Eldakak Amr, Hulett F Marion
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, IL 60607, USA.
J Bacteriol. 2007 Jan;189(2):410-21. doi: 10.1128/JB.01205-06. Epub 2006 Nov 3.
The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the resA promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR(C303A), exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR(C303A) and DTT are different. PhoR and PhoR(C303A) were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoR approximately P, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoR approximately P or PhoR(C303A) approximately P and PhoP occurred rapidly; most label from PhoR approximately P was transferred to PhoP, but only 10% of the label from PhoR(C303A) approximately P was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoP approximately P or PhoR autophosphatase activity was observed between PhoR and PhoR(C303A) that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR(C303) in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoR approximately P to PhoP, which is stabilization that is required for efficient production of PhoP approximately P.
PhoPR双组分系统激活或抑制Pho调控子基因，以克服磷酸盐缺乏。Pho信号转导网络由三个双组分系统PhoPR、ResDE和Spo0A组成。从resA启动子表达ResDE需要激活的PhoP，而ResD对于80%的Pho诱导至关重要，这在这两个双组分系统之间建立了一个正反馈回路，以放大Pho系统接收到的信号。ResD在Pho反应中的作用是通过产生末端氧化酶。还原型醌类在体外抑制PhoR自身磷酸化，有人提出末端氧化酶的表达导致醌池的氧化，从而解除抑制。我们在此表明，体内二硫苏糖醇（DTT）产生的还原环境以PhoR依赖的方式抑制Pho诱导，这与我们之前的体外数据一致。含有PhoR变体PhoR(C303A)的菌株表现出Pho诱导降低，并且对DTT抑制仍敏感，这表明通过PhoR(C303A)和DTT进行Pho还原的机制不同。PhoR和PhoR(C303A)在细胞浓度、有限蛋白水解模式、自身磷酸化速率、PhoRP的稳定性以及DTT对自身磷酸化的抑制方面相似。PhoRP或PhoR(C303A)P与PhoP之间的磷酸转移迅速发生；来自PhoRP的大部分标记转移到了PhoP，但来自PhoR(C303A)P的标记只有10%与PhoP相关，而90%以无机磷酸盐的形式释放。在PhoR和PhoR(C303A)之间未观察到PhoPP或PhoR自身磷酸酶活性的差异，这可以解释无机磷酸盐的释放。我们的数据与PhoR(C303)在PhoR活性中的作用一致，即在从PhoRP向PhoP的磷酸转移过程中通过稳定磷蛋白中间体来实现，而这是高效产生PhoPP所必需的稳定作用。
