TIME RESOLVED STUDIES OF CHEMOTACTIC EXCITATION

我们这项工作的长期目标是理解信号 细菌趋化性的过程。我们将制定方法，以 施加并监测对快速趋化刺激的反应， 并确定该激发信号通路的特性。 给出随机的鞭毛切换，为了定量 激发动力学中的摄动有必要进行时间分辨 大量细胞对刺激做出反应的行为 都是在时间上精确定义的。将有两种方法 综合：(1)趋化刺激的闪光光解跳跃 以及(2)计算机辅助的运动分析 自由游动细菌对17种细菌的行为反应 毫秒级分辨率。如此快速的化学效应器的产生将 确保受体结合事件不是速率限制，并且 监测系统中的分辨率足以登记 在已知响应延迟的情况下，响应开始。 化学效应器跳跃将由脉冲激光完成 不耐光笼前体物的激发。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/功能提供了一个强大的工具 关系，特别是当与分子结合时 转导和其他趋化作用的遗传操作 这些细菌可能含有的成分。发展后的 运动分析例程，野生型细胞中的激发时间 一些不同的换能器中介以及换能器- 独立的(例如质子力传感器)接收系统 将会被分析。转导子甲基化的作用，如果有的话 在两种转导分子的动力学中的甲基化机制- 独立激励和传感器独立激励将被评估。 我们将首先确定信号是受受体还是受限制 野生型细胞中的受体后反应。严格控制 质粒改变的菌株将被用来调节 单个或组合的信令组件。这一次，一起 随着温度和pH的变化，将使我们能够 信号通路限速中的不同反应， 提供订购已知产品的策略 该途径中的趋化基因。闪光光解技术 也将使离子和小分子在细胞内的快速跳跃成为可能 可能参与信号传递的代谢物(如环状 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).