CHEMOTACTIC SIGNALING BY THE P1 PHOSPHORYLATION DOMAIN LIBERATED FROMTHE CHEA HISTIDINE KINASE OF ESCHERICHIA-COLI

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli, CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adapta tion, Polypeptides encompassing the N-terminal P1 domain of CheA can b e transphosphorylated in vitro by the CheA catalytic domain and Jet ha ve no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells, To find out why, we examined the effects o f a purified P1 fragment, CheA[1-149], on CheA-related signaling activ ities in vitro and devised in vivo assays for those same activities. A lthough readily phosphorylated by CheA[260-537], the CheA catalytic do main, CheA[1-149], was a poor substrate for transphosphorylation by fu ll-length CheA molecules, implying that the resident P1 domain monopol izes the CheA catalytic center, CheA-H48Q, a nonphosphorylatable mutan t, failed to transphosphorylate CheA[1-149], suggesting that phosphory lation of the P1 domain in cis may alleviate the exclusion effect, In agreement with these findings, a 40-fold excess of CheA[1-149] fragmen ts did not impair the CheA autophosphorylation reaction, CheA[1-149] d id acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules, An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site, The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA auto phosphorylation most likely account for the failure of liberated P1 do mains to jam chemotactic signaling in wild-type cells, However, an exc ess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragment s have both transphosphorylation and phosphotransfer capability in viv o.