Protein-protein interactions that regulate the energy stress activation of sigma(B) in Bacillus subtilis.
Sigma(B) is an alternative sigma factor that controls the general stress response in Bacillus subtilis. In the absence of stress, sigma(B) is negatively regulated by anti-sigma 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 the K(m) for ATP and the K(i) for 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 sigma(B) with different stoichiometries, i.e., RsbW(2)-RsbV(2) and RsbW(2)-sigma(B)(1). As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-sigma(B) interactions 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 RsbW(2) for RsbV is much higher than that for sigma(B). The intracellular concentrations of RsbV, RsbW (as a monomer), and sigma(B) measured before stress were similar (1.5, 2.6, and 0.9 micro M, respectively). After ethanol stress they all increased. The increase was greatest for RsbV, whose concentration reached 13 micro M, while those of RsbW (as a monomer) and sigma(B) reached 11.8 and 4.9 micro M, respectively. We conclude that the higher affinity of RsbW for RsbV than for sigma(B), rather than a difference in the concentrations of RsbV and sigma(B), is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.
Delumeau
Olivier
O
Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Lewis
Richard J
RJ
Yudkin
Michael D
MD
D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases; D011485:Protein Binding; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C086522:RsbV protein, Bacteria; C080358:RsbW protein, bacteria; C079011:SigB protein, Bacteria; D012808:Sigma Factor; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; C091162:RsbU protein, Bacillus subtilis