Biomolecular and isotopic evidence reveals key cellular controls on methylmercury fate during bacterial mercury methylation.

Microbial mercury (Hg) methylation drives the formation of methylmercury (MeHg) hotspots in natural environments, but the cellular pathways that determine MeHg fate and isotopic signatures remain largely unresolved. In this study, we cultured the model sulfate-reducing bacterium Pseudodesulfovibrio hydrargyri BerOc1 anaerobically under fumarate respiration for 30 h and quantified Hg speciation and fraction- and species-specific Hg isotopic compositions. The results showed that intracellular MeHg was rapidly exported and dominated the extracellular pool by 30 h (> 80%). Exported MeHg bound to specific bioligands across size fractions, likely associated with its export mechanisms. Mass-dependent Hg isotope fractionation revealed temporal shifts in extracellular MeHg isotopic signatures (δ202HgMeHg= -1.10‰ to -0.82‰, 4-30 h), suggesting demethylation of bioligand-bound MeHg upon export. This isotopic observation refines previously reported suppressed mass-dependent fractionation signatures of MeHg in bulk bacterial cultures. Our results demonstrate that bacterially produced MeHg is rapidly exported from cells, potentially bound to specific biomolecules, and subsequently undergoes extracellular demethylation, thereby shaping aqueous MeHg isotopic signatures. These findings provide insight into the key factors that govern MeHg fate and accumulation during bacterial Hg methylation in aquatic ecosystems.