Microbial mercury (Hg) methylation transforms a toxic trace metal into the highly bioaccumulated neurotoxin methylmercury (MeHg). The lack of a genetic marker for microbial MeHg production has prevented a clear understanding of Hg-methylating organism distribution in nature. Recently, a specific gene cluster (hgcAB) was linked to Hg methylation in two bacteria.1 Here we test if the presence of hgcAB orthologues is a reliable predictor of Hg methylation capability in microorganisms, a necessary confirmation for the development of molecular probes for Hg-methylation in nature. Although hgcAB orthologues are rare among all available microbial genomes, organisms are much more phylogenetically and environmentally diverse than previously thought. By directly measuring MeHg production in several bacterial and archaeal strains encoding hgcAB, we confirmed that possessing hgcAB predicts Hg methylation capability. For the first time, we demonstrated Hg methylation in a number of species other than sulfate- (SRB) and iron- (FeRB) reducing bacteria, including methanogens, and syntrophic, acetogenic, and fermentative Firmicutes. Several of these species occupy novel environmental niches for Hg methylation, including methanogenic habitats such as rice paddies, the animal gut, and extremes of pH and salinity. Identification of these organisms as Hg methylators now links methylation to discrete gene markers in microbial communities.

Dissolved organic matter (DOM) is generally thought to lower metal bioavailability in aquatic systems due to the formation of metal-DOM complexes that reduce free metal ion concentrations. However, this model may not be pertinent for metal nanoparticles, which are now understood to be ubiquitous, sometimes dominant, metal species in the environment. The influence of DOM on Hg bioavailability to microorganisms was examined under conditions (0.5-5.0 nM Hg and 2-10 μM sulfide) that favor the formation of β-HgS(s) (metacinnabar) nanoparticles. We used the methylation of stable-isotope enriched (201)HgCl(2) by Desulfovibrio desulfuricans ND132 in short-term washed cell assays as a sensitive, environmentally significant proxy for Hg uptake. Suwannee River humic acid (SRHA) and Williams Lake hydrophobic acid (WLHPoA) substantially enhanced (2- to 38-fold) the bioavailability of Hg to ND132 over a wide range of Hg/DOM ratios (9.4 pmol/mg DOM to 9.4 nmol/mg DOM), including environmentally relevant ratios. Methylmercury (MeHg) production by ND132 increased linearly with either SRHA or WLHPoA concentration, but SRHA, a terrestrially derived DOM, was far more effective at enhancing Hg-methylation than WLHPoA, an aquatic DOM dominated by autochthonous sources. No DOM-dependent enhancement in Hg methylation was observed in Hg-DOM-sulfide solutions amended with sufficient l-cysteine to prevent β-HgS(s) formation. We hypothesize that small HgS particles, stabilized against aggregation by DOM, are bioavailable to Hg-methylating bacteria. Our laboratory experiments provide a mechanism for the positive correlations between DOC and MeHg production observed in many aquatic sediments and wetland soils.

Dissolved organic matter (DOM) is a key component of fate and transport models for most metals, including mercury (Hg). Utilizing a suite of diverse DOM isolates, we demonstrated that DOM character, in addition to concentration, influences inorganic Hg (Hg(II)i) bioavailability to Hg-methylating bacteria. Using a model Hg-methylating bacterium, Desulfovibrio desulfuricans ND132, we evaluated Hg-DOM-sulfide bioavailability in washed-cell assays at environmentally relevant Hg/DOM ratios (∼1-8 ng Hg/mg C) and sulfide concentrations (1-1000 μM). All tested DOM isolates significantly enhanced Hg methylation above DOM-free controls (from ∼2 to >20-fold for 20 mg C/L DOM solutions), but high molecular weight/highly aromatic DOM isolates and/or those with high sulfur content were particularly effective at enhancing Hg methylation. Because these experiments were conducted under conditions of predicted supersaturation with respect to metacinnabar (β-HgS(s)), we attribute the DOM-dependent enhancement of Hg(II)i bioavailability to steric and specific chemical (e.g., DOM thiols) inhibition of β-HgS(s) growth and aggregation by DOM. Experiments examining the role of DOM across a wide sulfide gradient revealed that DOM only enhances Hg methylation under fairly low sulfide conditions (≲30 μM), conditions that favor HgS nanoparticle/cluster formation relative to dissolved HgS species.

. We tested the hypothesis that differences in Hg(II) i sorption and/or uptake rates drive observed differences in methylation rates among Desulfovibrio species. Hg(II) i associated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ϳ3 h. All MeHg produced was rapidly exported. We also tested the idea that all Desulfovibrio species are capable of Hg(II) i methylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in the Desulfovibrio is not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylating Desulfovibrio species even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive crossspecies differences in Hg methylation rates. As part of this study, Microbial mercury methylation is the main driver of risk associated with Hg pollution. Methylmercury production is an anaerobic process that occurs in saturated soils and wetlands (26,44,45,53), decaying periphyton mats (1, 14, 31), aquatic bottom sediments (16,27,33,36), and anaerobic bottom waters (56). Early investigations, prior to the advent of modern methylmercury (MeHg) analyses, reported a wide variety of aerobic and anaerobic Gram-positive and Gram-negative bacteria (30,49,55,58) and fungi (55) to be capable of MeHg production. However, subsequent studies with pure cultures have conclusively demonstrated a role only for sulfate-reducing bacteria (SRB) (4,8,11,13,20,23,38,50) and iron-reducing bacteria (FeRB; principally Geobacter spp.) (21, 37), all belonging to the Deltaproteobacteria. Many field studies, using selective microbial stimulants (1, 10, 26, 44, 57), inhibitors (1, 16, 24, 26, 59), and biogeochemical correlates (6,39,40,45), have buttressed the paradigm of SRB and FeRB as the dominant Hg methylators in natural aquatic systems (16,24,59), though recent studies have hypothesized that methanogens may be significant in some systems (31).Only a subset of SRB and FeRB are capable of Hg methylation (11,23,37,50), but why this is the case remains unclear. Early work by Choi and Bartha (13) suggested that Hg methylation was a "metabolic mistake" of SRB utilizing the acetyl coenzyme A (acetyl-CoA) pathway for carbon metabolism. Subsequent studies, however, indicated that Hg methylation capability is not restricted to SRB possessing the acetyl-CoA pathway (20). At present, it is not possible to conclusively identify the methyltransferase or methyl donor in SRB (or other Deltaproteobacteria) responsible for in vivo Hg methylation. Hg methylation occurs intracellularly (23), and significant effort has therefore been devoted to elucidating the mechanism(s) of Hg uptake by Hg-methylating bacteria.Passive diffusion of neutral HgS species has been hypothesized to control Hg uptak...