RELATIVE LEVELS AND FRACTIONATION PROPERTIES OF BACILLUS-SUBTILIS SIGMA(B) AND ITS REGULATORS DURING BALANCED GROWTH AND STRESS

sigma(B) is a secondary sigma factor that controls the general stress response in Bacillus subtilis. sigma(B)-dependent genes are activated when sigma(B) is released from an inhibitory complex with an anti-sigm a(B) protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular A TP levels or to environmental stress (e.g., heat, ethanol, or salt), c ause the release of sigma(B) from RsbW. rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that incl udes sigB (sigma(B)) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stati onary-phase (i.e., ATP-dependent) activation of sigma(B), but rsbU and one or more of the genes contained within an rsbR,S,T deletion are ne eded for stress induction of sigma(B). In other experiments, Western b lot (immunoblot) analyses showed that the levels of RsbR, RsbS, RsbT, and RsbU, unlike those of the sigB operon's four downstream gene produ cts (RsbV, RsbW, RsbX and sigma(B)), are not elevated during sigma(B) activation. Gel filtration and immunoprecipitation studies did not rev eal the formation of complexes between any of the four upstream sigB o peron products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fracti onate as a large-molecular-mass (approximately 600-kDa) aggregate whic h was excluded from our gel filtration matrix. The downstream sigB ope ron products were not present in this excluded material. The unaggrega ted RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elute d 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, sugges ting a protein complex of approximately 90 to 100 kDa.