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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029481-18
Application #
6519041
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Shapiro, Bert I
Project Start
1981-09-28
Project End
2005-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
18
Fiscal Year
2002
Total Cost
$287,000
Indirect Cost
Name
Loma Linda University
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
City
Loma Linda
State
CA
Country
United States
Zip Code
92350
Watts, Kylie J; Johnson, Mark S (2018) Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation. Methods Mol Biol 1729:137-145
Garcia, Darysbel; Watts, Kylie J; Johnson, Mark S et al. (2016) Delineating PAS-HAMP interaction surfaces and signalling-associated changes in the aerotaxis receptor Aer. Mol Microbiol 100:156-72
Campbell, Asharie J; Watts, Kylie J; Johnson, Mark S et al. (2011) Role of the F1 region in the Escherichia coli aerotaxis receptor Aer. J Bacteriol 193:358-66
Watts, Kylie J; Johnson, Mark S; Taylor, Barry L (2011) Different conformations of the kinase-on and kinase-off signaling states in the Aer HAMP domain. J Bacteriol 193:4095-103
Watts, Kylie J; Taylor, Barry L; Johnson, Mark S (2011) PAS/poly-HAMP signalling in Aer-2, a soluble haem-based sensor. Mol Microbiol 79:686-99
Airola, Michael V; Watts, Kylie J; Bilwes, Alexandrine M et al. (2010) Structure of concatenated HAMP domains provides a mechanism for signal transduction. Structure 18:436-48
Campbell, Asharie J; Watts, Kylie J; Johnson, Mark S et al. (2010) Gain-of-function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer. Mol Microbiol 77:575-86
Watts, Kylie J; Johnson, Mark S; Taylor, Barry L (2008) Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer. J Bacteriol 190:2118-27
Taylor, Barry L; Watts, Kylie J; Johnson, Mark S (2007) Oxygen and redox sensing by two-component systems that regulate behavioral responses: behavioral assays and structural studies of aer using in vivo disulfide cross-linking. Methods Enzymol 422:190-232
Taylor, Barry L (2007) Aer on the inside looking out: paradigm for a PAS-HAMP role in sensing oxygen, redox and energy. Mol Microbiol 65:1415-24

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