Fournier B, Aras R, Hooper D C
Infectious Disease Division and Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114-2696, USA.
J Bacteriol. 2000 Feb;182(3):664-71. doi: 10.1128/JB.182.3.664-671.2000.
To dissect genetically the regulation of NorA, a multidrug transporter of Staphylococcus aureus, we analyzed the differential expression of the norA promoter using a transcriptional fusion with a beta-lactamase reporter gene. Expression studies with an arlS mutant revealed that the norA promoter is ArlS dependent. The arlR-arlS locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of norA by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein. From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of norA expression in the presence of the arlS mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the arlS mutation, and the arlS mutation altered the growth-phase regulation of NorA. These results indicate that expression of norA is modified by a two-component regulatory system.
为了从遗传学角度剖析金黄色葡萄球菌多药转运蛋白NorA的调控机制，我们使用β-内酰胺酶报告基因的转录融合分析了norA启动子的差异表达。对arlS突变体的表达研究表明，norA启动子依赖于ArlS。arlR-arlS基因座编码一个双组分调控系统。蛋白ArlR与应答调节因子有很强的相似性，而ArlS与蛋白组氨酸激酶有很强的相似性。我们还通过凝胶迁移率变动实验分析了norA的Shine-Dalgarno序列上游350 bp的区域。结果表明，启动子上游仅115 bp的区域对于18 kDa蛋白的多重结合是必需的。通过转录融合，我们定位了4个不同的6 bp推定框，它们似乎在18 kDa蛋白的结合以及arlS突变存在时norA表达的上调中发挥作用。此外，在arlS突变存在的情况下，18 kDa蛋白的凝胶迁移率发生了改变，并且arlS突变改变了NorA的生长阶段调控。这些结果表明，norA的表达受到一个双组分调控系统的修饰。
