Sensory Transduction in Bacterial Chemotaxis

描述（由申请人提供）：细胞感知其环境的变化并作出反应，通常以全有或全无的方式调节运动性、生长、发育命运或突触功效。细菌趋化性是研究感觉传导的一个杰出的模型系统，其中机制可以在原子细节上理解。E.大肠杆菌是奈米科技的奇迹，只有一微米大小的细胞由数条螺旋状细丝推动，每一条细丝的底部由一个直径为50奈米的旋转马达驱动，马达的动力来自质子流。当细丝逆时针（CCW）旋转时，细胞就会稳定地向前“运行”。当一个或多个细丝顺时针（CW）旋转时，细胞“翻滚”并改变路线。细胞计数其环境中感兴趣的分子并延长被认为有利的运行。计数是由调节激酶活性的受体完成的，激酶磷酸化反应调节因子，当磷酸化时，反应调节因子扩散穿过细胞质并结合到鞭毛马达的基部，增加它们顺时针旋转的可能性。激酶活性被引诱剂结合抑制，并在适应过程中通过甲基化恢复。使用荧光融合蛋白在体内之间的荧光共振能量转移（FRET），我们将研究细胞随时间变化的刺激作为一个模型，其中甲基化率由激酶活性指定的测试的反应，我们将评估电机开关的机制。使用荧光漂白，我们将研究内部运动部件的相对运动。使用从纳米金球附着在没有鞭毛丝的细胞中的马达上散射的光，我们将研究马达在零负载附近的行为，这是一个质子和机械组件运动速率限制的领域。通过分析突变体，允许鞭毛形状的转换，通常是禁止的，我们将了解更多关于鞭毛推进的机制。虽然这项工作与微生物毒力直接相关，但它意味着研究基本的生物过程：化学感受，细胞内信号传导和化学渗透能转化为机械功。 公共卫生相关性：运动细菌测量周围环境中化学物质的浓度，并向它们认为更有利的区域移动。有鞭毛的细胞通过控制驱动螺旋桨的马达的旋转方向来做到这一点。我们的目标是在分子细节上了解该系统的遗传学、生物化学和生理学，在一个简单的模型系统中了解细胞如何与环境相互作用，这是所有生物的基本过程。

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. 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.