Heterologous Expression of the <i>arsRhgcAB</i> Gene Cluster Confers Mercury Methylation and Alters CH ₃ Hg Demethylation in a Non-methylating Bacterium

Microbial inorganic mercury (iHg(II)) methylation, mediated by hgcAB genes, is a key process controlling the formation of neurotoxic methylmercury (CH 3 Hg) in the environment. In this study, the arsRhgcAB gene cluster from Pseudodesulfovibrio hydrargyri BerOc1, a well-known Hg-methylating bacterium, was heterologously expressed in the non-methylating sulfate reducer Oleidesulfovibrio alaskensis G20. The heterologous expression of the arsRhgcAB gene cluster conferred the ability to methylate mercury to O. alaskensis G20, supporting its sufficiency to induce CH 3 Hg production in a non-methylating sulfate-reducing host. Although CH 3 Hg production rates in the engineered O. alaskensis G20 strain were lower than those in P. hydrargyri BerOc1, both strains followed a saturation reaction trend. Additionally, the engineered O. alaskensis G20 strain exhibited lower demethylation rates than the wild-type one, with a saturable kinetic profile similar to that of P. hydrargyri BerOc1, indicating that a regulatory mechanism, likely mediated by ArsR, limits demethylation. The expression of arsRhgcAB not only enables iHg(II) methylation but also influences CH 3 Hg demethylation, unveiling regulated dynamics more complex than previously recognized. Understanding these pathways is essential to better predict cellular Hg pools, elucidating the fate of mercury in anoxic ecosystems and ultimately developing microbially based strategies to mitigate CH 3 Hg production.