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Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro. The activity of Spo0A is increased by phosphorylation. We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo. In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting. Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A. The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template. Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.

The spo0E locus of Bacillus subtilis codes for a negative regulator of sporulation that, when overproduced, represses sporulation and, if deleted, results in inappropriate timing of sporulation. The product of this locus, Spo0E, was purified and found to be a protein phosphatase, which specifically dephosphorylated the sporulation transcription factor Spo0A-P, converting it to an inactive form. Spo0E was not significantly active as a phosphatase on other components of the phosphorelay signal-transduction pathway producing Spo0A-P. A mutant Spo0E protein that results in sporulation deficiency was purified and found to be hyperactive as a phosphatase. The Spo0E phosphatase may provide an additional control point for environmental, metabolic, or cell-cycle regulation of phosphate flow in the phosphorelay. These results reinforce the concept that the phosphorelay is subject to a host of positive and negative signals for sporulation that are recognized and interpreted as signal integration circuit that has the role of regulating the cellular level of active phosphorylated Spo0A sporulation transcription factor.

The spoIIG operon of Bacillus subtilis codes for a sporulation-specific sigma factor, sigma E. In vivo expression of the spoIIG promoter is activated shortly after the onset of sporulation and is dependent on kinA, spo0F, spo0B and spo0A genes. The products of these genes have been shown to participate in a phosphorelay reaction in vitro, culminating in phosphorylation of the transcription factor, Spo0A. The effect of Spo0A phosphorylation on in vitro transcription from the spoIIG promoter was determined. Aliquots from phosphorelay reactions enhanced spoIIG promoter activity 10-fold in transcription assays and stimulation of transcription was dependent on Spo0A phosphorylation. Our results provide biochemical evidence that Spo0A and the phosphorelay form a signal transduction pathway which activates spoII gene expression in development.