The sigma B-dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock

Abstract

oE, a secondary sigma factor of Bacillus subtilis, was found to increase 5-to 10-fold when cultures were shifted from 37 to 48°C. Western blot (immunoblot) analyses, in which monoclonal antibodies specific for the sigB operon products RsbV, RsbW, and crB were used to probe extracts from wild-type and mutant B. subtilis strains, revealed that all three proteins increased coordinately after heat shock and that this increase was dependent on &rB but not RsbV, a positive regulator normally essential for &3-dependent sigB expression.Nuclease protection experiments of RNA synthesized after heat shock supported the notion that the shift to 48°C enhanced transcription from the sigB operon's o'B-dependent promoter. The level of mRNA initiating at the &B-dependent ctc promoter was also seen to increase approximately 5-to 10-fold after heat shock.Pulse-labeling of the proteins synthesized after a shift to 48°C demonstrated that sigB wild-type and mutant strains produced the maijor heat-inducible proteins in similar amounts; however, at least seven additional proteins were present after the temperature shift in the wild-type strain but absent in the sigB null mutant.Thus, although cr' is not required for the expression of essential heat shock genes, it is activated by heat shock to elevate its own synthesis and possibly the synthesis of several other heat-inducible proteins.a3 is a minor abundance sigma factor of Bacillus subtilis that is found associated with the RNA polymerase from vegetatively growing bacteria but which disappears from the extractable RNA polymerase population by the second hour after the onset of sporulation (12). Although ae3 was the first secondary sigma factor to be discovered in bacteria, its physiological role remains obscure (6, 9, 13). Null mutations in the aW structural gene (sigB) have no obvious effect on vegetative growth or sporulation .6, 9).Even though the function of a' is not yet known, it is a potent regulatory protein with an elaborate mechanism for keeping its activity in check. sigB is the third gene in a four-gene operon with the other genes, rsbV, rsbW, and rsbX (formerly called orfV, orfW, and orfX, respectively), involved in controlling ar3 activity (3, 4, 7). Previous work suggested that the three gene products operate in a pathway of negative control, with RsbW being the primary inhibitor of ar3 activity and the other two gene products (RsbV and RsbX) functioning upstream of RsbW in the regulatory pathway (3, 7). Mutations which reduce RsbW's inhibitory effects severely impair the growth of strains which carry them and readily give rise to suppressor mutations which reduce or eliminate the activity of in these strains (2,3,7,15). In addition to the sigB operon itself (3, 17), two genes (ctc and csbA) which depend on ae for a significant part of their expression have been identified (8,14,16 genes is temperature-sensitive oligosporageny which occurred upon disruption of ctc by an integrating plasmid (pAK-6/p1949) (28). Placement of this ctc null mutation into a strain that carri...

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals

“…The transcription factor B of B. subtilis controls a set of genes that are induced both upon entry into stationary phase and by a variety of physiological stresses encountered during logarithmic growth (4,6,7,9,25,29,51). Previous genetic analysis indicated that the products of the rsbV, rsbW, and rsbX genes in the sigB operon form a hierarchical regulatory pathway that controls B activity in response to stationary-phase signals as well as to signals of salt and ethanol stress (2,6,8,24,29).…”

“…Significantly, recent work has shown that B is also activated in logarithmically growing cells by a variety of environmental stresses that limit growth, including hyperosmosis, heat, oxidative stress, and ethanol shock (4,6,51). These findings have led to the proposal that B controls a general stress regulon (6).…”

The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of sigma B

“…There is a growing list of conditions, including entry into stationary phase, heat shock, osmotic shock, ethanol treatment, and anaerobiosis, etc., which lead to activation of B (2,4,7,9,16,26). In each instance, activation of B presumably involves its release from RsbW.…”

“…With the exception of heat shock activation, all known B activation processes require RsbV (2,3,4,7,9). Therefore, we wished to test whether the RsbU-dependent activation that occurs in the absence of RsbX also requires RsbV or whether it defines a novel B activation system.…”

“…Genes that rely on B for their expression are induced when B. subtilis ceases exponential growth in rich, sporulation-suppressing media or is exposed to any of several environmental insults (e.g., heat, salt, or ethanol) (2, 4, 7-10, 16, 25, 26). Although the circumstances which activate B -dependent transcription argue that B plays a role in B. subtilis's ability to adapt to various stresses, it is not essential for the bacterium's survival under any of these conditions (4,6,9,13). Null mutations in the B structural gene confer no obvious phenotype on the strains which carry them (6,13).…”