Evolutionary Advantages of Multiple Arsenic Binding Sites in an ArsR Transcriptional Repressor

Microbes evolved ArsR transcriptional repressors to regulate genes involved in arsenic detoxification, resistance, and biotransformations. ArsRs are homodimers, with cysteine residues from each subunit forming either intrasubunit or intersubunit arsenic binding sites. Here, we identified an ArsR from <i>Arcticibacter tournemirensis</i> R1 (AtArsR) with three vicinal cysteine pairs in each monomer. AtArsR has one high-affinity site for As(III)/MAs(III) and a second site with a high affinity for MAs(III) but lower affinity for As(III). In <i>A. tournemirensis</i> R1, analysis of gene expression showed that AtArsR and its regulated genes are induced to significantly higher levels by As(III) compared with an <i>ars</i> operon controlled by a more typical ArsR. AtArsR responds to both As(III)/MAs(III), but not As(V)/MAs(V). AtArsR has a binding affinity for As(III) that is higher than that of ArsR with a single arsenic binding site per subunit. Cells of <i>Escherichia coli</i> expressing AtArsR exhibited increased arsenic accumulation. Mutating two of the three cysteine pairs decreased the level of accumulation. Our results indicate that cysteines from each pair contribute to two binding sites for As(III) and MAs(III), suggesting that AtArsR provides an evolutionary advantage for competition in high arsenic environments. AtArsR is a promising candidate for engineering enhanced arsenic accumulation, which is a viable strategy for arsenic bioremediation.