SENSORY TRANSDUCTION IN BACTERIA

这些研究探讨了大肠杆菌对氧的趋化性机制和相关反应，如氧化还原趋化性和甘油趋化性。这些反应需要Aer或Tsr换能器，与其他趋化行为不同的是，它们需要一个功能性的电子传递系统。Aer是一种具有n端PAS输入域和c端信号域的黄蛋白。长期目标是在分子细节上解释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)定义的角色不是和链接器/ HAMP贷款领域的信号通路在爱尔兰不是域c端信号域。3)测量质子动力在大肠杆菌作为氧气浓度的函数和确定一个共同的信号(在细胞能量变化)可以调解嗜气的和aerophobic反应氧和4)调查爱尔兰的生理作用在氧气和氧化还原传感分析转录调控的爱尔兰基因。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，将用于检验每个目标的假设。通过半胱氨酸替代或低保真PCR扩增，Aer PAS和连接子/HAMP结构域中假定的蛋白-蛋白接触结构域将发生突变，并确定对趋氧性、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、交联和二位点抑制分析来确定。利用aer- lacz融合体作为报告基因，我们将确定大肠杆菌中aer基因和可氧性的调控是否与缺氧对代谢的控制相协调。阐明Aer的FAD-PAS结构域的氧传感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.