Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

Bacillus subtilis initiates a complex developmental program that leads to the differentiation of the cell into a dormant endospore as the final step in a series of responses to nutrient depletion (for a review, see reference 18). Initiation of this differentiation is dependent upon phosphorylation of a response regulator, Spo0A (6). A complex signal transduction network comprising multiple kinases and phosphatases controls the level of phosphorylated Spo0A (Spo0A∼P) in the cells (6, 16). Spo0A∼P directly regulates expression of about 121 genes through either repression or activation (14) by binding specifically to a 7-bp DNA element, referred to as an 0A box (5′-TGNCGAA-3′) (24, 31). For example, Spo0A∼P binding directly represses the promoters for abrB, fruR, flgB, ftsE, sdp, and rocD (14, 17, 20). Other genes are directly activated by Spo0A∼P (14, 17, 20), and still others are activated by Spo0A∼P indirectly (14, 24). For example, the spo0H gene is repressed by AbrB until Spo0A∼P produced at the end of the exponential-growth phase represses abrB, relieving spo0H from AbrB-mediated repression (28). Moreover, Spo0A-activated promoters include those used by RNA polymerase containing the primary sigma factor, σA (e.g., spoIIG and spoIIE promoters) (20, 30) and those used by RNA polymerase containing the secondary sigma factor, σH (e.g., spoIIA and racA promoters) (2, 3, 29), which introduces additional combinations of control. In a strain mutant with respect to both kinA and kinB, two of the kinases that control the level of Spo0A∼P, a low level of Spo0A∼P accumulates that is sufficient to repress abrB transcription but insufficient for the activation of other Spo0A∼P-dependent genes required for sporulation (25). Therefore, the responses of some Spo0A-regulated promoters to Spo0A∼P appear to be more sensitive to Spo0A∼P levels than are the responses of other Spo0A-regulated promoters. A progressive accumulation of Spo0A∼P at the end of the exponential-growth phase produces a temporal pattern of responses by Spo0A-regulated promoters that reflects the specific response of each promoter to low- or high-threshold levels of Spo0A∼P (9). Thus, in addition to the combinations of controls described above, Spo0A-regulated promoters can be classified as either high- or low-threshold activated or repressed promoters (9). High-threshold promoters require a high level of Spo0A∼P to be activated or repressed, probably in part because the regulatory regions for these genes have relatively weak affinities for Spo0A∼P, whereas low-threshold promoters respond to a low level of Spo0A∼P because their regulatory regions have relatively high-affinity binding sites for Spo0A∼P (9). The progressive accumulation of Spo0A∼P allows cells to try less-drastic responses to nutrient depletion before commitment to differentiation into a dormant cell type. Moreover, Spo0A phosphorylation occurs in only a fraction of the population (7, 9, 10). Activation of Spo0A in this fraction of the population enables these cells to delay progression into sporulation by activating two operons, the sporulation killing factor (skf) and the sporulation delaying protein (sdp) operons (10). The products of these two operons prevent non-Spo0A∼P-expressing sibling cells from sporulating (sdp) and cause them to lyse (skf). The cells that have activated Spo0A early are then able to feed off the nutrients released, allowing them to continue growing and thus to delay their differentiation into dormant endospores. The skf promoter must be highly active to drive production of the secreted toxin, and it must be activated early in response to low levels of phosphorylated Spo0A (9, 10). However, its expression must be tightly regulated to prevent premature expression of the killing factor. Here we investigate how this promoter can be both highly active in response to low levels of Spo0A∼P and tightly controlled to prevent premature expression of the toxin. The sequence of the skf promoter, which is activated by low levels of Spo0A∼P (9), appears to indicate that Spo0A∼P may directly activate this promoter by a mechanism that is different from that of the spoIIG promoter, which has been studied more extensively (4, 12, 19-21, 23). Comparison of the relative affinities of Spo0A for the high- and low-threshold promoters shows that spoIIG has a Kd of 1,700 nM whereas the skf promoter has an apparent Kd value of 26 nM (9). A bioinformatics search identified two Spo0A binding sites (14); however, the search did not reveal other features of the skf promoter that would contribute to its positive regulation by low levels of Spo0A∼P. Here we report that skf is a σA-dependent promoter with multiple Spo0A binding sites, two of which span the −35 region. Moreover, upstream of the mapped transcriptional start site is a sequence that is very similar to the TRTGn motif found in extended −10 promoters in Bacillus subtilis (13, 26, 27), another feature that is not present in the spoIIG promoter. We show that this sequence is necessary for the high level of Spo0A-dependent promoter activity. We also describe other features of this promoter that are necessary for its tight regulation by Spo0A.