Permeabilization of Bacillus subtilis to Chemotaxis

The net methylation of methyl-accepting chemotaxis proteins (MCPs) is the biochemical manifestation of physiological adaptation to amino acid chemoattractants by Escherichia coli (4). In Bacillus subtilis, demethylation of MCPs occurs during such adaptation (3). The activity of chemotaxis methyltransferase is detectable in vivo and in vitro. As an alternative assay, whole cells can be permeabilized, giving an essentially in vitro system but one which generally conserves in vivo-like spatial relationships and relative concentrations of the various cellular constituents (9, 10). In this study, cells permeabilized by toluene were used to study chemotactic methylation in unstimulated B. subtilis cells. Under the permeabilizing conditions described here, it was apparent that membrane holes generated by toluene treatment were large enough for the methylating enzyme to pass through them. This work reconfirms the notion of a divalent cation requirement for the MCP methylation reaction, and the lower doublet methylation is consistent with data suggesting the existence of a cytoplasmic substrate for methyltransferase II, whose function is not known (A. Burgess-Cassler and G. W. Ordal, submitted for publication). (The material presented here is part of a Ph.D. dissertation submitted by A.B.-C. to the University of Illinois, Urbana, 1983.) The permeabilization assay was based on a procedure developed by Paoni and Koshland (9) with modifications. B. subtilis OI 1085, the wild type for chemotaxis, and 01 1100, a chemotaxis methyltransferase II strain (11), were grown to 180 to 200 Klett units (red filter) in Luria broth, washed twice in chemotaxis buffer (8), and suspended in the same buffer at an absorbance at