Regulation of the cnt operon by the AraC-type regulator, Rsp
Fur is an iron-dependent transcriptional regulator that uses Fe (II) as a cofactor and negatively regulates transcription of iron transport genes by binding to the two Fur boxes present in the cnt operon . Besides the Fur-dependent regulation, a Fur-independent regulation by iron was also described, but these regulators have not been identified , and nothing is known about an iron-independent regulation of the cnt operon. In addition, no activators of the cnt system has been described.
In a screen for regulators of efflux pumps and/or exporters, using antibiotic selection in a previously reported phage-based ultra-high-density transposon library procedure  we identified Rsp, as a candidate regulator of the cntE exporter gene.
To evaluate further the role of Rsp in cnt expression, a rsp mutant (NE1304) and its parental strain (USA300 JE2) from the Nebraska Transposon Mutant Library  were used to measure the expression of all genes of the cnt operon by RT-qPCR. As shown in Table 2, the expression of all genes in cnt operon decreased by an average of two-fold in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator not only of cntE but also of the entire cnt operon. This was further supported by the results that restoration of the presence of Rsp via introducing plasmid encoding rsp gene (pRsp) reestablished the expression of cntK, cntA and cntE genes to the levels of wild type strain (USA300 JE2) with the empty plasmid (Fig. 1).
Rsp activation acts in iron-independent manner
To determine if the Rsp activation acts in iron-dependent manner, we measured the level of expression of cntK, cntA and cntE under varying metal conditions. As it is shown in Fig. 2A, the transcript levels of the three genes in the Drsp mutant strain decreased in the three conditions tested, rich media (TSB), metal-depleted (TSB + DIP) and metal-replete medium (TSB + DIP + Fe) relative to the wild type strain (USA300 JE2). A 2-fold change was detected in TSB for all the genes of the operon. A slightly smaller decrease (1.9-, 1.7- and 1.5-fold changes for cntK, cmtA and cntE, respectively) was observed under DIP conditions. Additionally, the decrease was amplified with addition of 50 µM FeSO4 to DIP media (4.1-, 5.3- and 6.3-fold changes for cntK, cntA and cntE respectively) (Fig. 2A), likely due to the maximum level of repression of Fur in an iron-rich media. These data suggest that Rsp activates the cnt operon independently of the iron-dependent repressor Fur, thereby supporting a basal level of the cnt gene expression in the cells. In our complementation assays in the iron-depleted media, we observed a larger decrease in the Drsp mutant strain carrying the empty plasmid pTZN10 than in the Drsp mutant strain alone, being 3.3-, 2- and 3.2-fold changes detected for cntK, cmtA and cntE, respectively. Those changes reached wild type levels when we introduced rsp gene in a plasmid (pRsp). Concretely 1.4-, 0.92- and 1.2-fold changes were observed for cntK, cmtA and cntE, respectively (Fig. 2B1). The same phenomenon was observed in the iron-repleted media, when pRsp plasmid was introduced into the Drsp mutant, the expression of the three genes comparing to the Drsp mutant strain with the empty plasmid pTZN10 changed from 3.47- to 1.35-fold for cntK, 3.3-to 0.96-fold for cntA and 2.7-to 0.9-fold for cntE, showing complementation of rsp and confirming the effect of the Rsp activation in an iron-independent manner (Fig. 2B2).
Relationship between the repressors Fur and Zur and the activator Rsp in the regulation of the cnt operon
As noted, Fur negatively regulates transcription of the cnt operon in an iron-dependent manner . In addition, zinc regulation of the cnt operon through the repressor Zur has been previously reported . To determine if the positive contribution of Rsp to cnt expression is dependent on the presence of the repressors Fur and Zur, we created single (Dfur, Dzur) and double mutants (Dfur Drsp, Dzur Drsp) in a US300 background strain via bacterial phage φ85 transduction as explained in Methods and measured the expression of cnt genes. The expression of cntK and cntA genes in the Dfur Drsp double mutant still decreased (1.5- and 1.6-fold respectively) relative to Dfur single mutant strain but was not statistically significant, indicating that Rsp does not act independently of the repressor Fur (Fig. 3). The same effect was not seen for cntE initially, despite the cntE gene being expressed from the cntA promoter. In the same manner, the expression of cntK, cntA, and cntE deceased 1.3-, 1.6-, and 1.6-fold, respectively (but not statistically significant) in the double mutant Dzur Drsp relative to single mutant Dzur, indicating that Rsp activation of cnt genes also does not act independently of Zur (Fig. 3).
Binding of Rsp to the promoter region of the cnt operon
To determine if Rsp acts directly to modulate expression of cnt genes, Rsp was expressed in a pQE-9 (His tag expression vector) previously constructed  using E. coli SG13009 containing the pREP4 plasmid as a host. After induction by IPTG and further purification on a nickel affinity column, an SDS-PAGE gel indicated a homogenous single protein band (data not shown). Two promoters have been already determined for the cnt operon, one within the upstream region of cntK (cntK promoter) and the second one within the intergenic region between cntM and cntA genes (named cntA promoter) . Incubation of Rsp with either the 494-bp cntK or the 381-bp cntA promoter fragments resulted in a DNA band shift (Fig. 4). These bands shifts were reduced in the presence of 200-fold excess unlabeled specific promoter region DNA and remained unchanged in the presence of a similar excess of salmon sperm DNA, indicating specific binding to the promoter DNA fragment.