GENETIC-ANALYSIS OF THE CATALYTIC DOMAIN OF THE CHEMOTAXIS-ASSOCIATEDHISTIDINE KINASE CHEA

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemota xis-associated response regulators, CheY and CheB. CheA(L) first autop hosphorylates amino acid His-48 before its phosphoryl group is transfe rred to these response regulators. The short form, CheA,, lacks the am ino-terminal 97 amino acids of CheA, and therefore does not possess th e site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, hi ghly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to hi ghly conserved regions of certain eukaryotic kinases, the G1 and G2 re gions are purported to be involved in the binding and hydrolysis of AT P. We report here that alleles mutated in the G1, G2, or F region synt hesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the non phosphorylatable CheA, protein mediates transphosphorylation of a CheA , variant defective in both G1 and G2. In contrast, CheA, variants def ective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by wh ich the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that d imer.