Characterization of the energy -sensing module of the Rsb cascade in Bacillus subtilis

The secondary sigma factor, σB, coordinates responses of Bacillus subtilis to environmental and energy stress conditions. A cascade of eight Rsb (Regulator of sigma B) proteins tightly regulate the activity of σB at the post-translational level. Current models of the Rsb cascade group the proteins into an environmental-stress sensing module and an energy-stress-sensing module that are joined to RsbW, the primary regulator of σB. We set out to more fully characterize the protein-protein interactions between three of the proteins of the energy stress-sensing module. RsbV, RsbW, and σ B were expressed in their native form and purified using standard chromatographic techniques. A crosslinking assay was then used to examine interactions between purified σB and the anti-sigma factor, RsbW. Components of the complexes were identified using monoclonal antibodies to each of the proteins. Small molecules, which can signal stress status of the cell (ATP, GTP, and acetyl phosphate) had no obvious effect on the formation of RsbW:σB complexes under the conditions that were tested. The crosslinking assay was then used to examine the interactions between RsbW and the anti-anti-sigma factor, RsbV. Small molecules were examined for their effect on RsbW:RsbV complex formation. Our studies showed that ATP inhibits complex formation between RsbV and RsbW and ADP counteracts the effect of ATP. Other small molecules had no effect on RsbW:RsbV complex formation. Crosslinking assays indicated that RsbW has a greater affinity for RsbV than it does for σ B in the absence of ATP and ADP. Kinetic analysis of the phosphorylation of RsbV via RsbW indicated a Km of 1.3 mM for ATP. ADP altered the apparent Km in a competitive-inhibition type mechanism indicating that the Ki for ADP is approximately 0.22 mM