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 i nclude: 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.
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