Liu W, Hulett F M
Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
J Bacteriol. 1997 Oct;179(20):6302-10. doi: 10.1128/jb.179.20.6302-6310.1997.
Several gene products, including three two-component systems, make up a signal transduction network that controls the phosphate starvation response in Bacillus subtilis. Epistasis experiments indicate that PhoP, a response regulator, is furthest downstream of the known regulators in the signaling pathway that regulates Pho regulon genes. We report the overexpression, purification, and use of PhoP in investigating its role in Pho regulon gene activation. PhoP was a substrate for both the kinase and phosphatase activities of its cognate sensor kinase, PhoR. It was not phosphorylated by acetyl phosphate. Purified phosphorylated PhoP (PhoPP) had a half-life of approximately 2.5 h, which was reduced to about 15 min by addition of the same molar amount of *PhoR (the cytoplasmic region of PhoR). ATP significantly increased phosphatase activity of *PhoR on PhoPP. In gel filtration and cross-linking studies, both PhoP and PhoPP were shown to be dimers. The dimerization domain was located within the 135 amino acids at the N terminus of PhoP. Phosphorylated or unphosphorylated PhoP bound to one of the alkaline phosphatase gene promoters, the phoB promoter. Furthermore, PhoP bound exclusively to the -18 to -73 region (relative to the transcriptional start site +1) of the phosphate starvation-inducible promoter (Pv) but not to the adjacent developmentally regulated promoter (Ps). These data corroborate the genetic data for phoB regulation and suggest that activation of phoB is via direct interaction between PhoP and the phoB promoter. Studies of the phosphorylation, oligomerization, and DNA binding activity of the PhoP protein demonstrate that its N-terminal phosphorylation and dimerization domain and its C-terminal DNA binding domain function independently of one another, distinguishing PhoP from other response regulators, such as PhoB (Escherichia coli) and NtrC.
包括三个双组分系统在内的几种基因产物构成了一个信号转导网络，该网络控制枯草芽孢杆菌中的磷酸盐饥饿反应。上位性实验表明，应答调节因子PhoP在调节Pho调控子基因的信号通路中，是已知调节因子中最下游的一个。我们报道了PhoP的过表达、纯化及其在研究其在Pho调控子基因激活中的作用方面的应用。PhoP是其同源传感激酶PhoR的激酶和磷酸酶活性的底物。它不会被乙酰磷酸磷酸化。纯化的磷酸化PhoP（PhoPP）的半衰期约为2.5小时，加入等量的PhoR（PhoR的细胞质区域）后，半衰期缩短至约15分钟。ATP显著增加PhoR对PhoPP的磷酸酶活性。在凝胶过滤和交联研究中，PhoP和PhoPP均显示为二聚体。二聚化结构域位于PhoP N端的135个氨基酸内。磷酸化或未磷酸化的PhoP与碱性磷酸酶基因启动子之一phoB启动子结合。此外，PhoP仅与磷酸盐饥饿诱导型启动子（Pv）的-18至-73区域（相对于转录起始位点+1）结合，而不与相邻的发育调控启动子（Ps）结合。这些数据证实了phoB调控的遗传数据，并表明phoB的激活是通过PhoP与phoB启动子之间的直接相互作用实现的。对PhoP蛋白的磷酸化、寡聚化和DNA结合活性的研究表明，其N端磷酸化和二聚化结构域以及C端DNA结合结构域彼此独立发挥作用，这使PhoP与其他应答调节因子如PhoB（大肠杆菌）和NtrC有所区别。
