Many species of bacteria swim by means of filamentous organelles called flagella. Flagellar motility contributes to the virulence of many disease-causing bacteria and is important to the normal functioning of many species in the environment. The flagellum consists of a slender helical filament that functions as a propeller, turned by a membrane-embedded motor that obtains its energy from the membrane ion gradient. Motors can turn either clockwise (CW) or counter- clockwise (CCW). Regulated reversals in motor direction are the basis for directed movement in response to particular stimuli such as nutrients or repellents. The direction of rotation is controlled by a large multi-protein complex called the switch complex. In E. coli and a number of other species, function of the flagellar motor is additionally regulated by two other proteins: YcgR, which binds the second messenger cyclic-di-GMP and inhibits motor function during the conversion from the motile to the biofilm (surface-associated) state, and H-NS, a protein that functions in binding DNA and regulating gene expression in addition to regulating the motor. Both YcgR and H-NS have been shown to act at the switch complex. Goals of the present research are to understand the direction-reversing mechanism of the flagellar switch complex, and the mechanisms of motor regulation by YcgR and H-NS.
Four aims are proposed. The first is to characterize the responses of the switch complex to the chemotactic signaling molecule CheY-phosphate. This will be accomplished using a combination of structural, biochemical, and advanced spectroscopic methods.
The second aim i s to elucidate the mechanisms of motor regulation by YcgR and H-NS. Interactions of these proteins with the motor will be examined using structural and biochemical methods, and effects on motor function will be studied using various measures of motor performance. Goals will be to understand how YcgR inhibits motility as cells begin entry into the biofilm state, which is a process important in many infections, and to clarify the logic underlying the dual functions of H-NS in both motility and gene regulation.
A third aim i s to extend studies of the switch complex, which have previously been carried out mainly in E. coli, to the gram-positive species B. subtilis. Gram-positive species are abundant in nature and of great importance in both medicine and ecology. Guided by information developed in the E. coli system, studies of the B. subtilis switch should proceed in a relatively efficient fashion and will greatly augment our understanding of motility in bacteria.
The final aim i s to begin development of a formal quantitative model of the flagellar switch. This will provide a framework for integrating the available structural and mechanistic information and for exploring the implications of the model in quantitative terms.

Public Health Relevance

Many bacteria swim by means of rotating filaments called flagella, and this ability to move is a factor in the virulence of many disease-causing species. The proposed research seeks to understand the structure and mechanism of a large protein complex that has central functions in powering and controlling bacterial flagella. The findings wil contribute to our understanding of the disease-causing abilities of E. coli, Salmonella, and many other motile, flagellated pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM064664-09A1
Application #
8693536
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Flicker, Paula F
Project Start
2001-12-01
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
9
Fiscal Year
2014
Total Cost
$397,690
Indirect Cost
$97,663
Name
University of Utah
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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Kim, Eun A; Panushka, Joseph; Meyer, Trevor et al. (2017) Biogenesis of the Flagellar Switch Complex in Escherichia coli: Formation of Sub-Complexes Independently of the Basal-Body MS-Ring. J Mol Biol 429:2353-2359
Kim, Eun A; Panushka, Joseph; Meyer, Trevor et al. (2017) Architecture of the Flagellar Switch Complex of Escherichia coli: Conformational Plasticity of FliG and Implications for Adaptive Remodeling. J Mol Biol 429:1305-1320
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Sircar, Ria; Borbat, Peter P; Lynch, Michael J et al. (2015) Assembly states of FliM and FliG within the flagellar switch complex. J Mol Biol 427:867-86
Boschert, Ryan; Adler, Frederick R; Blair, David F (2015) Loose coupling in the bacterial flagellar motor. Proc Natl Acad Sci U S A 112:4755-60
Kim, Eun A; Blair, David F (2015) Function of the Histone-Like Protein H-NS in Motility of Escherichia coli: Multiple Regulatory Roles Rather than Direct Action at the Flagellar Motor. J Bacteriol 197:3110-20
Sircar, Ria; Greenswag, Anna R; Bilwes, Alexandrine M et al. (2013) Structure and activity of the flagellar rotor protein FliY: a member of the CheC phosphatase family. J Biol Chem 288:13493-502
Paul, Koushik; Gonzalez-Bonet, Gabriela; Bilwes, Alexandrine M et al. (2011) Architecture of the flagellar rotor. EMBO J 30:2962-71

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