PH-DEPENDENCE OF CHEA AUTOPHOSPHORYLATION IN ESCHERICHIA-COLI

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium dep ends upon the ability of chemoreceptors called transducers to communic ate with snitch components of flagellar motors to modulate swimming be havior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheA(L), CheA(S), CheW, CheY, and CheZ. Of these, the autokinase CheA(L) is most central. Modi fications or mutations that affect the rate at which CheA(L) autophosp horylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheA(L) autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10. 0, consistent with the notion that two proton dissociation events affe ct CheA(L) autophosphorylation kinetics: one characterized by a pK(a) of about 8.1 and another exhibiting a pK(a) of about 8.9. These in vit ro results predict a decrease in the rate of CheA(L) autophosphorylati on in response to a reduction in intracellular pH, a decrease that sho uld cause increased counterclockwise flagellar rotation. We observed s uch a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when und issociated readily traverses the cytoplasmic membrane, causes a reduct ion of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheA(L), CheA(S), and CheY, but not transducers, exhi bited a small but reproducible increase in the fraction of time that t hey spun their flagellar motors counterclockwise. The added presence o f CheW and the transducers Tar and Trg resulted in a more dramatic res ponse. The significance of our in vitro results, their relationship to regulation of swimming behavior, and the mechanisms by which transduc ers might affect the pH dependence of CheA autokinase activity are dis cussed.