SummaryWe screened the putative rap-phr ( r esponse regulator a spartyl-phosphate p hosphatase-ph osphatase r egulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE ( alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE , which belongs to the regulon of DegU, the response regulator of the DegS-DegU two-component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE-lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1-30 nM of a synthetic pentapeptide (PhrG; NH 2 -EKMIG-COOH) to the phrG disruptant completely rescued aprE-lacZ expression, indicating that the PhrG peptide is indeed involved in aprE-lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100-300 nM) to the phrG cells decreased aprE-lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA-lacZ expression directed by ComA, the regulator of the ComP-ComA two-component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU-P (phosphorylated DegU) in vitro . RapG, however, did not affect dephosphorylation of DegU-P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.

The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development. Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively. Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity. This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter. We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions. Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation. Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE. Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.