EpsR^Ac is a copper-sensing MarR family transcriptional repressor from Acidithiobacillus caldus

The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsR^Ac) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsR^Ac monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsR^Ac protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsR^Ac could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsR^Ac negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsR^Ac did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: • Identification of a MarR family EPS-associated transcriptional regulator, named EpsR^Ac. • Cu(I) can bind to the EpsR^Ac protein with low affinity. • EpsR^Ac negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.