The RcsC/RcsD/RcsB system in Salmonella typhimurium: a bacterial two-hybrid analysis

Abstract

The Rcs phosphorelay is a adaptive response system composed of three proteins: the sensor RcsC, the cognate response regulator RcsB; and the intermediary in the phosphoryl transfer RcsD. Has been described that the flow of phosphoryl groups through the Rcs phosphorelay can occur in following way RcsC/RcsD/RcsB. At present, the signal that activates the Rcs system has not been determined. We have demonstrated in a previous study that the overexpression of rcsB modulates the RcsB-dependent genes in an rcsC or rcsD mutant but not in the doble mutant. These results suggest that only RcsB-P, the RcsB phosphorylated form, is able to induce this modulation, and we postulated that the RcsB-P would occur by two or more different pathways. The goal of present work was study the possible phosphorelay pathways. In this work we used the two-hybrid assay (BACTH), consisting of the reconstitution of the adenylate cyclase activity in E. coli when two target proteins interacts in vivo in bacteria, measured by b-galactosidase activity. In this assay we probe the RcsC, RcsD and RcsB interaction to define the Rcs phosphorelay transduction mechanism.