The two-component system PhoPR of Clostridium acetobutylicum is involved in phosphate-dependent gene regulation
The two-component system PhoPR of Clostridium acetobutylicum is involved in phosphate-dependent gene regulation
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DOI：
10.1128/jb.00574-08
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发表时间：
2008-10-01
影响因子：
3.2
通讯作者：
Fischer, Ralf-Joerg
中科院分区：
文献类型：
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作者：
Fiedler, Tomas;Mix, Maren;Fischer, Ralf-Joerg
乙酰丁酸梭菌ATCC 824的phoR基因位点由phoP和phoR两个基因组成。推断出的蛋白质被预测为一个磷酸盐依赖的双组分调节系统的反应调节器和传感器激酶。我们分析了phoPR在p -i限制的趋化培养和对Pi脉冲的响应中的表达模式。在高磷条件下显示出基本的转录水平，当外部Pi浓度低于0.3 mM时，mRNA转录水平显著增加。在二维凝胶电泳实验中，与P-i过量生长相比，在Pi限制生长条件下，PhoP的表达增加了2.5倍。引物延伸分析确定了phoP至少三个不同的转录起始点。PhoP蛋白和n端截断的*PhoR蛋白分别在大肠杆菌中异源表达并纯化。*PhoR的自磷酸化和PhoP的磷酸化均在体外显示。电迁移位移实验证明，PhoP与C. acetobutylicum的磷酸调控后操纵子的启动子区存在特异性结合。
The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in P-i-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of P-i. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.