Aiba H, Mizuno T
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Japan.
Mol Microbiol. 1994 Jul;13(1):25-34. doi: 10.1111/j.1365-2958.1994.tb00399.x.
In Synechococcus species PCC7942, the production of a subset of proteins is induced when it is grown in a phosphate-limited medium. We previously suggested that a pair of cyanobacterial two-component regulatory proteins, SphS (sensory-kinase) and SphR (response-regulator), may be involved in this particular response to phosphate limitation. Here it was found that a protein with an apparent molecular mass of 33 kDa became particularly abundant when the total cellular proteins from cells grown in a phosphate-limited medium were analysed by SDS-PAGE. A deletion mutant lacking both the sphS and the sphR genes failed to produce this 33 kDa protein in response to phosphate limitation. Thus it was reasonable to assume that this protein is a member of the group of proteins involved in the Synechococcus phosphate regulatory circuit (hence, it was named SphX). The SphX protein was purified to near homogeneity, and the corresponding structural gene was cloned. The determined nucleotide sequence revealed that the sphX gene encodes a novel protein with a calculated molecular mass of 36,374 Da, which was demonstrated to be located in the cytoplasmic membrane. Structural features of the sphX promoter were then clarified by determining its transcription start site, from which transcription was triggered in response to phosphate limitation. Furthermore, the putative response-regulator, SphR, was demonstrated to bind to the upstream region of the sphX promoter by means of in vitro DNase I footprinting. From these results, we conclude that the sphX gene is a member of the Synechococcus phosphate regulatory circuit, in which the two signal-transduction components, SphS and SphR, are crucially involved as transcriptional regulators. The SphX protein may play a role in phosphate assimilation in Synechococcus.
在聚球藻属PCC7942菌株中，当它在磷酸盐限制的培养基中生长时，会诱导产生一部分蛋白质。我们之前曾提出，一对蓝藻双组分调节蛋白，即SphS（传感激酶）和SphR（反应调节因子），可能参与了对磷酸盐限制的这种特定反应。在此发现，当通过SDS-PAGE分析在磷酸盐限制培养基中生长的细胞的总细胞蛋白时，一种表观分子量为33 kDa的蛋白质变得特别丰富。一个缺失sphS和sphR基因的缺失突变体在磷酸盐限制条件下无法产生这种33 kDa的蛋白质。因此，可以合理地假设这种蛋白质是参与聚球藻磷酸盐调节回路的蛋白质组的一员（因此，将其命名为SphX）。SphX蛋白被纯化至近乎均一，并克隆了相应的结构基因。所确定的核苷酸序列表明，sphX基因编码一种计算分子量为36374 Da的新型蛋白质，已证明该蛋白质位于细胞质膜中。然后通过确定其转录起始位点来阐明sphX启动子的结构特征，转录是从该位点响应磷酸盐限制而触发的。此外，通过体外DNase I足迹法证明，推定的反应调节因子SphR与sphX启动子的上游区域结合。从这些结果中，我们得出结论，sphX基因是聚球藻磷酸盐调节回路的成员，其中两个信号转导组分SphS和SphR作为转录调节因子至关重要地参与其中。SphX蛋白可能在聚球藻的磷酸盐同化中发挥作用。
