Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St, Boston, MA, 02114-2696, USA.
BMC Microbiol. 2020 Oct 28;20(1):327. doi: 10.1186/s12866-020-02013-0.
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 FeSO (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.
Cnt 系统对于在金属限制条件下必需金属的最佳导入至关重要，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文库的外排泵调节剂筛选中，我们发现 Rsp 是 cntE 基因的候选调节剂。
与亲本菌株相比，RT-qPCR 观察到 cnt 操纵子的所有基因表达减少了两倍，表明 Rsp 作为 cnt 操纵子的激活剂起作用。为了确定 Rsp 激活是否依赖于铁，我们比较了不同金属条件下突变体和亲本 cnt 的表达。在 TSB 中，rsp 突变体中 cnt 基因的表达减少了两倍，并且在用二吡啶处理后，观察到 cntK、cmtA 和 cntE 的表达分别减少了 1.9、1.7 和 1.5 倍。最大的减少发生在用 FeSO 添加时（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 抑制剂 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 基因表达均显著增加，fur rsp 双突变体也是如此。zur rsp 双突变体 cntK 表达也显著增加，cntA 和 cntE 表达略有增加，但未达到统计学意义。因此，Fur 和 Zur 抑制 cnt 基因表达的能力不会被 Rsp 消除。然而，与单突变体相比，双突变体中 cnt 基因表达的增加幅度明显较小，表明即使没有这些抑制剂，Rsp 激活仍可能发生。为了确定 Rsp 是否直接调节 cnt 基因的表达，纯化的 Rsp 孵育导致 cntK 和 cntA 启动子的 DNA 特异性带移动。
Rsp 激活可能会在需要时维持基础细胞水平的葡萄菌素以清除游离金属，除了 Fur 和 Zur 依赖金属的调节外。
