Activation of the spoIIG promoter at the onset of sporulation in Bacillus subtilis requires the regulatory protein, SpoOA, which binds to two sites in the promoter, sites 1 and 2. Phosphorylation of SpoOA is essential for the initiation of sporulation. Therefore, we examined the role of SpoOA phosphorylation in spoHG promoter activation. Phosphorylation of SpoOA stimulated transcription from the spoIIG promoter in vitro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of SpoOA increased its afinity for site 2 more than for site 1, which is the site to which nonphosphorylated SpoOA binds most avidly.This result could not be explained by increased cooperativity between SpoOA bound at sites 1 and 2 because the increased alfinity for site 2 by phosphorylated SpoOA was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located SpoOA-binding sequences in the spolHG promoter by DMS protection assays and mutational analysis, and found that site 1 contains one higher-afinity binding sequence whereas site 2 contains two weaker-binding sites. Two substitutions in site 2 of the spolIG promoter that change the sequence to be more like an optimal SpoOA-binding site were found to increase promoter activity. Moreover, phosphorylation of SpoOA was not required in vivo for activation of the spoIIG promoter containing these strong binding sites. The results suggest that the primary role for phosphorylation of SpoOA is to increase its affinity for specific sites rather than to activate an activity of SpoOA that acts on RNA polymerase at promoters.During endospore formation in Bacillus subtilis, transcription of sporulation-specific genes is regulated both temporally and spatially. Particularly at later times, this regulation is achieved largely by the sequential and, in some cases, compartment-specific appearance or activation of a series of secondary sigma factors that direct core RNA polymerase to different sets of promoters (reviewed in reference 6). However, at early times in the
The spoIIG promoter is used by RNA polymerase containing cA (E&r), the primary form of RNA polymerase found in vegetative cells in BaciUus subtilis. However, the spolIG promoter is active only after the onset of sporulation. Activation of the spoIIG promoter requires the product of the spoOA gene (SpoOA). SpoOA is a sequence-specific DNA-binding protein which binds to two sites in the spoIIG promoter that are essential for promoter activity. We found that single-base-pair substitutions in these two regions that reduced promoter activity in vivo caused reduced binding of SpoOA in vitro, and one substitution that increased promoter activity in vivo increased the affinity of SpoOA for this DNA in vitro. Furthermore, SpoOA stimulated transcription from the spolIG promoter by Ecr in vitro. These results support the model that binding of SpoOA activates EMA-dependent transcription from the spoIIG promoter after the onset of sporulation.Under conditions of nutrient deprivation the bacterium Bacillus subtilis undergoes complex morphological and physiological changes that result in the formation of an endospore (reviewed in reference 16). Endospore formation requires the expression of over 50 genetic loci, and these loci are transcribed in a precise temporal sequence. At least six of these genetic loci encode secondary sigma factors which confer on RNA polymerase the ability to transcribe different subsets of genes (reviewed in reference 10). The production of these sporulation-specific sigma factors plays a fundamental role in regulating the gene expression required for sporulation. However, RNA polymerase containing 0A (EoA), the primary sigma factor in vegetative cells, also plays a role in the expression of genes during sporulation by directing the transcription of at least two key operons during the early stages of sporulation (5, 9, 23). One of the promoters used by EoA after the onset of sporulation directs the expression of the spoIIG operon which encodes the structural gene for cE (8), a secondary sigma factor necessary for the transcription of later sporulation genes.
The transcriptional regulator Spo0A activates transcription from two types of promoters. One type of promoter is used by RNA polymerase containing sigma A, whereas the other type is used by RNA polymerase containing sigma H. There are Spo0A-binding sites near the -35 region of both types of promoters. It has been reported that some transcriptional regulators that bind near the -35 regions of promoters directly interact with the sigma subunit of RNA polymerase. Therefore, we looked for evidence that Spo0A interacts with both sigma factors by searching for single amino acid substitutions in these factors that specifically prevent expression from Spo0A-dependent promoters, but that do not decrease activity of Spo0A-independent promoters. Two such amino acid substitutions were isolated in sigma A and one was isolated in sigma H. The amino acid substitutions in sigma A prevented expression from the Spo0A-activated promoters, spoIIG and spoIIE, but expression was not impaired from the Spo0A-independent, sigma A-dependent promoter tms or from the Spo0A-activated, sigma H-dependent promoter, spoIIA. The amino acid substitution in sigma H prevented expression from the spoIIA promoter but not from the Spo0A-independent promoter, citGp2, which is used by sigma H-RNA polymerase. All of these amino acid substitutions occur in the carboxyl terminus of the sigma factors. These amino acid substitutions may define the sites of contact between the sigma factors and Spo0A. The ability of response regulators such as Spo0A to interact with multiple sigma factors may increase the variety of responses made by bacteria using a limited number of transcription factors.
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