Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C‐terminal HPt domains of the sensor proteins

Abstract

SummaryDespite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg twocomponent systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox twocomponent systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.

The Vibrio cholerae Hybrid Sensor Kinase VieS Contributes to Motility and Biofilm Regulation by Altering the Cyclic Diguanylate Level

“…Residues 968 to 1084 constitute a phosphoreceiver domain, characteristic of bacterial response regulators (33,39), with the conserved residue D1018 serving as the predicted target of phosphorylation by the HisKa domain. Finally, VieS contains a conserved histidine phosphotransferase domain (Hpt) at the C terminus, predicted to mediate the transfer of phosphate to its cognate response regulator and also to serve as the recognition site between the sensor and its cognate response regulator (21,27). Based on the homologies described above, we hypothesized that VieS acts as a periplasmic sensor of amino acids or small peptides which, upon activation, autophosphorylates and mediates phosphotransfer to the cognate response regulator VieA.…”

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli

“…This interaction appeared to be dependent on a functional histidine phosphoryl-transfer (HPt) domain of the sensor kinase ArcB and thus reinforces the suggestion that signalling domains within multi-component phosphorelay systems (Appleby et al, 1996 ;Yaku et al, 1997) are potential participants in physiological crosstalk. However, in contrast, no significant cross-talk was detected between the HPt domains of BvgS and EvgS and the non-cognate response regulators BvgA and EvgA, either in vitro or in vivo (Beier et al, 1995 ;Perraud et al, 1998Perraud et al, , 1999. To show the subtle effects of cross-talk, existing under various conditions, the DNA microarray method may become a powerful tool in future experiments, provided that it will allow sufficiently quantitative analysis.…”

Phosphate flow in the chemotactic response system of Helicobacter pylori

“…pQE plasmids with inserts encoding His 6 -fusion proteins were propagated in E. coli M15. Expression and purification of GST-and His 6 -fusion proteins was performed as described previously (Perraud et al, 1998;Beier & Frank, 2000).…”