Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX.

The alternate sigma factor sigmaS plays an important role in the survival of Salmonella typhimurium following sudden encounters with a variety of stress conditions. The level of sigmaS is very low in rapidly growing cells but dramatically increases as those cells encounter environmental stress or enter into stationary phase. This increase is due in large measure to the stabilization of sigmaS protein against degradation by the ClpXP protease. The MviA protein, also known as RssB or SprE in Escherichia coli, is a putative member of a two component signal transduction system that plays a central role in facilitating sigmaS degradation by ClpXP. In contrast to most two-component systems, MviA does not appear to regulate gene expression but is believed to interact directly with sigmaS and somehow facilitate degradation. We now provide evidence that MviA(RssB) directly interacts both with sigmaS and ClpX in vivo, presumably enabling presentation of sigmaS to the ClpP protease. Interactions were demonstrated using a bacterial two-hybrid system in which sigmaS, MviA, and ClpX were fused to separate moieties of Bordetella pertussis CyaA (adenylate cyclase). Paired hybrid plasmids containing Cya'-MviA/RpoS-'Cya or Cya'-MviA/ClpX-'Cya successfully reconstituted adenylate cyclase activity in both S. typhimurium and E. coli. However, no direct interactions were detected between ClpX and RpoS. A second series of experiments has indicated that the interaction between MviA and sigmaS requires the N-terminus but not the C-terminus of MviA. Cellular levels of MviA appear to be very low in the cell based on lacZ fusion, Western blot and Northern blot analyses suggesting a catalytic role for MviA in sigmaS degradation. Mutagenesis of MviA residue D58, a canonical residue subject to phosphorylation in many two-component systems, decreased the ability of MviA to facilitate sigmaS turnover in vivo confirming that phosphorylation of MviA increases MviA activity.