A novel antimony metallochaperone AntC in Comamonas testosteroni JL40 and its application in antimony immobilization

The microbial metabolism of toxic antimony (Sb) and the bioremediation of Sb-contaminated environments have attracted significant attention recently. This study identified an Sb(III) metallochaperone AntC in the Sb(III) efflux operon antRCA of Comamonas testosteroni JL40. The deletion of AntC significantly increased the intracellular Sb content in strain JL40 and concomitantly diminished resistance to Sb(III). By contrast, the complementary expression of AntC in the knockout strain resulted in a substantial recovery of Sb(III) resistance. The site-directed mutagenesis assay demonstrated the three conserved cysteine (Cys) residues (Cys³⁰, Cys³⁴, and Cys³⁶) play an essential role in the binding of Sb(III) to AntC and its transfer. The function of the metallochaperone AntC was further investigated in an Sb(III) sensitive bacterium Escherichia coli AW3110 (Δars). The co-expression of AntC and AntA in AW3110 cells resulted in a four-fold increase in minimum inhibitory concentrations (MICs) toward Sb(III), while the intracellular Sb content decreased five-fold compared to cells expressing AntA alone. In addition, a genetically modified E. coli strain was engineered to co-express AntC and the Sb uptake protein GlpF, showing an eight-fold increase in Sb absorption and achieving a remarkable 90% removal of Sb from the solution. This engineered strain was also applied in a hydroponic experiment, displaying a significant 80% reduction in Sb uptake by rice seedlings. This finding provides new insights into the mechanisms of bacterial Sb detoxification and a potential bioremediation strategy for Sb pollution.