Sensory Transduction in Bacterial Chemotaxis

Harvard University, Cambridge MA

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Abstract

? DESCRIPTION (provided by applicant): 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.