Relative levels and fractionation properties of Bacillus subtilis σ(B) and its regulators during balanced growth and stress

σ(B) is a secondary sigma factor that controls the general stress response in Bacillus subtilis. σ(B)-dependent genes are activated when σ(B) is released from an inhibitory complex with an anti-σ(B) protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of σ(B) from RsbW, rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (σ(B)) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of σ(B), but rsbU and one or more of the genes contained within an rsbR, S, T deletion are needed fur stress induction of σ(B). In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, RsbT, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and σ(B)), are not elevated during σ(B) activation. Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600- kDa) aggregate which was excluded from our gel filtration matrix. The downstream sigB operon products were not present in this excluded material. The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, eluted from the column earlier than expected from their molecular weights. The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.