My research focuses on the regulation of chemotaxis in bacteria. Environmental signals are detected by the chemotactic receptors, located in the cytoplasmic membrane, and transduced via several proteins to the flagella which eventually affect the movement of the cell. The receptor can be reversibly methylated and induces reversible phosphorylation of a cytoplasmic protein, CheA, which appears to be the major regulatory site in chemotaxis. It appears that CheA contains a potential methylation site homologous to one of the known loci of the bacterial chemoreceptor, and recent results indicate that it can be methylated, presumably by CheR. Addition of purified CheR to purified CheA in the presence of S-adenosyl-L-[methyl3H]methionine results in an increase in methylation. This increased level of methylation was not observed when the CheA extract was omitted. The proteins were subjected to SDS/poly acrylamide gel electrophoresis and analyzed using fluorography. The results show that only CheA was capable of becoming methylated. Western blot examination of the methylated CheA using CheA antibodies showed only one band which corresponded to the migration pattern that was expected for CheA. The role of CheA methylation, which could be regulatory in nature, will be verified and explored by the following experiments: Determine the significance of CheA methylation. Measure the rate of CheA methylation and its effect on phosphorylation, and vice versa. Determine the effect of CheA methylation on its ability to transfer phosphate to CheY or CheB. Identify the proteins that are involved in CheA methylation and demethylation. Investigate the nature of the methylated residue.?
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