SENSORY TRANSDUCTION IN BACTERIA

这些研究探讨了大肠杆菌对氧气的趋化机制以及相关的反应，如氧化还原趋化和甘油趋化。这些反应需要Aer或TSR转导，与其他趋化行为不同的是需要一个功能正常的电子传输系统。Aer是一种黄素蛋白，具有N端PAS输入域和C端信号域。长期目标是从分子上详细解释Aer的信号转导。具体目标包括：1)确定Aer与电子传递系统之间的信号通路。2)明确PAS和接头/HAMP结构域在Aer中从PAS结构域到C-末端信号域的信号通路中的作用。3)测量大肠杆菌中的质子动力作为氧浓度的函数，并确定共同的信号(细胞能级的变化)是否可以介导对氧的嗜氧和厌氧反应；4)通过分析Aer基因的转录调控来研究Aer在氧和氧化还原反应中的生理作用。一种跨学科的方法，结合分子生物学、遗传学和生物化学的当代方法与本实验室开发的新技术，将被用来检验每个目标的假设。通过半胱氨酸置换或低保真的PCR扩增，Aer Pas和连接子/HAMP结构域中可能的蛋白质-蛋白质接触结构域将发生突变，并确定其对趋气性、FAD结合、蛋白质折叠和中点电位的影响。Aer PAS结构域与电子传输系统和RAMP结构域的相互作用将通过酵母双杂交、交联和第二位点抑制物分析来定义。以Aer-LacZ融合为记者，我们将确定在大肠杆菌中Aer基因和趋气性的调节是否与低氧对新陈代谢的控制相协调。阐明Aer的FAD-PAS结构域对氧敏感的机制可能有助于深入了解PAS结构域在人体氧传感器中的作用。

These studies investigate the Escherichia coli mechanism of chemotaxis to oxygen (aerotaxis) and related responses, such as redox taxis and glycerol taxis. The responses require the Aer or Tsr transducer, and differ from other chemotactic behaviors in requiring a functional electron transport system. Aer is a flavoprotein that has a N-terminal PAS input domain-and a C-terminal signaling domain. The long-term goal is to explain, in molecular detail, signal transduction by Aer. The specific aims include: 1) Determine the signaling pathway between Aer and the electron transport system. 2) Define the roles of the PAS and linker/HAMP domains in the signaling pathway from the PAS domain to the C-terminal signaling domain in Aer. 3) Measure the proton motive force in E. coli as a function of the oxygen concentration and determine whether a common signal (change in cell energy level) can mediate the aerophilic and aerophobic responses to oxygen and 4) Investigate the physiological role of Aer in oxygen and redox sensing through analysis of the transcriptional regulation of the aer gene. An interdisciplinary approach, combining contemporary methods of molecular biology, genetics and biochemistry with novel techniques developed in this laboratory, will be used to test the hypothesis for each aim. Putative protein-protein contact domains in the Aer PAS and linker/HAMP domains will be mutated by cysteine replacement or low-fidelity PCR amplification, and the effect on aerotaxis, FAD-binding, protein-folding and mid-point potential determined. Interactions of the Aer PAS domain with the electron transport system and RAMP domains will be defined by yeast two-hybrid, cross linking and second-site suppressor analyses. Using aer-lacZ fusions as reporters, we will determine whether regulation of the aer gene and aerotaxis in E. coli is coordinated with control of metabolism by hypoxia. Elucidating the mechanism of oxygen sensing by the FAD-PAS domain of Aer may provide insight into the role of PAS domains in human oxygen sensors.