (colicin E1 ), as well as to examine its interactions with other proteins of the transport complexes. Identification of functionally critical TolC residues and the various protein-protein interactions that must occur to accomplish the diverse transport activities will be achieved through employing a combination of genetic, structural, and biochemical approaches; available preliminary data concerning all aspects of the project strongly reflect their feasibility.The presence of TolC and its homologues in other Gram-negative bacteria demonstrates the broader relevance of this unique multifunctional protein. Besides the obvious role of TolC in antibiotic resistance, its ability to mediate the secretion of a variety of virulence factors in pathogenic bacteria makes it a medically important subject of investigation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Chin, Jean
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Arizona State University-Tempe Campus
Schools of Arts and Sciences
United States
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Weeks, Jon W; Bavro, Vassiliy N; Misra, Rajeev (2014) Genetic assessment of the role of AcrB ?-hairpins in the assembly of the TolC-AcrAB multidrug efflux pump of Escherichia coli. Mol Microbiol 91:965-75
Weeks, Jon W; Celaya-Kolb, Teresa; Pecora, Sara et al. (2010) AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening. Mol Microbiol 75:1468-83
Misra, Rajeev; Bavro, Vassiliy N (2009) Assembly and transport mechanism of tripartite drug efflux systems. Biochim Biophys Acta 1794:817-25
Bavro, Vassiliy N; Pietras, Zbigniew; Furnham, Nicholas et al. (2008) Assembly and channel opening in a bacterial drug efflux machine. Mol Cell 30:114-21
Masi, Muriel; Vuong, Phu; Humbard, Matthew et al. (2007) Initial steps of colicin E1 import across the outer membrane of Escherichia coli. J Bacteriol 189:2667-76
Augustus, Anne Marie; Celaya, Teresa; Husain, Fasahath et al. (2004) Antibiotic-sensitive TolC mutants and their suppressors. J Bacteriol 186:1851-60
Gerken, Henri; Misra, Rajeev (2004) Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. Mol Microbiol 54:620-31
Husain, Fasahath; Humbard, Matthew; Misra, Rajeev (2004) Interaction between the TolC and AcrA proteins of a multidrug efflux system of Escherichia coli. J Bacteriol 186:8533-6