Grkovic S, Brown M H, Roberts N J, Paulsen I T, Skurray R A
School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
J Biol Chem. 1998 Jul 17;273(29):18665-73. doi: 10.1074/jbc.273.29.18665.
The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of the qacA and divergently transcribed qacR mRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of the qacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump.
金黄色葡萄球菌QacA蛋白是一种多药转运蛋白，它通过质子动力依赖的化合物外排赋予对多种抗菌剂的抗性。进行引物延伸分析以定位qacA和反向转录的qacR mRNA的转录起始点。每个基因利用单个启动子元件，其位置通过定点诱变得到证实。将qacA和qacR启动子与氯霉素乙酰转移酶报告基因融合，以证明QacR是qacA转录的反式作用阻遏物，它不会自动调节自身表达。与qacA转录起始位点重叠的反向重复序列被证明是控制qacA基因表达的操纵序列。去除操纵序列的一半可防止QacR介导的qacA启动子抑制。在DNA酶I足迹实验中，纯化的QacR蛋白特异性结合该操纵序列。重要的是，通过凝胶迁移率变动分析表明，添加多种QacA底物可在体内诱导qacA表达，并在体外抑制QacR与操纵DNA的结合。因此，QacR似乎直接与作为QacA多药外排泵底物的结构不同的诱导化合物相互作用。
