Protein-Protein Interactions That Regulate the Energy Stress Activation of σBinBacillus subtilis

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Abstract
σBis an alternative σ factor that controls the general stress response inBacillus subtilis. In the absence of stress, σBis negatively regulated by anti-σ factor RsbW. RsbW is also a protein kinase which can phosphorylate RsbV. When cells are stressed, RsbW binds to unphosphorylated RsbV, produced from the phosphorylated form of RsbV by two phosphatases (RsbU and RsbP) which are activated by stress. We now report the values of theKmfor ATP and theKifor ADP of RsbW (0.9 and 0.19 mM, respectively), which reinforce the idea that the kinase activity of RsbW is directly regulated in vivo by the ratio of these nucleotides. RsbW, purified as a dimer, forms complexes with RsbV and σBwith different stoichiometries, i.e., RsbW2-RsbV2and RsbW2-σB1. As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-σBinteractions were found to be similar (63 and 92 nM, respectively). Nonetheless, an analysis of the complexes by nondenaturing polyacrylamide gel electrophoresis in competition assays suggested that the affinity of RsbW2for RsbV is much higher than that for σB. The intracellular concentrations of RsbV, RsbW (as a monomer), and σBmeasured before stress were similar (1.5, 2.6, and 0.9 μM, respectively). After ethanol stress they all increased. The increase was greatest for RsbV, whose concentration reached 13 μM, while those of RsbW (as a monomer) and σBreached 11.8 and 4.9 μM, respectively. We conclude that the higher affinity of RsbW for RsbV than for σB, rather than a difference in the concentrations of RsbV and σB, is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.