Abstract

In Escherichia coli, chemotactic movement towards an energetically-favorable environment is mediated by five transmembrane chemoreceptors. These sensory proteins respond to numerous environmental signals, including amino acids, sugars, and pH. The aerotaxis receptor, Aer, is an intracellular sensor that responds to oxygen concentration and other parameters affecting cellular energy levels. Bacterial chemotaxis receptors form dimeric signaling units that organize into tetramers and hexamers and form large clusters at the cell poles. A cluster arrangement could increase receptor sensitivity by lateral communication between dimers, thus amplifying the signal from one dimer. We have shown that Aer forms dimers, trimer of dimers, and localizes to the poles. To form polar clusters, chemoreceptors require a highly conserved domain which binds to the cytoplasmic signaling components, CheA and CheW, to form a stable ternary complex. In this study, determinants influencing the localization of Aer were studied, specifically the absence/presence of other chemoreceptors and the Aer signaling domain. I visualized this receptor by creating EGFP-Aer fusions and examining the spatial distribution in vivo using fluorescence microscopy. No distinct polar localization was observed when Aer was expressed as the sole chemoreceptor, or in the presence of a low-abundance chemoreceptor. However, Aer localized at the poles when expressed along with the high abundance receptors, Tsr or Tar. Phenotypic analysis of Aer using a swarm plate assay revealed that an aerotactic ring was only observed in conditions that retained Aer polar localization. A truncated Aer homodimer that lacked the signaling domain displayed no distinct fluorescent foci at the poles but appeared to be distributed throughout the inner bacterial membrane. This suggests that the signaling domain is required for targeting Aer to the cell poles. Similar to other chemoreceptors, the absence of cheA and cheW reduced Aer clustering at the poles, indicating that these chemotaxis proteins are required for maximal polar clustering. The synthesis of lipoprotein was also required for polar localization of Aer E. coli lacking lipoprotein (lpp) formed Aer clusters, but they did not localize exclusively at the poles. Sparse clustering was evident in cells missing anionic phospholipids. This study was the first to quantify the copy number of the Aer receptor in four E. coliK-12 derived strains wild type for chemotaxis. Native expressions levels of Aer were measured from cells grown in rich media (LB and Tryptone) and minimal media (H1 supplemented with various carbon sources) under aerobic and anaerobic conditions. Aer expression varied markedly depending on the growth medium and strain, with values ranging from < 10 to 1,800 Aer monomers per cell. The median Aer value was 4% of the total receptor pool, and approximately 12% of all trimers-of-dimer signaling squads would contain an Aer dimer. In conclusion this work establishes the polar distribution of Aer with native expressions levels ranging between 2.5% to 5.5% dependant upon the strain and growth conditions.

LLU Discipline

Microbiology and Molecular Genetics

School

Graduate Studies

First Advisor

Barry L. Taylor

Second Advisor

Penelope J. Duerksen-Hughes

Third Advisor

Mark S. Johnson

Fourth Advisor

William Langridge

Fifth Advisor

Subburaman Mohan

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2009

Date (Title Page)

6-2009

Language

English

Library of Congress/MESH Subject Headings

Escherichia coli -- enzymology; Escherichia coli Proteins; Aer protein, E coli; Mutant Proteins -- genetics; Chemotaxis; DNA Mutational Analysis; Molecular Sequence Data; Models, Molecular

Type

Dissertation

Page Count

xvii; 123

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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