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Journal of Bacteriology, October 2002, p. 5583-5589, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5583-5589.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Protein-Protein Interactions That Regulate the Energy Stress Activation of {sigma}B in Bacillus subtilis

Olivier Delumeau,1 Richard J. Lewis,2 and Michael D. Yudkin1*

Microbiology Unit,1 Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom2

Received 25 March 2002/ Accepted 16 July 2002

{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 Km for ATP and the Ki 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., RsbW2-RsbV2 and RsbW2-{sigma}B1. 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 RsbW2 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 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 {sigma}B reached 11.8 and 4.9 µM, 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.

* Corresponding author. Mailing address: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Phone: 44 1865 275302. Fax: 44 1865 275297. E-mail: mdy{at}bioch.ox.ac.uk.

Journal of Bacteriology, October 2002, p. 5583-5589, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5583-5589.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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