Reactive sulfur species mediated persulfidation cascade regulation mechanism of novel transcription factor SscR<i>Ac</i> in extremophile <i>Acidithiobacillus caldus</i> under extreme copper stress

Severe environmental conditions enhance the resilience of biomining microorganisms to elevated metal ion concentrations. The mechanism of how biomining microorganisms resist metal ions is poorly understood. We identified a novel reactive sulfur species (RSS)-sensitive MarR family transcription factor (SscRAc) in Acidithiobacillus caldus by persulfidation proteomics and observed increase in RSS and protein persulfidation levels under 250 mM Cu2+ stress. The deletion of sscRAc gene via CRISPR-Cas9 and conjugative transfer technology enhanced copper sensitivity in A. caldus. ChIP-seq/qRT-PCR revealed that SscRAc regulates copper detoxification by blocking efflux pumps and stimulating RSS metabolism. LC-MS/MS analysis revealed that both Cys74 and Cys78 in SscRAc interact with RSS and undergo persulfidation, resulting in the dissociation of the protein from the promoter-DNA of target genes. Upstream signaling analysis indicated that copper-sensitive repressor CsoRAc, regulated by SscRAc, inversely regulates SscRAc, thereby jointly enabling copper-RSS signal transduction. In conclusion, we identify SscRAc as the first RSS-dependent transcriptional switch directly linking copper toxicity with the persulfidation signaling pathway in extremophiles.