Motile bacteria possess a stimulus-response system in which the input is the local concentration of a chemical attractant and the output is the motion of the cell. In Escherichia coli, the input is determined by the occupancy of a number of specific receptors, while the output is determined by the sense of rotation of about six flagellar filaments. When the filaments turn counter- clockwise, the cell swims steadily along a smooth path (it runs); when they turn clockwise, the cell moves in a highly erratic manner with little net displacement (it tumbles). Normally, these modes alternate, and the cell executes a three-dimensional random walk. The receptor occupancy over the past second is compared to the receptor occupancy over the previous three seconds, and the cell responds to the difference: runs are extended that carry the cell in a favorable direction. The present research focuses on the molecular events that couple the receptors to the flagella, i.e., the events required for chemotactic signaling. Four gene products are known to be involved, CheW, CheA, CheY and CheZ. CheY appears to be a second messenger that is activated by the receptors and diffuses to the flagella, where it enhances clockwise rotation (tumbles). Evidence from in vitro studies suggests that this activation results from phosphorylation, catalyzed by CheA. However, in vivo, CheY can be activated in the absence of CheA following the addition of acetate, via a pathway that appears to involve acetyl-CoA synthetase. Reconstitution experiments will be carried out in cells deleted for most of the chemotaxis genes to determine optimum stoichiometries for signaling for CheW, CheA and CheY, and to learn more about the kinetics of interaction of CheY, CheZ and the flagellar switch. Behavioral responses will be monitored in a tethered-cell assay. The acs gene will be clone and sequenced and its gene product characterized. Evidence for interaction with other components will be sought by a variety of means, including an in vitro search for acetylation or adenylylation. An in vitro motor assay will be developed that will allow rapid comparisons of the effects on switching of purified cytoplasmic components. Assays for judging the motility and chemotaxis of cell populations will be improved. Finally, the rotational behavior of tethered minicells will be analyzed to determine the switching statistics relevant when motors run at high speed. This will improve our understanding of the synchronization of different flagella on the same cell and allow more accurate predictions of swimming behavior based on the results of tethered-cell assays. The long-range goal is a description at the molecular level of sensory transduction in bacterial chemotaxis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI016478-12
Application #
3480871
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1994-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Hosu, Basarab G; Berg, Howard C (2018) CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy. Biophys J 114:641-649
Lele, Pushkar P; Roland, Thibault; Shrivastava, Abhishek et al. (2016) The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backward. Nat Phys 12:175-178
Hosu, Basarab G; Nathan, Vedavalli S J; Berg, Howard C (2016) Internal and external components of the bacterial flagellar motor rotate as a unit. Proc Natl Acad Sci U S A 113:4783-7
Shrivastava, Abhishek; Roland, Thibault; Berg, Howard C (2016) The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins. Biophys J 111:1008-13
Lele, Pushkar P; Shrivastava, Abhishek; Roland, Thibault et al. (2015) Response thresholds in bacterial chemotaxis. Sci Adv 1:e1500299
Lele, Pushkar P; Berg, Howard C (2015) Switching of bacterial flagellar motors [corrected] triggered by mutant FliG. Biophys J 108:1275-80
Shrivastava, Abhishek; Lele, Pushkar P; Berg, Howard C (2015) A rotary motor drives Flavobacterium gliding. Curr Biol 25:338-341
Shrivastava, Abhishek; Berg, Howard C (2015) Towards a model for Flavobacterium gliding. Curr Opin Microbiol 28:93-7
Branch, Richard W; Sayegh, Michael N; Shen, Chong et al. (2014) Adaptive remodelling by FliN in the bacterial rotary motor. J Mol Biol 426:3314-3324
Wang, Fangbin; Yuan, Junhua; Berg, Howard C (2014) Switching dynamics of the bacterial flagellar motor near zero load. Proc Natl Acad Sci U S A 111:15752-5

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