| Abstract: | Using a modification of the EGTA treatment of Oishi and Smith Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine. |
