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 [8]. Besides the Fur-dependent regulation, a Fur-independent regulation by iron was also described, but these regulators have not been identified [8], 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 ciprofloxacin selection in a previously reported phage-based ultra-high-density transposon library procedure [14] we identified Rsp, as a candidate regulator of the cntE exporter gene. The screen used a selection at varying concentrations of ciprofloxacin, a substrate of both the NorA and NorB efflux pumps, and identified differences in knockouts of genes of transcriptional regulators.
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 [9] 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, being statistically significant for all genes of the operon, indicating that Rsp acts as an activator not only of cntE but also of the entire cnt operon. This finding 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 as the control (Fig. 1).
Table 2 Relative expression of the cnt operon in a rsp mutant strain
Genes
|
Fold change relative to USA300 JE2 (WT strain)
|
Mean
|
SEM
|
StP biosynthesis
|
|
|
cntK
|
0.477
|
0.190
|
cntL
|
0.166
|
0.081
|
cntM
|
0.711
|
0.167
|
StP importer
|
|
|
cntA
|
0.712
|
0.175
|
cntB
|
0.439
|
0.117
|
cntC
|
0.571
|
0.129
|
cntD
|
0.504
|
0.145
|
cntF
|
0.439
|
0.051
|
StP exporter
|
|
|
cntE
|
0.377
|
0.052
|
SEM: error standard of the mean.
Rsp activation acts in iron-independent manner
To determine if the Rsp activation acts in iron-independent 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 rsp 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 can activate the cnt operon under varying levels of iron-dependent repression by Fur, thereby supporting a basal level of the cnt gene expression in the cells. In complementation assays in the iron-depleted media, the decrease in cnt gene expression observed in the rsp mutant strain carrying the empty plasmid pTZN10 (3.3-, 2- and 3.2-fold changes detected for cntK, cmtA and cntE, respectively)was complemented to wild type levels with the plasmid containing rsp (pRsp). (1.4-, 0.92- and 1.2-fold changes for cntK, cntA and cntE, respectively) (Fig. 2B1). The same complementation was observed in the iron-repleted media, when pRsp was introduced into the rsp mutant. Compared to the rsp mutant strain with the empty plasmid pTZN10, the rsp strain with pRsp exhibited changes of 3.47- vs 1.35-fold for cntK, 3.3-vs 0.96-fold for cntA and 2.7-vs 0.9-fold for cntE, further confirming the effect of the Rsp activation in varying iron conditions (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 [8]. In addition, zinc regulation of the cnt operon through the repressor Zur has been previously reported [8]. To determine the possible interaction of Rsp with Fur and Zur in controlling cnt expression , we created single (fur, zur) and double mutants (fur rsp, zur rsp) in a USA300 background and measured the expression of cnt genes. As expected, the expression of cntK, cntA and cntE genes was increased substantially in the fur mutant (23- ,14-, and 7-fold, respectively) as well as the zur mutant (15-, 17-, and 13-fold, respectively) (Fig. 3A). Although these increases were reduced in the rsp double mutants (by1.5- to 1.6-fold for fur rsp and by 1.3- to 1.6-fold for zur rsp) relative to single mutants there remained substantial increases in cnt gene expression relative to the parental strain the double mutants (7- to 15-fold for fur rsp and 7- to 12-fold for zur rsp).Thus, repression by Fur and Zur can occur in the presence and absence of Rsp activation, however, was sufficient to significantly partially counter Fur repression for cntK and cntE and to partially counter Zur repression for cntA and cntE (Fig. 3B).
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 [12] 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 previously 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) [8]. 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.