Ogura Mitsuo, Shimane Kana, Asai Kei, Ogasawara Naotake, Tanaka Teruo
Department of Marine Science and Technology, Tokai University, 3-20-1 Orido, Shimizu, Shizuoka 424-8610, Japan.
Mol Microbiol. 2003 Sep;49(6):1685-97. doi: 10.1046/j.1365-2958.2003.03665.x.
We screened the putative rap-phr (response regulator aspartyl-phosphate phosphatase-phosphatase regulator) 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; NH2-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.
我们利用多拷贝质粒，在枯草芽孢杆菌基因组中筛选了假定的rap-phr（应答调节因子天冬氨酰磷酸磷酸酶-磷酸酶调节因子）系统，寻找影响aprE（碱性蛋白酶基因）表达的rap基因。我们发现rapG参与了aprE的调控，aprE属于DegS-DegU双组分系统的应答调节因子DegU的调控子。rapG和phrG的缺失分别导致aprE-lacZ表达增强和降低，这表明PhrG抑制RapG的活性。向phrG缺失突变体中添加1-30 nM的合成五肽（PhrG；NH2-EKMIG-COOH）可完全恢复aprE-lacZ的表达，这表明PhrG肽确实参与了aprE-lacZ的表达。令人惊讶的是，向phrG细胞中引入多拷贝phrG或添加高浓度（100-300 nM）的PhrG肽都会降低aprE-lacZ的表达。这些结果让人想起之前的观察结果，即较高浓度的PhrC肽会抑制ComP-ComA双组分系统的调节因子ComA所指导的srfA-lacZ表达。由于Rap蛋白属于天冬氨酰蛋白磷酸酶家族，我们试图在体外研究RapG对DegU-P（磷酸化的DegU）去磷酸化的可能影响。然而，在采用的实验条件下，RapG并不影响DegU-P的去磷酸化。因此，我们推测RapG可能抑制DegU与靶启动子的结合活性。我们使用aprE启动子和另一个靶标comK启动子分析了DegU和RapG的相互作用。凝胶迁移分析表明，RapG作为DegU与aprE和comK启动子区域结合的抑制剂，并且这种抑制作用被PhrG肽抵消。
