Department of Pharmacy, College of Pharmacy, Laboratory of Microbiology, Pusan National University, Research Building 537, San 30, Jangjun-Dong, Geumjung-Gu, Busan 609-735, South Korea.
Res Microbiol. 2013 Jun;164(5):375-81. doi: 10.1016/j.resmic.2013.02.001. Epub 2013 Feb 9.
In Pseudomonas aeruginosa, a multi-host pathogen, quorum sensing (QS) plays an essential role in pathogenesis, wherein LasR, QscR and RhlR, the QS regulators, control the expression of many virulence factors. In this study, we constructed a signal-binding-domain (SBD)-deleted QscR (QscR 160-237) to make a signal-independently-active form of QscR. However, QscR 160-237 that has only a DNA binding domain (DBD) was not fully active. It was able to bind to the target site in a signal-independent manner, but was not able to activate transcription of the target promoter. Since QscR 160-237 could interfere with binding of wild-type QscR (QscR wt) to its QscR binding site, we investigated the competition between QscR 160-237 and QscR wt on the QscR binding site in vivo and in vitro. When QscR wt and QscR 160-237 were independently co-expressed by two different inducers, increasing expression of QscR 160-237 interfered with QscR wt activity. This was verified by a competitive gel shift assay in vitro using purified QscR wt and QscR 160-237. Our results show that the SBD deletion makes QscR a partially active form that has only DNA binding ability, but it can interfere with QscR wt by competitive binding.
在多宿主病原体铜绿假单胞菌中，群体感应（QS）在发病机制中起着至关重要的作用，其中 QS 调节剂 LasR、QscR 和 RhlR 控制着许多毒力因子的表达。在这项研究中，我们构建了一个信号结合域（SBD）缺失的 QscR（QscR 160-237），以使其成为信号独立激活的形式。然而，只有 DNA 结合域（DBD）的 QscR 160-237 并不完全活跃。它能够以信号独立的方式结合靶位点，但不能激活靶启动子的转录。由于 QscR 160-237 可以干扰野生型 QscR（QscR wt）与其 QscR 结合位点的结合，我们研究了 QscR 160-237 和 QscR wt 在体内和体外竞争 QscR 结合位点的情况。当 QscR wt 和 QscR 160-237 分别由两个不同的诱导物独立共表达时，增加 QscR 160-237 的表达会干扰 QscR wt 的活性。这在体外使用纯化的 QscR wt 和 QscR 160-237 进行竞争性凝胶迁移实验中得到了验证。我们的结果表明，SBD 缺失使 QscR 成为一种仅有 DNA 结合能力的部分活性形式，但它可以通过竞争性结合来干扰 QscR wt。
