A novel insight on signal transduction mechanism of RcsCDB system in Salmonella enterica serovar typhimurium

Abstract

The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the RcsB regulator, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response.

Figure 1. Modulation of cps and flhDC operons by the RcsC- or RcsD-dependent presence.

The β-galactosidase activity (Miller units), produced by rcsB overexpression, from cps::MudJ (A) and flhDC::MudJ (B) lac transcriptional fusions was investigated in the following genetic backgrounds: wild-type, rcsD, rcsC and rcsD rcsC, harboring the prcsB (black bars) or prcsBop (grey bars) plasmids. These strains were grown at 37°C in LB medium supplemented with 0.35 mM IPTG. The osmotic shock effect by sucrose addition (striped bars) on cps::MudJ (C) and flhDC::MudJ (D) expression was investigated in the wild-type, rcsD, rcsC and rcsD rcsC strains; and compared with control without sucrose (empty bars). These assays were performed as described in Material and Methods. The error bars correspond to the standard deviation of three independent experiments done in duplicate.

Figure 2. Effect of acetyl phosphate on cps expression in RcsC or RcsD-deficient cells.

The transcriptional activity of cps::MudJ fusion, measured as β-galactosidase activity (Miller units), was investigated in five genetic backgrounds: wild-type (EG13384), ackA (MDs1557), ackA rcsD (MDs1559), ackA rcsC (MDs1558) and ackA rcsD rcsC (MDs1562) strains, carrying the prcsB (black bars) or prcsBop (grey bars) plasmids. The strains were grown at 37°C in LB medium supplemented with 0.35 mM as described in Material and Methods. The error bars correspond to the standard deviation of three independent experiments done in duplicate.

Figure 3. In vivo interaction analysis of the Rcs components: Bacterial two-hybrid assay.

The ß-galactosidase activity expressed by E. coli DHM1 harboring derivatives plasmids encoding the rcsCcyt, rcsDcyt or rcsB genes fused to C-terminal (top panel) or N-terminal (bottom panel) of T25 fragments (in the top of each box) and these genes fused to N-terminal or C-terminal of the T18 fragment (under each bar), was measured from bacteria growing in stationary phase in LB medium containing 0.5 mM IPTG. Co-transformed cells with empty pKT25 and pUT18 vector plasmids producing basal ß-galactosidase activity levels served as negative control (−). Co-transformed cells with pKT25-zip and pUT18C-zip plasmid gave high ß-galactosidase activity levels served as positive control (+). The error bars correspond to the standard deviation of three independent experiments done in duplicate.

Figure 4. Autophosphorylation of RcsC and RcsD.

The RcsCcyt-His₆ (A) or RcsDcyt-His₆ (B) proteins were incubated with [γ-³²P] ATP as described in Material and Methods. The phosphorylation was stopped at different time points (0 to 30 min) by addition of 4× SDS sample buffer, and subjected to electrophoresis analysis in a 12% SDS-PAGE and them exposed to autoradiography.

Figure 5. RcsC or RcsD catalyze phosphorylation of RcsB.

The RcsB regulator was mixed with γ-³²P-RcsDcyt-His₆ (A) or γ-³²P-RcsCcyt-His₆ (B) as described in Material and Methods. The reaction was stopped at indicated times (10 to 30 min) by adding 4× SDS sample buffer, followed by analysis on 12% SDS-PAGE and exposed to autoradiography.

Figure 6. Signal transduction model of the RcsCDB phosphorelay system.

In response to cell envelop stress, the Rcs system activation can proceed at least in three pathways: (A) After that an specific stimulus is sensed by RcsC, this protein is autophosphorylated and able to interacts with RcsD, leading to the transfer of phosphate group to the RcsB regulator. The RcsB phosphorylated form is able to bind the promoter region of those target genes required to produce an instant response. (B) Same or different stimulus can produce the RcsC autophosphorylation forming a complex with other no phosphorylated RcsC monomer, and then the phosphate group is transferred to RcsB to modulate the gene expression. (C) In different growth condition, other stimulus could be recognized by RcsD, which after autophosphorylation interacts with other no phosphorylated-RcsD monomer. Then, the phosphate group is transferred to RcsB in order to regulate the expression of those genes necessary for bacteria adaptation. H, histidine; P, phosphate group; KD, kinase domain; p-KD, pseudo kinase domain; RD, receiver domain; HPt, histidine phosphotransfer domain.