Our long term objective is to understand the mechanism of sensory transduction during chemotaxis in Bacillus subtilis. The chemotaxis systems of Bacillus subtilis and Escherichia coli share some features. However, there are several basic differences. Methyl transfer reactions occur between proteins upon excitation by both attractants and repellents and methanol is released to bring about adaptation to both these stimuli. In Halobacterium halobium, one of the archebacteria, methanol release also occurs for all types of stimuli. E. coli does not use methyl transfer between proteins. The methyl-accepting chemotaxis proteins (MCPs), the receptors, in E. coli are around 60 kD, but the major MCPs of B. subtilis are 79, 87, and 97 kD. These are more similar in size to those of Halobacterium halobium. Thus, there is reason to believe that the underlying methylation mechanism of chemotaxis differs between B. subtilis and E. coli but there may be more similarity between B. subtilis and H. halobium. This similarity to H. halobium may have far-reaching significance. The archebacteria are more closely related to eucaryotic cells than to true bacteria; so that there is the possibility that methyl transfer reactions might play unsuspected roles in sensory transduction in eucaryotic cells. We soon will be finishing the cloning and sequencing of che/fla genes. We will then inactivate them to define from in vivo experiments the roles of the corresponding proteins in chemotaxis. We will express them in an E. coli expression system and purify them. We have purified the three major MCPs of B. subtilis, reconstituted them into lipid vesicles, and methylated them. We will reconstitute phosphoryl and methyl transfer pathways and observe effect of attractant and repellent on enzymatic activities and stoichiometry of complexes of chemotaxis proteins. We will use site-directed mutagenesis and casette mutagenesis to delineate structure/function relationships among chemotaxis proteins. Finally, we will study regulation of the major che/fla operon. This operon is over 26 kb and contains at least 27 genes. We will also examine the mechanism by which both hag and mot transcription and methanol formation on chemotactic stimulation depend on the morphology of the basal body.
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