Independent and cooperative regulation of staphylopine biosynthesis and trafficking by Fur and Zur

Abstract

Staphylococcus aureus expresses the Cnt system implicated in the active transport of trace metals by synthesizing (CntKLM) and exporting (CntE) staphylopine, a metallophore chelating metals and then taken up by an ABC-transporter (CntABCDF). This machinery is encoded in the cntKLMABCDFE operon, preceded by a non-coding region (PcntK) and containing an internal promoter region (PcntA). PcntK comprises a Fur box followed by a Zur box, a sRNA transcription start and a repeated region, while PcntA comprises a Fur box that overlaps a Zur box. We found that PcntK promoter activity is attenuated by the repeated sequence and strictly controlled by Fur or Zur binding to its respective target sequences. Interestingly, we discovered a cooperative regulation of the PcntA activity by both Fur and Zur binding to the Fur/Zur box, by identifying a tripartite complex with DNA. Repression of PcntA is less sensitive to metal concentration and therefore loosely repressed as compared to PcntK activity. Furthermore, the Cnt system is essential for the optimal import of zinc, thereby linking regulation and function of Cnt. Overall, our results highlight the need for fine and differential tuning of staphylopine biosynthesis and trafficking in order to efficiently respond to metal starvation and optimize metal recovery.

Intracellular accumulation of staphylopine impairs the fitness of Staphylococcus aureus cntE mutant

“…The fitness defect of DcntE mutant associated with intracellular StP accumulation occurred in both metal-depleted laboratory media as well as in sites of infection. Our work and that of others showed that Fe or Zn availability repressed the expression of cntKLM [17][18][19]. Besides StP, S. aureus produces other siderophores including SA and SB [11].…”

“…Disruption of the StP biosynthesis gene cntL eliminated intracellular StP accumulation and alleviated the growth defect of DcntE mutant. Under metal-rich conditions, activated Fur or Zur binds to the fur/zur box at the promoter regions of cnt operon and mediates repression of transcription of StP synthesis genes cntKLM and thus prevents the overproduction of StP [19]. SA accumulates in the cytoplasm of S. aureus in the absence of SA exporter SfaA, but unlike StP, the accumulation of SA does not cause a fitness defect or other apparent adverse effects in S. aureus [11].…”

“…The expression of the cnt operon in S. aureus is upregulated under metal starvation conditions and in infection sites that have low levels of available free iron, zinc, and manganese [16][17][18][19][20]. Thus, it was interesting to evaluate if the expression of StP synthesis genes in the DcntE mutant was altered under metaldepleted conditions compared with the WT.…”

“…The ninegene cntKLMABCDFE operon is required for StP synthesis and trafficking. The cnt operon expression is upregulated in metal-depleted conditions [17][18][19][20] and repressed by Fur and Zur in the presence of iron and zinc, respectively [19]. The cnt operon expression is upregulated in metal-depleted conditions [17][18][19][20] and repressed by Fur and Zur in the presence of iron and zinc, respectively [19].…”

“…The first three genes cntKLM are responsible for StP biosynthesis; cntA-F encode the importer of the StP-metal complex; and CntE, an MFS efflux pump is involved in the export of StP [16]. The cnt operon expression is upregulated in metal-depleted conditions [17][18][19][20] and repressed by Fur and Zur in the presence of iron and zinc, respectively [19]. The Cnt-StP system plays an important role in the virulence of S. aureus by facilitating the capture of essential metals, such as zinc and iron, from the host.…”