Abstract

Escherichia coli swims toward niches ideal for generating maximum energy by monitoring the environment with the aerotaxis sensor, Aer. This receptor has an N-terminal PAS domain, which is a sensor module present in a variety of proteins from all kingdoms of life (including humans). To date, the mechanism of signaling from a PAS sensor domain to a single signaling receiver domain has not been resolved. In Aer, the PAS sensor is separated from the signaling region by a membrane anchor. Most "transmembrane prediction" programs forecast a single membrane span for this membrane anchor, but such a topology is inconsistent our previous studies indicating the PAS and HAMP domains, which flank the membrane region, are in contact with one another.

To determine the general topology of the membrane anchor, I used a cysteine scanning approach. I estimated the proximity of 57 cognate cysteine replacements by measuring the rate and extent of dimer formation after adding an oxidant at 23°C, and at 4°C to limit random collisions. I found that the Aer membrane anchor formed two membrane-spanning segments that flank a central periplasmic loop. To map the membrane boundaries, I used hydrophilic sulfhydryl-reactive agents to measure the surface accessibility of these introduced cysteines. I then expanded the cysteine scanning method to include binary, ternary and quaternary combinations of introduced cysteines. Binary combinations were used to differentiate intra- from inter-dimeric collisions and thereby map the faces of Aer monomers. Ternary and quaternary combinations were used to study larger complexes. Aer formed hexamers at both the periplasmic and cytosolic ends, but this stable structure was orchestrated by the cytosolic signaling domain, because C-terminal truncations missing the signaling region altered the periplasmic neighborhood; here, octamers and decamers were trapped, indicating that the signaling region had limited the degrees of freedom in the periplasmic region. Lastly, strategic cross-linking combinations pairing periplasmic and cytosolic cysteine replacements suggested that different Aer trimers-of-dimers could be trapped if the oxidant copper phenanthroline were added at high concentrations. However, the myriad of complexes formed suggested that these interactions were not specific.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology

School

School of Medicine

First Advisor

Mark S. Johnson

Second Advisor

Katherine A. Borkovich

Third Advisor

Hansel M. Fletcher

Fourth Advisor

Lawrence C. Sowers

Fifth Advisor

Barry L. Taylor

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2006

Date (Title Page)

6-2006

Language

English

Library of Congress/MESH Subject Headings

Escherichia coli -- genetics; Escherichia coli -- chemistry; Chemotaxis; Cell membrane.

Type

Dissertation

Page Count

xi; 167

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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