Abstract

This study investigated the mechanism of signal transduction by Aer, the aerotaxis transducer in E.coli. A computerized search of GenBank revealed an open reading frame at 69.1 min on the E.co//chromosome that had a C-terminal fragment with 96.7% identity to the highly conserved domain of Tsr. The open reading frame was renamed aer for aerotaxis and energy responses. When the aer gene was inactivated, approximately 50% of aerotaxis responses were eliminated. When both aer and fsr were inactivated, aerotaxis, redox taxis and glycerol taxis were completely abolished. Expressing Aer in aer fsr cells restored aerotaxis responses and the duration of responses progressively lengthened with the amount of Aer overexpression. These results confirmed that Aer and Tsr are independently sense intracellular energy levels and transduce oxygen, redox and energy signals.

Sequence analysis of Aer revealed a PAS domain in the N-terminus of the protein. PAS domains are input modules found in a large family of light, oxygen, voltage and redox sensors. Forty two conserved and non-conserved Aer PAS domain residues were serially replaced with cysteine to measure their effect on sensing and signaling by Aer. This study identified Aer PAS domain residues important for FAD binding, sensing, signal bias and signal output. A 3D model of the Aer PAS domain was generated using PYP as the structural template. Important residues were mapped on the Aer PAS model and an active site for FAD binding and signal transduction was proposed.

Different approaches were used to identify the upstream component in the electron transport system that signals to Aer. The yeast two hybrid system was utilized to assay interactions between Aer and all other proteins encoded by a E.coli genomic library. The role of NADH dehydrogenase I in aerotaxis was also investigated by measuring responses of nuo fsr cells. The study indicated that Aer can function in the absence of NADH dehydrogenase I.

We sought to isolate and purify the soluble N-terminus of Aer. However, the N-terminus peptide formed inclusion bodies. Co-expressing the GroESL chaperonins alleviated inclusion body formation and increased the solubility of Aer.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Barry L. Taylor

Second Advisor

Hansel Fletcher

Third Advisor

John J. Rossi

Fourth Advisor

Igor B. Zhulin

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2000

Date (Title Page)

6-2000

Language

English

Library of Congress/MESH Subject Headings

Penicillinase; Signal Transduction; Gene Expression Regulation, Bacterial; Escherichia Coli.

Type

Dissertation

Page Count

xii; 303

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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