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, 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.