Cells sense changes in their environment and respond, often in an all-or-none fashion, modulating motility, growth, developmental fate, or synaptic efficacy. Bacterial chemotaxis is a pre-eminent model system for studies of sensory transduction, where mechanisms can be understood in atomic detail. E. coli is a nanotechnological marvel, with cells only a micron in size propelled by several helical filaments, each driven at its base by a rotary motor 50 nm in diameter powered by a proton flux. When the filaments spin counterclockwise (CCW), a cell "runs" steadily forward. When one or more filaments spin clockwise (CW), the cell "tumbles" and changes course. A cell counts molecules of interest in its environment and extends runs deemed favorable. The counting is done by receptors that regulate the activity of a kinase that phosphorylates a response regulator that, when phosphorylated, diffuses across the cytoplasm and binds to the base of the flagellar motors, increasing the probability that they spin CW. The kinase activity is depressed by attractant binding and restored, during adaptation, by methylation. Using fluorescence resonance energy transfer (FRET) between fluorescent fusion proteins in vivo, we will study responses of cells to time-varying stimuli as a test of a model in which methylation rate is specified by kinase activity, and we will assess mechanisms for motor switching. Using fluorescence bleaching, we will study the relative motion of internal motor components. Using light scattered from nano-gold spheres attached to motors in cells without flagellar filaments, we will study the behavior of the motor near zero load, a domain in which movement of protons and mechanical components are rate limiting. By analyzing mutants that allow transformations in flagellar shape that are normally forbidden, we will learn more about the mechanics of flagellar propulsion. While this effort is directly relevant to microbial virulence, it is meant as a study of fundamental biological processes: chemoreception, intracellular signaling, and conversion of chemiosmotic energy to mechanical work.

Public Health Relevance

Motile bacteria measure the concentrations of chemicals in their surroundings and move toward regions that they deem more favorable. Cells with flagella do this by controlling the direction of rotation of motors that drive helical propellers. The goal is to understand the genetics, biochemistry, and physiology of this system in molecular detail, i.e., to understand in a simple model system how cells interact with their environment, a process basic to all living things.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI016478-33
Application #
8204853
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Korpela, Jukka K
Project Start
1986-12-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
33
Fiscal Year
2012
Total Cost
$752,954
Indirect Cost
$304,767
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Branch, Richard W; Sayegh, Michael N; Shen, Chong et al. (2014) Adaptive remodelling by FliN in the bacterial rotary motor. J Mol Biol 426:3314-24
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
Stern, Alan S; Berg, Howard C (2013) Single-file diffusion of flagellin in flagellar filaments. Biophys J 105:182-4
Yuan, Junhua; Berg, Howard C (2013) Ultrasensitivity of an adaptive bacterial motor. J Mol Biol 425:1760-4
Lele, Pushkar P; Hosu, Basarab G; Berg, Howard C (2013) Dynamics of mechanosensing in the bacterial flagellar motor. Proc Natl Acad Sci U S A 110:11839-44
Wu, Yilin; Hosu, Basarab G; Berg, Howard C (2011) Microbubbles reveal chiral fluid flows in bacterial swarms. Proc Natl Acad Sci U S A 108:4147-51
Yuan, Junhua; Berg, Howard C (2010) Thermal and solvent-isotope effects on the flagellar rotary motor near zero load. Biophys J 98:2121-6
Shimizu, Thomas S; Tu, Yuhai; Berg, Howard C (2010) A modular gradient-sensing network for chemotaxis in Escherichia coli revealed by responses to time-varying stimuli. Mol Syst Biol 6:382
Yuan, Junhua; Fahrner, Karen A; Turner, Linda et al. (2010) Asymmetry in the clockwise and counterclockwise rotation of the bacterial flagellar motor. Proc Natl Acad Sci U S A 107:12846-9
Yuan, Junhua; Fahrner, Karen A; Berg, Howard C (2009) Switching of the bacterial flagellar motor near zero load. J Mol Biol 390:394-400

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