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Journal of Bacteriology, January 2003, p. 35-40, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.35-40.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Binding of ^A and ^B to Core RNA Polymerase after Environmental Stress in Bacillus subtilis

Claudia Rollenhagen,^1,2, Haike Antelmann,¹ Janine Kirstein,¹ Olivier Delumeau,² Michael Hecker,¹ and Michael D. Yudkin^2*

Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität, 17487 Greifswald, Germany,¹ Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OXI 3QU, United Kingdom²

Received 15 July 2002/ Accepted 16 September 2002

In Bacillus subtilis, the alternative sigma factor ^B is activated in response to environmental stress or energy depletion. The general stress regulon under the control of ^B provides the cell with multiple stress resistance. Experiments were designed to determine how activated ^B replaces ^A as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the ^A-dependent stress gene clpE under ^B-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of ^A and ^B for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of ^B for E was 60-fold lower than that of ^A. Western blot analyses with antibodies against ^A, ^B, and E showed that, after exposure to ethanol stress, the concentration of ^B was only twofold higher than those of ^A and E. Thus, the concentration of ^B after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of ^B to E after stress.

Present address: Abteilung Molekulare Biologie, Max-Planck-Institut für Infektionsbiologie, 10117 Berlin, Germany.

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