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, noted for its relative simplicity, high sensitivity, wide dynamic range, and robustness. 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 (Fig. 2) 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 a switch complex at the cytoplasmic face of the flagellar motors (Fig. 1), increasing the probability that the motors spin CW. The kinase activity is depressed by attractant binding and restored, during adaptation, by methylation. Motors adapt on a longer time scale, adding or subtracting components in order to remain sensitive to changes in the output of the chemotaxis signaling pathway or to provide adequate torque. We will use total internal reflection fluorescence (TIRF) microscopy to monitor changes in the makeup of the motor of a tethered cell as a function of CW bias, measured by phase-contrast imaging. How precise is motor adaptation? We will use electro-rotation to change the load on a tethered cell, so that we can learn more about stator remodeling: do torque-generating units leave the motor when the load decreases, and if so, at what rate? We will use TIRF microscopy to learn whether changes in CW bias observed during stator remodeling are due to recruitment of switch components. Are there other mechanisms used by the motor to remain sensitive to changes in the output of the chemotaxis signaling pathway? We will use fluorescence bleaching to confirm that the switch complex rotates and fluorescence anisotropy as an assay for energy transfer between identical fluorophores (homo-FRET) in order to monitor changes in conformation of multimeric motor components. We will try to label motor components that malfunction when fused to fluorescent proteins with fluorescent amino acids. How does motor remodeling affect other factors known to modulate motor function, e.g., H-NS and YcgR, and how do these factors affect motor remodeling? We will use differential interference contrast (DIC) microscopy to confirm that flagellar filaments grow at a constant rate and to learn whether there is a limit to filament growt. While this effort is directly relevant to microbial behavior and virulence, it is meant as a study f fundamental biological processes: chemoreception, intracellular signaling, and effector remodeling.
Motile bacteria measure the concentrations of chemicals in their surroundings and move toward regions that they deem more favorable, a strategy that enhances virulence. Cells with flagella do this by controlling the direction of rotation of motors that drive helical flagellar filaments (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 sense and respond to changes in their environment, a process basic to all living things.
| Turner, Linda; Berg, Howard C (2018) Labeling Bacterial Flagella with Fluorescent Dyes. Methods Mol Biol 1729:71-76 |
| 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 |
| Shrivastava, Abhishek; Patel, Visha K; Tang, Yisha et al. (2018) Cargo transport shapes the spatial organization of a microbial community. Proc Natl Acad Sci U S A 115:8633-8638 |
| Ko, William; Lim, Sookkyung; Lee, Wanho et al. (2017) Modeling polymorphic transformation of rotating bacterial flagella in a viscous fluid. Phys Rev E 95:063106 |
| Hughes, Kelly T; Berg, Howard C (2017) The bacterium has landed. Science 358:446-447 |
| Berg, Howard C (2017) The flagellar motor adapts, optimizing bacterial behavior. Protein Sci 26:1249-1251 |
| 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 |
| Turner, Linda; Ping, Liam; Neubauer, Marianna et al. (2016) Visualizing Flagella while Tracking Bacteria. Biophys J 111:630-639 |
| 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 |
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