SPECIFICITY OF THE BVGAS AND EVGAS PHOSPHORELAY IS MEDIATED BY THE C-TERMINAL HPT DOMAINS OF THE SENSOR PROTEINS

Despite the presence of highly conserved signalling modules, significa nt cross-communication between different two-component systems has onl y rarely been observed. Domain swapping and the characterization of li berated signalling modules enabled us to characterize in vitro the pro tein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-compon ent systems. Under equimolar conditions, significant in vitro phosphor ylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine ki nase consisting of the BvgS transmitter and HPt domains and of the Evg S receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS trans mitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unab le to phosphorylate BvgA but efficiently phosphorylated EvgA. These re sults demonstrate that the C-terminal HPt domains of the sensor protei ns endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein. In the case of the response r egulators, the receiver but not the output domains contribute to the s pecific interaction with the histidine kinases, because a hybrid prote in consisting of the EvgA receiver and the BvgA output domain could on ly be phosphorylated by the EvgS protein.