RSS mediated persulfidation cascade regulation mechanism of novel transcription factor SscRAc in extremophile Acidithiobacillus caldus under extreme copper stress

Biomining microorganisms are classified as extremophiles due to their chronic exposure to elevated metal ion concentrations, which drives evolutionary adaptations for environmental stress resistance. This study investigates the copper tolerance mechanisms in Acidithiobacillus caldus CCTCC M 2018727, a strain demonstrating exceptional metal resistance through long-term adaptive laboratory evolution (survival at 250 mM Cu²?). Comparative analysis revealed a 350.6% increase in intracellular reactive sulfur species (RSS) levels under 250 mM Cu²? stress. Sulfur-related post-translational modification proteomics demonstrated system-wide persulfidation enhancement and identified SscRAc, a novel RSS-sensitive MarR family transcription factor. Genetic deletion of sscRAc increased copper susceptibility and disrupted cellular redox homeostasis. Chromatin immunoprecipitation and qRT-PCR analyses revealed SscRAc's dual regulatory mechanism: suppressing copper efflux systems while activating RSS metabolic pathways to maintain intracellular equilibrium. 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. Furthermore, upstream signaling analysis uncovered a reciprocal regulatory relationship between SscRAc and the copper-sensitive transcription factor CsoRAc, establishing a coordinated copper-RSS signaling network. This work identifies SscRAc as a pivotal regulator of copper tolerance in A. caldus and delineates a novel metal stress response pathway connecting copper toxicity with sulfur redox biochemistry. Overall design: ChIP-seq profiling of SscRAc transcription factor binding sites in Acidithiobacillus caldus strain CCTCC M 2018727. Constructed recombinant plasmid p-sscRAc-Flag expressing 3×Flag-tagged SscRAc, which was transformed into donor strain E. coli SM10. The A. caldus p-sscRAc-Flag strain was subsequently generated via conjugation transfer.