Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation

The pivotal event establishing sporulation-specific gene expression in Bacillus subtilis is the reprogramming of the bacterium’s RNA polymerase (RNAP) (52). This occurs when the principal promoter recognition subunit (ςA) of the vegetative cell RNAP is replaced by analogous sporulation-specific subunits (ςE, ςF, ςG, and ςK) (18, 52). These alternative ς factors control both the timing and localization of spore gene expression. Early in sporulation, B. subtilis partitions itself into two unequal compartments with unique developmental fates. The smaller, forespore compartment is eventually engulfed by the larger, mother cell compartment, which nurtures the forespore as it develops into a mature endospore. Each of the sporulation-specific ς factors is active in only one of these two compartments. Mother cell gene expression is controlled by the sequential appearance of ςE followed by ςK, while forespore-specific genes are activated first by ςF and then by ςG (7, 8, 10, 18, 20, 30, 37, 41, 42, 44, 52, 60). The genes encoding each of the early sporulation ς factors (ςE and ςF) are expressed at the onset of sporulation (31, 36, 56); however, neither of these factors is active until later in development, when the two separate compartments are formed. ςE and ςF are each kept silent by a unique means. ςF is bound to an inactivating anti-ςF protein (SpoIIAB), while ςE is formed as an inactive proprotein, pro-ςE (9, 13, 36, 43, 51). Pro-ςE becomes active only after 27 amino acids are cleaved from its amino terminus (36). The septation event initiates a process that leads to the release of ςF from its antagonist in the forespore (1, 3, 12). ςF then directs the transcription of a gene, spoIIR, whose product in turn triggers pro-ςE processing in the mother cell (23, 29, 39). Once active in their particular compartments, ςE and ςF induce expression of the genes for the sigma factors which will ultimately replace them (ςK and ςG, respectively) (14, 34, 35, 44, 53). ςK and ςG, like their predecessors, are initially inactive. ςK is formed as a proprotein which is processed and activated in response to a signal from the developing forespore (7, 8, 41). ςG’s activity is restricted by SpoIIAB, the same anti-ς protein that inhibited ςF (16, 30). Turnover of SpoIIAB in the forespore is thought to be an important factor in activating ςG (30, 33). Although much is known about the mechanisms by which the sporulation-specific ς factors are expressed and activated, the process by which they replace ςA on RNAP is still unclear. ςA persists in sporulating B. subtilis (54), and yet by 2 to 3 h after the onset of sporulation, the RNAP extracted from these bacteria is virtually devoid of ςA (19, 38). Early studies of ςA’s displacement from RNAP revealed that treatment of a sporulating B. subtilis culture with the protein synthesis inhibitor chloramphenicol allowed ςA to again become extractable as an RNAP subunit (47). This result was interpreted as evidence for the existence of a short-lived protein inhibitor of ςA that appears in sporulating B. subtilis to block ςA’s binding to RNAP. Anti-ς factor proteins are now known to restrict the ability of several B. subtilis factors to form RNAP holoenzymes. In addition to ςF and ςG (9, 13, 16, 30, 33), the general stress response (ςB) and motility (ςD) ς factors of B. subtilis are controlled by inhibitory binding proteins (4, 6, 11). It is plausible that a similar protein could be involved in restricting ςA’s access to RNAP in sporulating B. subtilis. Alternatively, the presence of the sporulation-specific ς factors themselves could be the basis for ςA’s exclusion from RNAP. If the sporulation ς factors are effective competitors for a limited pool of core RNAP, their mere presence might be sufficient to deny ςA access. Using velocity sedimentation techniques to separate RNAP from unassociated ς factors and Western blot analyses to locate RNAP, ςA, ςE, and ςK in these gradients, we revisited the problem of ςA release during sporulation. The pattern of ς factor association with RNAP in wild-type and mutant B. subtilis was found to be consistent with a model in which competition for RNAP by sporulation ς factors is necessary for E-ςA decrease and the changeover from E-ςE to E-ςK that occurs later in development. If unknown sporulation factors facilitate this process, they do not appear to be adequate for separating the preexisting ς factors from RNAP in the absence of the ς proteins that will replace them.