Unique Regulation of Carbohydrate Chemotaxis in Bacillus subtilis by the Phosphoenolpyruvate-Dependent Phosphotransferase System and the Methyl-Accepting Chemotaxis Protein McpC

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of d-glucose or d-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate d-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of d-glucose, a decrease in d-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.  In enteric bacteria, chemotaxis toward many carbohydrate attractants is dependent upon components of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (1, 9, 15). This carbohydrate transport system consists of an autophosphorylating histidine kinase, EnzymeI, a common phosphocarrier protein, HPr, and a number of substrate-specific transporters, the EnzymeII complexes. At the expense of PEP, EnzymeI autophosphorylates on a histidine residue and transfers this phosphoryl group to a histidine residue on HPr. HPr-P then donates this phosphoryl group to a carbohydrate-specific EnzymeII complex. The carbohydrate substrate is the final phosphoryl group acceptor, as it is transported into the cell and is concomitantly phosphorylated by EnzymeII (13).Chemotaxis is also controlled by a phosphoryl transfer cascade. CheA, in response to an attractant- or repellent-bound receptor (methyl-accepting chemotaxis protein [MCP]), alters its rate of autophosphorylation appropriately to transiently increase or decrease the intracellular CheY-P pool and thereby modulate swimming behavior (4, 16). In enteric bacteria, increased CheY-P leads to tumbling (19). In Bacillus subtilis, increased CheY-P leads to smooth swimming (3). In enteric bacteria, chemotaxis toward PTS substrates requires CheA, CheY, EnzymeI, and HPr but does not depend on the presence of an MCP (12, 18). These observations have led investigators to suggest that the changes in the phosphorylation state of PTS components that accompany carbohydrate transport regulate CheA activity (10).Recent work has provided the following model for the role of the PTS in chemotaxis toward its substrates in Escherichia coli. As the bacteria encounter a PTS carbohydrate, HPr dephosphorylates EnzymeI faster than the latter protein can be rephosphorylated. The resulting increase in unphosphorylated EnzymeI and the resulting decrease in PEP both function to decrease the rate of CheA autophosphorylation. This is believed to lead to a transient decrease in the CheY-P pool that suppresses tumbling, allowing the bacteria to move up the carbohydrate gradient (10).This article describes studies on the process of carbohydrate chemotaxis in B. subtilis. In particular, we provide evidence that McpC is absolutely required for any response to all of the PTS carbohydrates tested. This is surprising considering the fact that McpC has previously been shown to also mediate chemotaxis toward eight different amino acids (11). McpA has previously been shown to partially mediate chemotaxis toward glucose (7). This result is confirmed in the present study with the use of direct behavioral assays. Our results suggest the existence of a multidimensional signaling mechanism involving both the PTS and specific MCPs, an unprecedented finding in the study of the molecular control of bacterial carbohydrate chemotaxis.