SENSORY TRANSDUCTION IN BACTERIA

我们的研究重点是大肠杆菌的趋化机制 对氧（嗜氧性）和相关反应，如氧化还原性 和甘油趋化性。 他们的反应与其他人不同。 趋化行为需要功能性电子传递 系统 Aer，一种新的黄素蛋白，Tsr，丝氨酸 化学感受器，最近被这个实验室鉴定为 用于氧、氧化还原和甘油（能量）趋化的传感器。 长期的目标是从分子的细节上了解 通过三个传感器进行信号处理。 具体目标包括： 1)验证黄素腺嘌呤二核苷酸（FAD）在 Aer senses氧化还原变化发布日期：05/07/1997打印日期： 10/22/1997在电子传输系统中，Tsr感测质子 原动力 2)研究结构与 Aer和Tsr在氧化还原电位和质子传感中的作用 原动力 3)验证质子动机 力/氧化还原电位是疏氧和氧化还原的共同信号。 E.大肠杆菌，将细菌引导到最佳的 质子动力最大的环境。 一个 跨学科方法-结合当代方法， 分子生物学、遗传学和生物化学， 本实验室开发的技术-将用于解决 为每个目标确定的关键研究问题。 的 电子传递系统在E.新用大肠杆菌 可用的结构。 Aer中FAD结合的推定残基 将通过定点诱变进行突变， 趋气性、FAD结合和中点还原潜力 测定 膜中Aer的拓扑结构将是 使用夹心Aer融合蛋白测定，其中PhoA或 LacZ被插入Aer的N-末端结构域内的读码框中。 H328的半胱氨酸扫描，选择组氨酸残基， 周质结构域和K1卷曲螺旋结构域周围的残基 的Tsr将用于鉴定Ph受体及其对 Tsr的质子动力（氧化还原）传感。的假设 趋氧性将细菌引导到氧气浓度， 最高的质子动力将被测试，同时使用 测量氧浓度和膜电位， 在氧浓度可以紧密地 控制。 阐明这些机制将提供重要的 对趋化性的原始途径的深入了解， 氧传感的一般原理（其中一些可能 适用于人的颈动脉体化学感受器）。

Our studies focus of the Escherichia coli mechanism of chemotaxis to oxygen (aerotaxis) and related responses, such as redox taxis and glycerol taxis. The responses are different from other chemotactic behaviors in requiring a functional electron transport system. Aer, a novel flavoprotein, and Tsr, the serine chemoreceptor, have been recently identified by this laboratory as the transducers for oxygen, redox and glycerol (energy) taxis. The long term goal is to understand, in molecular detail, the signal processing by thee transducers. The specific aims include: 1) Test the hypothesis that flavin adenine dinucleotide (FAD) in Aer senses redox changes Date Released:05/07/1997 Date Printed: 10/22/1997 in the electron transport system, and Tsr senses proton motive force. 2) Investigate the relationship between structure and function of Aer and Tsr in sensing redox potential and proton motive force. 3) Test the hypothesis that proton motive force/redox potential is a signal that is common to aerophobic and aerophilic responses in E. Coli, guiding bacteria to an optimal environment where the proton motive force is maximal. An interdisciplinary approach - combining contemporary methods of molecular biology, genetics and biochemistry with novel techniques developed in this laboratory - will be used to address the critical research questions identified for each aim. The electron transport system will be perturbed in E. Coli using newly available constructs. Putative residues for FAD binding in Aer will be mutated by site directed mutagenesis and the effect on aerotaxis, FAD binding and mid-point reduction potential determined. The topology of Aer in the membrane will be determined using a sandwich Aer fusion protein in which PhoA or LacZ is inserted in frame within the N-terminal domain of Aer. Cysteine scanning of H328, selected histidine residues in the periplasmic domain and residues around the K1 coiled coil domain of Tsr will be used to identify the Ph receptors and their effect on proton motive force (redox) sensing by Tsr. The hypothesis that aerotaxis guides bacteria to the oxygen concentration that supports the highest proton motive force will be tested using simultaneous measurement of oxygen concentration and membrane potential, under conditions where the oxygen concentration can be tightly controlled. Elucidating these mechanisms will provide important insights into a primordial pathway for chemotaxis, and also into general principles of oxygen sensing (some of which may be applicable to carotid body chemoreceptors in humans).