Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions
Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions
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DOI：
10.1099/mic.0.2007/007070-0
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发表时间：
2007-10-01
期刊：
影响因子：
2.8
通讯作者：
Martin, Juan F.
中科院分区：
文献类型：
--
作者：
Apel, Alexander K.;Sola-Landa, Alberto;Martin, Juan F.
通过与灰色链霉菌PhoA蛋白的氨基酸序列同源性分析，在天蓝色链霉菌基因组中鉴定出3个碱性磷酸酶基因phoA、phoC和phoD。PhoA和PhoC对应于广谱碱性磷酸酶，而PhoD类似于色褐链霉菌的Ca 2+依赖性磷脂酶D。phoA和phoD基因在磷酸盐限制条件下在R5培养基中有效表达，如使用xylE报告基因的研究所示，而phoC在相同条件下转录较差。phoA的表达明显依赖于PhoP，因为它在S. coelicolor Delta phoP突变体，并在低磷酸盐浓度下强烈激活。类似地，phoD的表达是PhoP依赖性的，并且对磷酸盐的可用性高度敏感。相比之下，phoC的表达不是PhoP依赖性的。电泳迁移率变动分析表明，PhoP结合的phoA和phoD的启动子，但不是phoC。GST-PhoP的足迹法研究揭示了phoA启动子中存在一个PHO盒（两个11 nt的直接重复），phoD启动子中存在两个PHO盒。通过引物延伸鉴定了三个启动子的转录起始点。phoD的转录起始点与其翻译起始密码子的G相一致，表明该基因转录为无前导的mRNA。推导的-10和-35区域的phoD（而不是那些phoA）重叠的PHO盒在这个启动子，这表明过量的PhoP干扰RNA聚合酶的结合到这个启动子。总之，三种启动子在PhoP对其表达的调节中显示出明显的差异。
Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca2+-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor Delta phoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.