Primary energy transduction in biological systems is derived from two coupled sources. Oxidative phosphorylation (and substrate level phosphorylation) ultimately generate both an ion motive force and ATP in reactions where the reactants and products are physically and spatially coupled. Both of these energy sources are used to drive active transport of nutrients across the cytoplasmic membrane. The outer membranes of Gram-negative bacteria present a special problem: they are unenergized and they are impermeant to molecules of greater than 600 Da. In order to obtain vital nutrients, these organisms have developed a sophisticated system whereby energy generated at the cytoplasmic membrane (in the form of proton motive force) can be transduced to proteins in the outer membrane for active transport of these vital nutrients into the periplasmic space between the two membranes. Using E. coli as a model system, it has become clear that several proteins are involved in this process. The core of the energy transducing system is TonB protein, which can contact proteins in both the cytoplasmic and outer membranes. Two cytoplasmic membrane proteins, ExbB and ExbD appear to be required to energize TonB and then recycle it, following energy transduction. A variety of TonB-dependent outer membrane receptors exist. The mechanism of energy transduction across space--which may constitute a new paradigm--is not well understood. Current data suggest that TonB shuttles back and forth between the cytoplasmic and outer membranes. The roles that ExbB and ExbD play in this process are not well understood. Furthermore, there is evidence that additional uncharacterized proteins are involved in this process.
The aims of this proposal are directed toward investigating these areas. Specifically, we will 1) resolve the composition and stoichiometry of these energy-transducing complexes, 2) determine if the physical shuttling of TonB between membranes is an essential feature of energy transduction, 3) elucidate the roles of ExbB and ExbD, 4) define the primary interactions of TonB with the outer membrane receptors and other outer membrane components, 5) determine the mechanism by which TonB physically stores energy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042146-10
Application #
6179702
Study Section
Special Emphasis Panel (ZRG1-BM-1 (01))
Program Officer
Shapiro, Bert I
Project Start
1991-08-01
Project End
2003-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
10
Fiscal Year
2000
Total Cost
$327,716
Indirect Cost
Name
Washington State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Gresock, Michael G; Postle, Kathleen (2017) Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo. J Bacteriol 199:
Baker, Kristin R; Postle, Kathleen (2013) Mutations in Escherichia coli ExbB transmembrane domains identify scaffolding and signal transduction functions and exclude participation in a proton pathway. J Bacteriol 195:2898-911
Bulathsinghala, Charles M; Jana, Bimal; Baker, Kristin R et al. (2013) ExbB cytoplasmic loop deletions cause immediate, proton motive force-independent growth arrest. J Bacteriol 195:4580-91
Ollis, Anne A; Postle, Kathleen (2012) ExbD mutants define initial stages in TonB energization. J Mol Biol 415:237-47
Ollis, Anne A; Kumar, Aruna; Postle, Kathleen (2012) The ExbD periplasmic domain contains distinct functional regions for two stages in TonB energization. J Bacteriol 194:3069-77
Ollis, Anne A; Postle, Kathleen (2012) Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo. J Bacteriol 194:3078-87
Ollis, Anne A; Postle, Kathleen (2011) The same periplasmic ExbD residues mediate in vivo interactions between ExbD homodimers and ExbD-TonB heterodimers. J Bacteriol 193:6852-63
Jana, Bimal; Manning, Marta; Postle, Kathleen (2011) Mutations in the ExbB cytoplasmic carboxy terminus prevent energy-dependent interaction between the TonB and ExbD periplasmic domains. J Bacteriol 193:5649-57
Swayne, Cheryl; Postle, Kathleen (2011) Taking the Escherichia coli TonB transmembrane domain ""offline""? Nonprotonatable Asn substitutes fully for TonB His20. J Bacteriol 193:3693-701
Postle, Kathleen; Kastead, Kyle A; Gresock, Michael G et al. (2010) The TonB dimeric crystal structures do not exist in vivo. MBio 1:

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