INTERACTIONS BETWEEN A BACILLUS-SUBTILIS ANTI-SIGMA FACTOR (RSBW) AND ITS ANTAGONIST (RSBV)

The activity of sigma(B), a secondary sigma factor of Bacillus subtilis, is primarily controlled by an anti-sigma factor protein (RsbW) that binds to sigma(B) and blocks its ability to form an RNA polymerase holoenzyme (E-sigma(B)). Inhibition of sigma(B) by RsbW is counteracted by RsbV, a protein that is essential for the activation of sigma(B)-dependent transcription. When crude B. subtilis extracts were fractionated by gel filtration chromatography or electrophoresis through nondenaturing polyacrylamide gels, a complex composed of RsbW and RsbV that is distinct from the previously observed RsbW-sigma(B) complex was detected. In analogous experiments, RsbX, an additional regulator of sigma(B)-dependent transcription that is thought to act independently of RsbV-RsbW, was not found to associate with any of the other sigB operon products. Two forms of RsbV were visualized when crude cell extracts of B. subtilis were subjected to isoelectric focusing (IEF), with the more negatively charged RsbV species absent from extracts prepared from RsbW- strains. In vitro, RsbV became phosphorylated when incubated with ATP and RsbW but not with ATP alone. The phosphorylated RsbV species comigrated during IEF with the RsbW-dependent form of RsbV found in crude cell extracts. These results suggest that the modified RsbV, present in crude cell extracts, is phosphorylated. When gel filtration fractions containing RsbV-RsbW complexes or RsbV alone were subjected to IEF, only the unmodified form of RsbV was found associated with RsbW. The presumed phosphorylated variant of RsbV was present only in fractions that did not contain RsbW. The data support a model whereby RsbV binds directly to RsbW and blocks its ability to form the RsbW-sigma(B) complex. This activity of RsbV appears to be inhibited by RsbW-dependent phosphorylation.