TIME RESOLVED STUDIES OF CHEMOTACTIC EXCITATION

我们在这项工作中的长期目标是了解信号 细菌趋化性的过程。 我们将开发方法， 应用并监测对快速趋化刺激的反应， 并确定兴奋信号通路的特性。 考虑到鞭毛的随机转换，为了定量 在激发动力学的扰动，有必要时间分辨 大量细胞对刺激的反应行为， 在时间上是精确定义的。 将采用两种方法 结合：（1）闪光光解跳跃的趋化刺激， 微秒时间，和（2）计算机辅助运动分析， 自由游动细菌的行为反应， 毫秒分辨率 这种快速的化学效应物产生将 确保受体结合事件不是速率限制， 监测系统的分辨率足以记录 已知缓解延迟，缓解开始。 化学效应器跳跃将通过脉冲激光完成 激发光不稳定的笼状前体。 直接测量 鼠伤寒沙门氏菌和大肠杆菌的激发时间 coli为建立结构/功能提供了一个强有力的工具 关系，特别是当结合分子 对转换器和其他趋化性的遗传操纵 可能与这些细菌有关。 发展后， 运动分析程序，野生型细胞中的激发时间， 许多不同的转导介导的以及转导- 独立的（例如质子动力传感器）接收系统 将被分析。 如果有的话，转导甲基化的作用， 甲基化机制的动力学， 将评估依赖性和不依赖于换能器的激发。 我们将首先确定信号传导是否受受体或 野生型细胞中的受体后反应。 严格控制 质粒改变的菌株将用于调节 单独地或组合地发送信号部件。 连同 随着温度和pH值的变化， 不同的反应在信号通路限速， 提供用于订购已知产品的策略 趋化性基因的作用。 闪光光解技术 也将使细胞内离子的快速跳跃和小的 可能参与信号传导的代谢物（例如环 核苷酸和二价阳离子）。

Our long term objective in this work is to understand the signaling process in bacterial chemotaxis. We will develop methods for applying, and monitoring responses to, rapid chemotactic stimuli, and determine properties of the excitation signalling pathway. Given that flagella switch randomly, in order to quantitate perturbations in excitation kinetics it is necessary to time-resolve the behavior of large number of cells in response to stimuli which are precisely defined temporally. Two methodologies will be combined: (1) Flash photolytic jumps of chemotactic stimuli in microsecond times, and (2) computer-assisted motion analysis of the behavioral responses of free-swimming bacteria with 17 millisecond resolution. Such rapid chemoeffector generation will ensure that receptor binding events are not rate limiting and the resolution in the monitoring system is sufficient to register the onset of the response, given known response latencies. Chemoeffector jumps will be accomplished by pulse laser excitation of photoabile caged precursors. Direct measurement of the excitation time of Salmonella typhimurium and Escherichia coli provides a powerful tool for establishing structure/function relationships, especially when combined with the molecular genetic manipulations of the transducers and other chemotaxis components possible with these bacteria. After developement of the motion analysis routines, excitation times in wildtype cells for a number of different transducer-mediated as well as transducer- independent (e.g. protonmotive force sensor) reception systems will be analyzed. The role, if any, of transducer methylation and of the methylation machinery in the kinetics of both transducer- dependent and transducer-independent excitation will be assessed. We will first determine whether signaling is limited by receptor or post-receptor reactions in wildtype cells. Stringently controlled plasmid-altered strains will be used to modulate the levels of the signaling components singly or in combination. This, together with changes of temperature and pH, will allow us to make different reactions in the signaling pathway rate-limiting, providing a strategy for ordering products of the known chemotaxis genes in the pathway. The flash-photolytic technique will also make possible rapid intracellular jumps of ions and small metabolites potentially involved in signaling (e.g. cyclic nucleotides and divalent cations).