Signal transduction mechanism of the RcsCDB system in Salmonella Typhimurium

PESCARETTI, MARIA DE LAS MERCEDES; FARIZANO, JUAN VICENTE; TORREZ LAMBERTI, MONICA FLORENCIA; MORERO, ROBERTO; DELGADO, MONICA ALEJANDRA

Santos, San Pablo

Congreso; XXI Congreso Latinoamericano de Microbiología (XXI ALAM); 2012

Asociacion Latino Americana de Microbiologia

The RcsCDB phosphorelay system controls the expression of cps and flhDC operons, which are involved in the colanic acid and flagellar biosynthesis respectively. Rcs is an unusual phosphorelay system composed of the sensor RcsC, the response regulator RcsB; and the RcsD protein reported as intermediary in the transduction signal. The described multi-step phosphorelay mechanism for the Rcs system involves the corresponding sequence RcsC→RcsD→RcsB. Even though at present the signal that activates the Rcs system has not been determined, we have previously demonstrated that the Rcs regulon is modulated by the overproduction of the RcsB response regulator in an rcsC or rcsD mutant but not in the doble mutant. This fact, allowed us to assume that only the RcsB phosphorylated form (RcsB-P) is able to produce this modulation, and we postulated that the phosphorylation of the RcsB regulator would take place by different pathways. The goal of present study was determinate the putative phosphorelay pathways. In this work we used the two-hybrid assay (BACTH) to probe the RcsC, RcsD and RcsB interaction. To determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, we here purified all the Rcs system components. For this purpose, we constructed plasmids containing the full-length of the RcsB regulator and the cytoplasmic domain of the RcsC and RcsD sensors, because is well known that the alone HK cytoplasmic domain can retain the activity of autophosphorylation and trans-phosphorylation to the response regulator. To facilitate the protein purification procedure, a 6His-tag sequence was added in the amino terminus of each protein. We here achieved for the first time the expression and purification of the RcsC and RcsD cytoplasmic domain. These domains were recovered in soluble fractions and purified by a single step of affinity chromatography, allowed us to use it in the self- and trans-phophorylation in vitro assays.