Apel Alexander K, Sola-Landa Alberto, Rodríguez-García Antonio, Martín Juan F
Área de Microbiología, Fac. CC. Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071, León, Spain.
Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real 1, 24006 León, Spain.
Microbiology (Reading). 2007 Oct;153(Pt 10):3527-3537. doi: 10.1099/mic.0.2007/007070-0.
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 Ca(2+)-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 DeltaphoP 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.
通过与天蓝色链霉菌 PhoA 蛋白的氨基酸序列同源性比对,在天蓝色链霉菌基因组中鉴定出三个假定的碱性磷酸酶基因,即phoA、phoC 和 phoD。PhoA 和 PhoC 对应于广谱碱性磷酸酶,而 PhoD 与暗产色链霉菌的一种 Ca(2+) 依赖性磷脂酶 D 相似。如使用 xylE 报告基因的研究所表明的,phoA 和 phoD 基因在磷酸盐限制条件下于 R5 培养基中高效表达,而phoC 在相同条件下转录较差。phoA 的表达明显依赖于 PhoP,因为它在天蓝色链霉菌 DeltaphoP 突变体中不转录,并且在低磷酸盐浓度下被强烈激活。同样,phoD 的表达也依赖于 PhoP,并且对磷酸盐的可利用性高度敏感。相比之下,phoC 的表达不依赖于 PhoP。电泳迁移率变动分析表明,PhoP 与 phoA 和 phoD 的启动子结合,但不与phoC 的启动子结合。用 GST-PhoP 进行的足迹分析表明,phoA 启动子中存在一个 PHO 框(两个直接的 11 核苷酸重复序列),phoD 启动子中有两个 PHO 框。通过引物延伸确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 重合,表明该基因转录为无先导 mRNA。phoD 的推导 -10 和 -35 区域(但不是 phoA 的)与该启动子中的 PHO 框重叠,这表明过量的 PhoP 会干扰 RNA 聚合酶与该启动子的结合。总之,这三个启动子在 PhoP 对其表达的调控方面表现出明显差异。