Environmental stress activates B , the general stress response factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a B -dependent promoter, stress activation of B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived B activation. The data suggest that RsbX is not a direct participant in the B stress induction process but rather serves as a device to limit the magnitude of the stress response. B, the general stress response factor of Bacillus subtilis (20), is activated by a drop in intracellular ATP levels (1,22) or by any of a number of diverse environmental insults (e.g., heat shock, ethanol treatment, or salt shocks) (5, 7,22,23).B is held inactive by a binding protein (RsbW) which can form a complex with either B or an alternative target (RsbV) (6, 8, 9). RsbW is also a protein kinase which can phosphorylate RsbV and convert it into a form (RsbV-P) that no longer binds RsbW (8). The relatively high intracellular ATP levels that occur during growth favor the phosphorylation of RsbV, the formation of RsbW-B complexes, and the inhibition of B -dependent transcription (1,22). Upon entry into stationary phase, a drop in intracellular ATP levels leads to ineffective phosphorylation of RsbV, the formation of RsbV-RsbW complexes, and the release of B (1,22). Environmental stress activation of B occurs irrespective of intracellular ATP levels and the activity of the RsbW kinase (22). Instead, it involves the reactivation of RsbV-P by an RsbV-P-specific phosphatase (21). The existence of such a phosphatase was anticipated following the discovery that a phosphatase (SpoIIE) is involved in the reactivation of the phosphorylated form of the RsbV homolog (SpoIIAA) in the F system (2, 10). RsbV-P phosphatases participate in both stress-induced and stationaryphase activation of B (21). Phosphatase activity is essential for stress activation of B ; however, it merely enhances the stationary-phase response (21). Both the stationary-phase-and stress-induced dephosphorylation reactions require one or more of the products of the B regulators RsbR, RsbS, and RsbT (13,21,23). The RsbU protein is also needed for stressdependent, but not stationary-phase-dependent, RsbV-P dephosphorylation (21). Recent experiments have shown that RsbU can dephosphorylate RsbV-P in vitro and that this activity is enhanced by RsbT (15,24). It is likely that RsbU is a stress-activated phosphatase with RsbT providing the activation signal (24). The stationary-phase-specific phosphatase is unknown.An additional protein (RsbX) is a negative regulator of RsbU-dependent RsbV-P dephosphorylation. B. subtilis strains with null mutations in RsbX h...

Endospore formation in Bacillus subtilis begins with an asymmetric cell division that partitions the bacterium into mother cell and forespore compartments. Mother cell-specific gene expression is initiated by E , a transcription factor that is active only in the mother cell but which existed as an inactive precursor (pro-E ) in the predivisional cell. Activation of pro-E involves the removal of 27 amino acids from its amino terminus. A chimera of pro-E and the green fluorescent protein (GFP) was expressed from either the normal sigE promoter (P spoIIG ), which places pro-E ::GFP in both mother cell and forespore compartments, or the forespore-specific promoter (P dacF ), which produces pro-E ::GFP only in the forespore compartment. The pro-E ::GFP expressed from P spoIIG , but not P dacF , was converted to a lower-molecular-weight form by a mechanism dependent on gene products (SpoIIGA and F

ςE is a mother cell-specific transcription factor of sporulating Bacillus subtilis that is derived from an inactive precursor protein (pro-ςE). To examine the process that prevents ςE activity from developing in the forespore, we fused the ςE structural gene (sigE) to forespore-specific promoters (PdacF and PspoIIIG ), placed these fusions at sites on the B. subtilis chromosome which translocate into the forespore either early or late, and used Western blot analysis to monitor SigE accumulation and pro-ςE processing. sigE alleles, placed at sites which entered the forespore early, were found to generate more protein product than the same fusion placed at a late entering site. SigE accumulation and processing in the forespore were enhanced by null mutations in spoIIIE, a gene whose product is essential for translocation of the distal portion of the B. subtilischromosome into the forespore. In other experiments, a chimera of pro-ςE and green fluorescence protein, previously shown to be unprocessed if it is synthesized within the forespore, was found to be processed in this compartment if coexpressed with the gene for the pro-ςE-processing enzyme, SpoIIGA. The need forspoIIGA coexpression is obviated in the absence of SpoIIIE. We interpret these results as evidence that selective degradation of both SigE and SpoIIGA prevent mature ςE from accumulating in the forespore compartment of wild-type B. subtilis. Presumably, a gene(s) located at a site that is distal to the origin of chromosome transfer is responsible for this phenomenon when it is translocated and expressed in the forespore.

No abstract