Rsp Activates Expression of the Cnt System in Staphylococcus aureus
Abstract Background 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 and other exporters in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE exporter gene. Results 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 both low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. A decrease in the expression of cntK (1.5-fold) and cntA (1.6-fold) was seen for Δfur Δrsp compared to Δfur strain. Similarly decreased expression of cntK (1.3-fold), cntA (1.6-fold) and cntE (1.6-fold) genes was seen comparing Δzur Δrsp and Δzur mutants but statistically not significant, indicating that Rsp is dependent on Fur and Zur. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for both the cntK and cntA promoters. Conclusions 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.