The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

中文翻译：

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Rsp 激活金黄色葡萄球菌中 Cnt 系统的表达

Cnt 系统对于在金属限制条件下最佳导入必需金属至关重要，并有助于金黄色葡萄球菌的毒力。在基于噬菌体的超高密度转座子库中外排泵调节器的筛选中，我们将 Rsp 确定为 cntE 基因的候选调节器。与亲本菌株相比，通过 RT-qPCR 在 rsp 突变体中观察到 cnt 操纵子的所有基因的表达降低了两倍，表明 Rsp 充当 cnt 操纵子的激活剂。为了确定 Rsp 激活是否取决于铁，我们比较了不同金属条件下的突变体和亲本 cnt 表达。在 TSB 的 rsp 突变体中检测到 cnt 基因表达降低了 2 倍，并且在添加联吡啶后观察到的降低幅度略小（cntK、cmtA 和 cntE 分别降低了 1.9、1.7 和 1.5 倍）。添加 FeSO4 后降低幅度最大（cntK、cmtA 和 cntE 的变化分别为 4.1、5.3 和 6.3 倍）。这些发现表明 Rsp 在低铁和高铁条件下激活 cnt 操纵子。为了研究 Rsp 与 cnt 抑制因子 Fur 和 Zur 之间的关系，我们创建了单突变体和双突变体。与亲本菌株相比，fur 和 zur 单突变体的 cnt 基因表达显着增加，fur rsp 双突变体也是如此。zur rsp 双突变体的 cntK 表达也有显着增加，并且 cntA 和 cntE 表达的增加趋势略低于统计显着性。因此，Rsp 的存在并没有消除 Fur 和 Zur 抑制 cnt 基因表达的能力。然而，与单突变体相比，双突变体中 cnt 基因表达的增加显着较小，这表明在没有这些阻遏物的情况下仍然可以发生 Rsp 激活。为了确定 Rsp 是否直接调节 cnt 基因的表达，纯化的 Rsp 的孵育导致 cntK 和 cntA 启动子的 DNA 特异性条带偏移。除了通过 Fur 和 Zur 进行金属依赖性调节外，Rsp 激活还可用于维持葡萄碱的基础细胞水平，以在需要时清除游离金属。