For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.Chemotaxis is the process by which bacteria sense their chemical environment and migrate toward more favorable conditions. In Bacillus subtilis, chemotaxis toward the attractant asparagine has been shown to be mediated by the methylaccepting chemotaxis protein McpB (1). When asparagine is added to membranes containing McpB in vitro, the rate of autophosphorylation of the CheA autokinase increases (2). The phosphorylated form of CheA transfers a phosphoryl group to CheY to produce CheY-P (2, 3), which then interacts with switch proteins to cause CCW 1 rotation of the flagella, resulting in smooth swimming behavior (3). CheA-P also donates phosphoryl groups to CheB, 2 which thereby becomes activated to demethylate the MCPs and produce methanol (4, 5). Methylation of the MCPs is known to occur on glutamate side chains (6) through the action of CheR, the chemotactic methyltransferase, which utilizes AdoMet as a substrate (7).The B. subtilis chemotactic machinery also includes CheW, CheC, CheD, and CheV. CheW and CheV are thought to couple CheA activity to the MCPs (8 -11). CheC inhibits methylation of the MCPs by an unknown mechanism but does not interfere with the methylesterase, CheB (12, 13). CheD is required to produce a normal prestimulus bias, normal methylation, and azetidine-2-COOH-induced activation of CheA in vivo (12). How these proteins interact to regulate the chemotactic response in B. subtilis remains unknown.The chemotaxis system in Escherichia coli has been well characterized and has served as a paradigm for our studies (for reviews, see Refs. 14 -17). The E. coli system includes homologs of the MCPs, CheA, CheB, CheR, CheW, and CheY. The E. coli system also includes CheZ, which facilitates dephosphorylation of CheY-P (18 -21), but does not include homologs to CheC, CheD, or...