Our studies focus of the Escherichia coli mechanism of chemotaxis to oxygen (aerotaxis) and related responses, such as redox taxis and glycerol taxis. The responses are different from other chemotactic behaviors in requiring a functional electron transport system. Aer, a novel flavoprotein, and Tsr, the serine chemoreceptor, have been recently identified by this laboratory as the transducers for oxygen, redox and glycerol (energy) taxis. The long term goal is to understand, in molecular detail, the signal processing by thee transducers.
The specific aims i nclude: 1) Test the hypothesis that flavin adenine dinucleotide (FAD) in Aer senses redox changes Date Released:05/07/1997 Date Printed: 10/22/1997 in the electron transport system, and Tsr senses proton motive force. 2) Investigate the relationship between structure and function of Aer and Tsr in sensing redox potential and proton motive force. 3) Test the hypothesis that proton motive force/redox potential is a signal that is common to aerophobic and aerophilic responses in E. Coli, guiding bacteria to an optimal environment where the proton motive force is maximal. An interdisciplinary approach - combining contemporary methods of molecular biology, genetics and biochemistry with novel techniques developed in this laboratory - will be used to address the critical research questions identified for each aim. The electron transport system will be perturbed in E. Coli using newly available constructs. Putative residues for FAD binding in Aer will be mutated by site directed mutagenesis and the effect on aerotaxis, FAD binding and mid-point reduction potential determined. The topology of Aer in the membrane will be determined using a sandwich Aer fusion protein in which PhoA or LacZ is inserted in frame within the N-terminal domain of Aer. Cysteine scanning of H328, selected histidine residues in the periplasmic domain and residues around the K1 coiled coil domain of Tsr will be used to identify the Ph receptors and their effect on proton motive force (redox) sensing by Tsr. The hypothesis that aerotaxis guides bacteria to the oxygen concentration that supports the highest proton motive force will be tested using simultaneous measurement of oxygen concentration and membrane potential, under conditions where the oxygen concentration can be tightly controlled. Elucidating these mechanisms will provide important insights into a primordial pathway for chemotaxis, and also into general principles of oxygen sensing (some of which may be applicable to carotid body chemoreceptors in humans).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029481-16
Application #
6179472
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Shapiro, Bert I
Project Start
1981-09-28
Project End
2001-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
16
Fiscal Year
2000
Total Cost
$260,882
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
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
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
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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