Abstract

B. subtilis, like Escherichia coli and Salmonella typhimurium, responded to a step increase in oxygen concentration by swimming smoothly (~ 35 s duration) and to a decrease in oxygen by tumbling. In a spatial gradient of oxygen in liquid media, a band of cells congregated near the air interface. Aerotaxis required a functioning respiratory chain. Adaptation of B. subtilis, E. coli and S. typhimurium to media containing amino acids requires methylation of receptors/methyl-accepting chemotaxis proteins. Adaptation to oxygen and phosphotransferase sugar substrates in B. subtilis was dependent on methylation, unlike similar adaptation in E. coli and S. typhimurium. B. subtilis cheR (methyltransferase) mutants demonstrated impaired aerotaxis under the best of conditions and methionine starvation (which depletes S-adenosylmethionine) lengthened the time of adaptation to oxygen. Normal adaptation times were restored after the addition of methionine to methionine-starved cells. A modified flow assay was developed to measure demethylation of methyl-accepting chemotaxis proteins. Methanol evolution was observed after the addition or removal of air-equilibrated buffer to B. subtilis. Thin layer chromatographic analysis of 3,5-dinitrobenzylchloride derivatized fractions confirmed that these peaks were exclusively methanol. No methanol was evolved in a B. subtilis cheR mutant or in wild type E. coli and S. typhimurium cells under similar conditions. The effect of oxygen stimulation on the amount of methylation in methyl-accepting chemotaxis-proteins was investigated. Quantitative changes in alkali-labile methyl groups reflected the amount of methanol produced in the aerotaxis flow assay. Phosphotransferase chemotaxis is also methylation-dependent. This study offers conclusive evidence that methylation is required for aerotaxis and phosphotransferase chemotaxis in B. subtilis but not in enteric bacteria, and indicates that a different molecular mechanism of aerotaxis may be involved. Methylation-dependent aerotaxis was first suggested in Halobacterium halobium.

Earlier reports indicating that methanol is produced in B. subtilis after the addition of attractant or repellent were found to be in error. Methanol was released during the attractant response and methionine was released during the repellent response, unlike aerotactic stimulation. Methionine degradation released an artifactual volatile methanethiol peak after prolonged incubation. This artifact was not related to chemotaxis. As a result, some of the earlier evidence for a putative intermediate methyl-carrier protein and the involvement of methylation in excitation may be incorrect. Histidine starvation, which depletes ATP, caused B. subtilis cells to tumble, in contrast to S. typhimurium which swim smoothly when ATP is depleted. In addition, aerotactic and chemotactic responses were shorter in histidine-starved cells. It is concluded that ATP is required for generating the smooth swimming behavior in B. subtilis unlike S. typhimurium which requires ATP for tumbling.

The interaction of ATP with E. coli and S. typhimurium CheY was investigated and shown to be non-specific. In addition to behavioral studies, the nucleotide sequence of a previously cloned S. typhimurium Hindlll-mtlA fragment and of an overlapping Accl clone was determined. The clones showed 87% and 95% identity with the E. coli sequence at the nucleotide and amino acid level, respectively.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Barry L. Taylor

Second Advisor

E. Clifford Herrmann

Third Advisor

Mark S. Johnson

Fourth Advisor

Lawrence B. Sandberg

Fifth Advisor

R. Bruce Wilcox

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1993

Date (Title Page)

8-1993

Language

English

Library of Congress/MESH Subject Headings

Bacillus subtilis -- physiology; Cell Movement; Oxygen -- chemistry; Methylation -- physiology

Type

Dissertation

Page Count

2 xii; 289

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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