Interplay of RsbM and RsbK controls the σ^B activity of Bacillus cereus

The alternative transcription factor σ^B of Bacillus cereus controls the expression of a number of genes that respond to environmental stress. Four proteins encoded in the sigB gene cluster, including RsbV, RsbW, RsbY (RsbU) and RsbK, are known to be essential in the σ^B-mediated stress response. In the context of stress, the hybrid sensor kinase RsbK is thought to phosphorylate the response regulator RsbY, a PP2C serine phosphatase, leading to the dephosphorylation of the phosphorylated RsbV. The unphosphorylated RsbV then sequesters the σ^B antagonist, RsbW, ultimately liberating σ^B. The gene arrangement reveals an open reading frame, bc1007, flanked immediately downstream by rsbK within the sigB gene cluster. However, little is known about the function of bc1007. In this study, the deletion of bc1007 resulted in high constitutive σ^B expression independent of environmental stimuli, indicating that bc1007 plays a role in σ^B regulation. A bacterial two-hybrid analysis demonstrated that BC1007 interacts directly with RsbK, and autoradiographic studies revealed a specific C¹⁴-methyl transfer from the radiolabelled S-adenosylmethionine to RsbK when RsbK was incubated with purified BC1007. Our data suggest that BC1007 (RsbM) negatively regulates σ^B activity by methylating RsbK. Additionally, mutagenic substitution was employed to modify 12 predicted methylation residues in RsbK. Certain RsbK mutants were able to rescue σ^B activation in a rsbK-deleted bacterial strain, but RsbK_E439A failed to activate σ^B, and RsbK_E446A only moderately induced σ^B. These results suggest that Glu439 is the preferred methylation site and that Glu446 is potentially a minor methylation site. Gene arrays of the rsbK orthologues and the neighbouring rsbM orthologues are found in a wide range of bacteria. The regulation of sigma factors through metylation of RsbK-like sensor kinases appears to be widespread in the microbial world.