SENSORY TRANSDUCTION IN BACTERIA

这些研究探讨了大肠杆菌对氧的趋化性（嗜氧性）机制和相关反应，如氧化还原趋化性和甘油趋化性。这些反应需要Aer或Tsr转换器，并且与其他趋化行为不同，需要功能性电子传递系统。Aer是一种黄素蛋白，具有N-末端PAS输入结构域和C-末端信号结构域。长期的目标是解释，在分子上的细节，信号转导的艾尔。具体目的包括：1）确定Aer与电子传递系统之间的信号通路。2)定义PAS和接头/HAMP结构域在Aer中从PAS结构域到C-末端信号传导结构域的信号传导途径中的作用。3)测量E中的质子动力。大肠杆菌作为氧浓度的函数，并确定共同信号（细胞能量水平的变化）是否可以介导对氧的好氧和疏氧反应;以及4）通过分析aer基因的转录调控来研究Aer在氧和氧化还原传感中的生理作用。一个跨学科的方法，结合现代分子生物学，遗传学和生物化学的方法与本实验室开发的新技术，将用于测试每个目标的假设。Aer PAS和接头/HAMP结构域中推定的蛋白质-蛋白质接触结构域将通过半胱氨酸置换或低保真PCR扩增进行突变，并确定对嗜氧性、FAD结合、蛋白质折叠和中点电位的影响。Aer PAS结构域与电子传递系统和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子分析来定义。利用aer-lacZ融合基因作为报告基因，我们将确定是否调控aer基因和嗜氧性在大肠杆菌中。大肠杆菌的代谢与低氧控制相协调。阐明Aer的FAD-PAS结构域的氧感受机制可能有助于深入了解PAS结构域在人体氧感受器中的作用。

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.