Abstract

Chemotaxis is the migrational response of bacteria to chemical gradients. Aerotaxis, the response to oxygen, was among the earliest observed tactic phenomena, but progress in the study of aerotaxis was previously limited by the available assays. These assays relied upon oxygen depletion by the bacteria to generate the oxygen gradient which elicits the behavioral response. Such assays are unsuitable for analyzing the role of the electron transport system in aerotaxis. In this study the biochemical mechanism of aerotaxis and its relationship to other chemotaxis pathways in Salmonella typhimurium was explored with the aid of two new assays which imposed an oxygen gradient independently of bacterial respiration. In the spatial assay, bacteria accumulated at the mouth of an oxygen-filled capillary. In the temporal assay, cells in anaerobic medium under the microscope were stimulated with a preset step increase in oxygen concentration, and the duration of the response was measured. Both assays could be used in the presence of respiratory inhibitors. Aerotaxis in Escherichia coli and Bacillus cereus was also assayed to corroborate the evidence obtained with S. typhimurium.

The receptor for aerotaxis was the major terminal oxidase of the electron transport system. The evidence supporting this conclusion was 1) the K0.5 of the aerotaxis receptor (K0.5 = 0.7 μM) and the Km of the terminal oxidase (Km = 0.7 μM) were similar in S. typhimurium. They were also similar in E. coli and in B. cereus. 2) Aerotaxis and respiration were inhibited by the respiratory inhibitors cyanide and 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO). 3) The alternative electron acceptors nitrate and fumarate partially blocked aerotaxis. In S. typhimurium, the major terminal oxidase in log-phase cells had not been identified previously. The presence of cytochrome o was confirmed by low temperature difference spectroscopy.

The signal from classical chemotaxis receptors passes directly to the signal processing proteins of the chemotaxis pathway. The aerotaxis receptor did not function in this manner. The inhibitor HOQNO, which inhibits respiration at cytochrome b, was a noncompetitive inhibitor of aerotaxis and respiration with respect to oxygen. Therefore the electron transport system and not just the terminal oxidase were required for aerotaxis. Intermediates modulated by changes in the rate of electron transport were evaluated for their role in aerotaxis. The response was independent of adenosine triphosphate formation via oxidative phosphorylation, because a S. typhimurium unc- mutant displayed normal aerotaxis. The proton motive force fulfilled the requirements for the signal carrier. A temporal correlation between the response to an oxygen stimulus and changes in membrane potential was measured with the fluorescent cyanine dye diS-C3-(5) in a permeable strain of S. typhimurium. Smooth swimming accompanied membrane hyperpolarization and tumbling accompanied depolarization. Corresponding changes in behavior are induced by artificial perturbation of the proton motive force.

When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis initially supported slow swimming but not tumbling. Tumbling was eventually restored while the anaerobic swimming speed remained the same. These observations suggested that the proton motive force in anaerobic cells was insufficient to permit tumbling, but that the tumble control mechanism adapted to restore tumbling while the proton motive force remained depressed. The ability of the cells to adapt to lowered proton motive force, in addition to adaptation to sensory stimuli, indicated that the cells possess at least two mechanisms which compensate for changes in the proton motive force. Neither involved the methylation-dependent adaptation mechanism which operates for most attractants. Aerotaxis was normal in E. coli mutants with defects in the genes for the methyl-accepting chemotaxis proteins MCP I, MCP II, or MCP III. Aerotaxis was postulated to be methylation independent. However, aerotaxis and chemotaxis are both mediated by the cheC protein, because a cheC mutant which shows an inverse response to chemo-affectors also gave an inverse response to oxygen.

These findings establish the mechanism of aerotaxis as fundamentally distinct from the mechanism of chemotaxis to most other nutrients. Aerotaxis was distinguished by the requirement for metabolism of the attractant for sensing, methylation-independent adaptation, and the signalling role of the proton motive force.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Barry L. Taylor

Second Advisor

R. Bruce Wilcox

Third Advisor

W. B. Rippon

Fourth Advisor

Charles W. Slattery

Fifth Advisor

David A. Hessinger

Degree Name

Doctor of Philosophy (Medical Science)

Degree Level

Ph.D.

Year Degree Awarded

1981

Date (Title Page)

6-1981

Language

English

Library of Congress/MESH Subject Headings

Salmonella typhimurium; Oxygen -- physiology

Type

Dissertation

Page Count

x; 153

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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