Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli.
Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli.
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启动子交换技术的应用表明，PhoU 可调节大肠杆菌中 PstSCAB2 ABC 转运蛋白的活性。
DOI：
10.1128/aem.01046-08
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发表时间：
2009
影响因子：
4.4
通讯作者：
McCleary,WilliamR
中科院分区：
文献类型：
--
作者：
Rice,ChristopherD;Pollard,JacobE;Lewis,ZacheryT;McCleary,WilliamR
大肠杆菌中Pho调节子的表达受低水平环境磷酸盐（Pi）的诱导。在这些条件下，高亲和力PstSCAB 2蛋白（即，具有两个PstB蛋白）是主要的Pitransporter。pstSCAB-phoU操纵子的表达受PhoB/PhoR双组分调控系统的调控。PhoU是Pho调节子的负调节子;然而，PhoU实现这一点的机制目前尚不清楚。phoU的遗传学研究已被证明是困难的，因为phoU基因的缺失会导致严重的生长缺陷，并产生强选择的补偿突变，导致混淆数据。为了克服phoU缺失的不稳定性，我们采用了一种启动子交换技术，将phoBR双组分系统的表达置于Ptac启动子和lacoid调控模块的控制下。该技术可普遍应用于控制其他染色体基因在大肠杆菌中的表达。杆菌在这里，我们利用PphoB：：Ptac和PpstS：：Ptac菌株来表征各种Δ phoU突变导致的表型。我们的研究结果表明，PhoU控制的PstSCAB 2转运蛋白的活性，以及其在细胞内的丰度。此外，我们使用PphoB：：PtacΔ phoU菌株作为平台，开始表征质粒中的新phoU突变。
Expression of the Pho regulon inEscherichia coliis induced in response to low levels of environmental phosphate (Pi). Under these conditions, the high-affinity PstSCAB2protein (i.e., with two PstB proteins) is the primary Pitransporter. Expression from thepstSCAB-phoUoperon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies ofphoUhave proven to be difficult because deletion of thephoUgene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability ofphoUdeletions, we employed a promoter-swapping technique that places expression of thephoBRtwo-component system under control of thePtacpromoter and thelacOIDregulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes inE. coli. Here we utilizedPphoB::PtacandPpstS::Ptacstrains to characterize phenotypes resulting from various ΔphoUmutations. Our results indicate that PhoU controls the activity of the PstSCAB2transporter, as well as its abundance within the cell. In addition, we used thePphoB::PtacΔphoUstrain as a platform to begin characterizing newphoUmutations in plasmids.