Abstract
In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of sigma(A) and sigma(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 sigma(B) for E was 60-fold lower than that of sigma(A). Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those of sigma(A) and E. Thus, the concentration of sigma(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 sigma(B) to E after stress.