Microbial Mercury Methylation Research Articles

The origin of methyl group in methanogen-mediated mercury methylation: From the Wolfe cycle

Methylmercury (MeHg) is a bioaccumulating neurotoxin mainly produced by anaerobic microorganisms, with methanogen being one of the important methylators. A critical aspect for understanding the mechanism for microbial mercury (Hg) methylation is the origin of the methyl group. However, the origin of methyl group in methanogen-mediated Hg methylation remains unclear. This study aims to identify the source of methyl group for MeHg synthesis in methanogens. Our study revealed that Hg methylation in Methanospirillum hungatei JF-1 is closely related to methanogenesis process, according to the results of proteomic study and substrate limitation study. Next, we proved that nearly all methyl group in MeHg derives from the Wolfe cycle in this species, rather than the previously demonstrated acetyl-coenzyme A pathway, based on the results of 13C labeling study. We then proposed the Wolfe cycle-dependent Hg methylation mechanism in this species. Further genome analyses and 13C labeling experiments indicated that the involvement of the Wolfe cycle in Hg methylation is probably a universal feature among Hg-methylating methanogens. These findings reveal a unique Hg methylation mechanism in methanogens. Our study broadens the carbon substrates and controlling factors for MeHg synthesis in the environment, which can inform the prediction of MeHg production potential and remediation strategies for MeHg contamination.

Biogeochemical controls on methylmercury distribution in a subtropical wetland ecosystem

Wetlands are widely regarded as biogeochemical hotspots of mercury methylation but little is known regarding such roles of mangrove forests. Here, we examined the detailed depth profile of mercury, methylmercury, and organic matter in surface sediments within an estuarine pond at Mai Po Nature Reserve in Hong Kong, China. There is a progressive enrichment of organic matter in sites closer to mangrove forests, and methylmercury showed a significantly positive correlation with organic matter content (p < 0.001). Methylmercury in sediments is significantly (p < 0.05) higher in the summertime when the temperature is elevated but salinity is reduced. Further, sediments at or near the mangrove forest have lower carbon to nitrogen ratio, which may imply more labile organic matter in these organic-rich sediments that can promote microbial mercury methylation. In summary, mangrove forests can enhance net methylmercury production and increase the risk to the migratory birds overwintering in this internationally important wetland.

Crystalline Phase Regulates Microbial Methylation Potential of Mercury Bound to MoS2 Nanosheets: Implications for Safe Design of Mercury Removal Materials.

Transition-metal dichalcogenides (TMDs) have shown great promise as selective and high-capacity sorbents for Hg(II) removal from water. Yet, their design should consider safe disposal of spent materials, particularly the subsequent formation of methylmercury (MeHg), a highly potent and bioaccumulative neurotoxin. Here, we show that microbial methylation of mercury bound to MoS2 nanosheets (a representative TMD material) is significant under anoxic conditions commonly encountered in landfills. Notably, the methylation potential is highly dependent on the phase compositions of MoS2. MeHg production was higher for 1T MoS2, as mercury bound to this phase primarily exists as surface complexes that are available for ligand exchange. In comparison, mercury on 2H MoS2 occurs largely in the form of precipitates, particularly monovalent mercury minerals (e.g., Hg2MoO4 and Hg2SO4) that are minimally bioavailable. Thus, even though 1T MoS2 is more effective in Hg(II) removal from aqueous solution due to its higher adsorption affinity and reductive ability, it poses a higher risk of MeHg formation after landfill disposal. These findings highlight the critical role of nanoscale surfaces in enriching heavy metals and subsequently regulating their bioavailability and risks and shed light on the safe design of heavy metal sorbent materials through surface structural modulation.

Structural incorporation of iron influences biomethylation potential of mercury sulfide

Mercury and ferrous iron commonly co-exist in anaerobic environments, the hotspots of microbial mercury methylation that leads to formation of neurotoxic methylmercury. However, the influence of ferrous iron on mercury bioavailability and methylation remains unclear. Here, we discover that microbial methylmercury production is inhibited by iron addition in a non-monotonical manner, reaching the minimal level at the initial molar ratio of dissolved Fe/Hg as 1/3, when highly aggregated Hg–S–Fe nanoparticles occur and prevent the nanoparticle–bacteria interactions. The dominant mercury species formed upon Hg–S–Fe co-precipitation is iron-incorporated nano-metacinnabar with short-range structural disorder. The methylation potential of this mercury species is inversely correlated with the aggregation state of nanoparticles. The non-monotonical trends of the aggregation state and subsequent methylation of nano-metacinnabar are due to the opposite effects of ferrous iron on the nucleation and particle attachment that determine the formation of nanoparticle aggregates. Increased Fe/Hg ratio hinders particle nucleation and thus decreases the density of particle monomers for aggregation. However, Fe substitution of sub-surface Hg weakens the inner-sphere coordination of surface Hg with natural thiol ligands and consequently enhances nano-metacinnabar aggregation. This new mechanistic insight is important for assessing and minimizing the risks of mercury methylation, and informs how intertwined iron and sulfur cycling may influence metal bioavailability in the changing environment.

Transcriptional Control of hgcAB by an ArsR-Like Regulator in Pseudodesulfovibrio mercurii ND132.

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg methylator Pseudodesulfovibrio mercurii ND132, we evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and cotranscribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosylhomocysteine (SAH)-responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR proteins in P. mercurii. Using quantitative PCR (qPCR) and RNA sequencing (RNA-seq) transcriptome analyses, we confirmed this ArsR regulates hgcAB transcription and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations, with significant upregulation of other ArsR-regulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to As(V) (but not As(III)) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted rates of Hg methylation by ND132; however, responses varied with culture conditions. Differences in growth and metabolic activity did not account for arsenic impacts on methylation. While arsenic significantly increased hgcAB expression, hgcAB gene and transcript abundance was not a good predictor of Hg methylation rates. Taken together, these results support the idea that Hg and As cycling are linked in P. mercurii ND132. Our results may hold clues to the evolution of hgcAB and the controls on Hg methylation in nature. IMPORTANCE This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosylhomocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression, which is needed to better predict environmental methylmercury production.

On the Origin and Evolution of Microbial Mercury Methylation.

The origin of microbial mercury methylation has long been a mystery. Here, we employed genome-resolved phylogenetic analyses to decipher the evolution of the mercury-methylating gene, hgcAB, constrain the ancestral origin of the hgc operon, and explain the distribution of hgc in Bacteria and Archaea. We infer the extent to which vertical inheritance and horizontal gene transfer have influenced the evolution of mercury methylators and hypothesize that evolution of this trait bestowed the ability to produce an antimicrobial compound (MeHg+) on a potentially resource-limited early Earth. We speculate that, in response, the evolution of MeHg+-detoxifying alkylmercury lyase (encoded by merB) reduced a selective advantage for mercury methylators and resulted in widespread loss of hgc in Bacteria and Archaea.

Amendments of nitrogen and sulfur mitigate carbon-promoting effect on microbial mercury methylation in paddy soils

The imbalance of nutrient elements in paddy soil could affect biogeochemical processes; however, how the key elements input influence microbially-driven conversion of mercury (Hg) to neurotoxic methylmercury (MeHg) remains virtually unknown. Herein, we conducted a series of microcosm experiments to explore the effects of certain species of carbon (C), nitrogen (N) and sulfur (S) on microbial MeHg production in two typical paddy soils (yellow and black soil). Results showed that the addition of C alone into the soils increased MeHg production approximately 2–13 times in the yellow and black soils; while the combined addition of N and C mitigated the C- promoting effect significantly. Added S also had a buffering effect on C-facilitated MeHg production in the yellow soil despite the extent being lower than that of N addition, whereas this effect was not obvious for the black soil. MeHg production was positively correlated with the abundance of Deltaproteobactera-hgcA in both soils, and the changes in MeHg production were related to the shifts of Hg methylating community resulting from C, N, and S imbalance. We further found that the changes in the proportions of dominant Hg methylators such as Geobacter and some unclassified groups could contribute to the variations in MeHg production under different treatments. Moreover, the enhanced microbial syntrophy with adding N and S might contribute to the reduced C-promoting effect on MeHg production. This study has important implications for better understanding of microbes-driven Hg conversion in paddies and wetlands with nutrient elements input.

Extracellular polymeric substances enhance dissolution and microbial methylation of mercury sulfide minerals.

Due to the extremely low solubility, mercury sulfide minerals, as the major environmental mercury sinks, are generally considered to be inert mercury species with minimal bioavailability. Here, we demonstrate that extracellular polymeric substances (EPS), continuously secreted and released by anaerobic methylating bacteria, enhance the dissolution processes of cinnabar (α-HgS) minerals. The enhancing effects of EPS occur to a greater extent in the dissolution of nanoparticulate α-HgS compared to the bulk-scale counterpart. The released EPS-Hg(II) species are available for microbial methylation to produce bioaccumulative neurotoxin, methylmercury. This is probably due to the abundant aromatic proteins in EPS that strongly interact with surface Hg(II) via inner-sphere complexation as well as cation-π interaction. Our study discovers the potential environmental risks of "inert" mercury sulfide minerals in natural microbial habitats, particularly benthic biofilms with abundant microbial EPS, transformed to the severely toxic methylmercury. The mechanistic findings will facilitate an accurate understanding of the interactions between soft and transition metals and microorganism-derived organics, which may dictate the environmental fate and impact of these elements.

Ferrous sulfide nanoparticles control mercury speciation and bioavailability to methylating bacteria in contaminated groundwater: Impacts of mercury species

Mercury speciation in groundwater affects its removal effectiveness and methylation potential. Yet, most studies focus on the removal of inorganic dissolved Hg(II) and few studies explored the mercury methylation before and after the treatment. This study comprehensively explored the removal performance of three model mercury species, namely, dissolved inorganic divalent Hg (Hg(II), including free Hg2+ and Hg2+ complexes with Cl− and OH−), Hg2+ bound to dissolved organic matter (Hg-DOM), and HgS nanoparticles by FeS nanoparticles and further investigated the resultant impacts on the microbial methylation of Hg. Among three different stabilizers (starch, carboxymethyl cellulose (CMC), carboxymethyl starch (CMS)), CMC stabilized FeS nanoparticles (CMC-FeS) demonstrated best physical stability and highest mercury uptake. The CMC-FeS nanoparticles efficiently immobilized the three mercury species within 20 h. The sorption isotherm data of Hg(II) and Hg-DOM were well fitted by the dual-mode isotherm model and the maximum sorption capacities were 3358.28 and 2396.38 mg/g, respectively. Hg(II) and Hg-DOM were predominantly removed via ion exchange, chemical precipitation, and surface complexation whereas HgS was mainly immobilized through heteroaggregation. The simple treatment greatly reduced the bioavailable Hg species, thereby diminishing the net MeHg production by 70.2 %, 32.7 %, and 11.3 %, respectively. This study provides compelling evidence that FeS nanoparticles efficiently removed various mercury species in groundwater and remarkably inhibited the microbial methylation of mercury.

Inputs of Terrestrial Dissolved Organic Matter Enhance Bacterial Production and Methylmercury Formation in Oxic Coastal Water.

Methylmercury (MeHg) is a potent neurotoxin commonly found in aquatic environments and primarily formed by microbial methylation of inorganic divalent mercury (Hg(II)) under anoxic conditions. Recent evidence, however, points to the production of MeHg also in oxic pelagic waters, but the magnitude and the drivers for this process remain unclear. Here, we performed a controlled experiment testing the hypothesis that inputs of terrestrial dissolved organic matter (tDOM) to coastal waters enhance MeHg formation via increased bacterial activity. Natural brackish seawater from a coastal area of the Baltic Sea was exposed to environmentally relevant levels of Hg(II) and additions of tDOM according to climate change scenarios. MeHg formation was observed to be coupled to elevated bacterial production rates, which, in turn, was linked to input levels of tDOM. The increased MeHg formation was, however, not coupled to any specific change in bacterial taxonomic composition nor to an increased abundance of known Hg(II) methylation genes. Instead, we found that the abundance of genes for the overall bacterial carbon metabolism was higher under increased tDOM additions. The findings of this study may have important ecological implications in a changing global climate by pointing to the risk of increased exposure of MeHg to pelagic biota.

Increased water inputs fuel microbial mercury methylation in upland soils

Mercury (Hg) can be converted to neurotoxic methylmercury (MeHg) by certain microbes typically in anaerobic environments, threatening human health due to its bioaccumulation in food webs. However, it is unclear whether and how Hg can be methylated in legacy aerobic uplands with increasing water. Here, we conducted a series of incubation experiments to investigate the effects of increased water content on MeHg production in two typical upland soils (i.e., long-term and short-term use). Results showed that marked MeHg production occurred in water-saturated upland soils, which was strongly correlated with the proportions of significantly stimulated Hg methylating taxon (i.e., Geobacter). Elevated temperature further enhanced MeHg production by blooming proportions of typical Hg methylators (i.e., Clostridium, Acetonema, and Geobacter). Water saturation could also enhance microbial Hg methylation by facilitating microbial syntrophy between non-Hg methylators and Hg methylators. Taken together, the present work suggests that uplands could turn into a potential MeHg reservoir in response to water inputs resulting from rainfall or anthropogenic irrigation.

Microbial mercury methylation potential in a large-scale municipal solid waste landfill, China

Landfills harbor ideal conditions for microbial mercury methylation. However, the levels and distribution of mercury (Hg) and methylmercury (MeHg), potential microbial Hg methylation, and their linkage within landfills are largely unknown. In the present study, total mercury (THg), MeHg, the Hg methylation gene (hgcA) and mer operon were quantified in 30 waste samples from different depths (0–30 m) at 5 locations within a large-scale landfill in China. The average concentrations of THg and MeHg in the solid waste samples were 1422.91 ng/g and 3.15 ng/g, respectively. THg (up to 14405.29 ng/g) and MeHg (up to 10.42 ng/g) have high concentrations in the middle part (10–15 m) along the depth profiles. The concentration of THg was strongly positively (both p < 0.05) correlated with the MeHg concentration and the relative abundance of hgcA, indicating that the THg concentration can play an important role in microbial Hg methylation. The hgcA genes were detected in most samples and mer operon were detected in all samples. Combined hgcA qPCR and metagenomics data showed that Archaea Methanofollis may mainly account for Hg methylation within landfills. These findings provide fundamental knowledge on Hg cycles in landfills.

The relative importance of mercury methylation and demethylation in rice paddy soil varies depending on the presence of rice plants

Neurotoxic methylmercury (MeHg) accumulates in rice grain from paddy soil, where its concentration is controlled by microbial mercury methylation and demethylation. Both up- and down-regulation of methylation is known to occur in the presence of rice plants in comparison to non-vegetated paddy soils; the influence of rice plant presence/absence on demethylation is unknown. To assess the concurrent influence of rice plant presence/absence on methylation and demethylation, and to determine which process was more dominant in controlling soil MeHg concentrations, we maintained six rhizoboxes of paddy soil with and without rice plants. At the peak of plant growth, we simultaneously measured ambient MeHg, ambient inorganic mercury (IHg), and potential rate constants of methylation and demethylation (Kmeth and Kdemeth) in soil using stable isotope tracers and ID-GC-ICPMS. We also measured organic matter content, elemental S, and water-extractable sulfate. We found MeHg concentrations were differentially controlled by MeHg production and degradation processes, depending on whether plants were present. In non-vegetated boxes, MeHg concentration was controlled by Kmeth, as evidenced by a strong and positive correlation, while Kdemeth had no relation to MeHg concentration. These results indicate methylation was the dominant driver of MeHg concentration in non-vegetated soil. In vegetated boxes, Kdemeth strongly and negatively predicted MeHg concentration, indicating that demethylation was the dominant control in soil with plants. MeHg concentration, Kmeth, and % MeHg all had significantly less variance in vegetated than in non-vegetated soils due to a consistent elimination of greater values. This pattern suggests that reduced MeHg production capacity was a secondary control on MeHg concentrations in vegetated soils. We observed no difference in the magnitude or variance of Kdemeth between treatments, suggesting that demethylation was robust to soil chemical conditions influenced by the plant, perhaps because of a wider taxonomic diversity of demethylators. Our results suggest that methylation and demethylation processes could both be leveraged to alter MeHg concentrations in rice paddy soil.

Nutrient Inputs Stimulate Mercury Methylation by Syntrophs in a Subarctic Peatland

Climate change dramatically impacts Arctic and subarctic regions, inducing shifts in wetland nutrient regimes as a consequence of thawing permafrost. Altered hydrological regimes may drive changes in the dynamics of microbial mercury (Hg) methylation and bioavailability. Important knowledge gaps remain on the contribution of specific microbial groups to methylmercury (MeHg) production in wetlands of various trophic status. Here, we measured aqueous chemistry, potential methylation rates (kmeth), volatile fatty acid (VFA) dynamics in peat-soil incubations, and genetic potential for Hg methylation across a groundwater-driven nutrient gradient in an interior Alaskan fen. We tested the hypotheses that (1) nutrient inputs will result in increased methylation potentials, and (2) syntrophic interactions contribute to methylation in subarctic wetlands. We observed that concentrations of nutrients, total Hg, and MeHg, abundance of hgcA genes, and rates of methylation in peat incubations (kmeth) were highest near the groundwater input and declined downgradient. hgcA sequences near the input were closely related to those from sulfate-reducing bacteria (SRB), methanogens, and syntrophs. Hg methylation in peat incubations collected near the input source (FPF2) were impacted by the addition of sulfate and some metabolic inhibitors while those down-gradient (FPF5) were not. Sulfate amendment to FPF2 incubations had higher kmeth relative to unamended controls despite no effect on kmeth from addition of the sulfate reduction inhibitor molybdate. The addition of the methanogenic inhibitor BES (25 mM) led to the accumulation of VFAs, but unlike molybdate, it did not affect Hg methylation rates. Rather, the concurrent additions of BES and molybdate significantly decreased kmeth, suggesting a role for interactions between SRB and methanogens in Hg methylation. The reduction in kmeth with combined addition of BES and molybdate, and accumulation of VFA in peat incubations containing BES, and a high abundance of syntroph-related hgcA sequences in peat metagenomes provide evidence for MeHg production by microorganisms growing in syntrophy. Collectively the results suggest that wetland nutrient regimes influence the activity of Hg methylating microorganisms and, consequently, Hg methylation rates. Our results provide key information about microbial Hg methylation and methylating communities under nutrient conditions that are expected to become more common as permafrost soils thaw.

Algal Organic Matter Drives Methanogen-Mediated Methylmercury Production in Water from Eutrophic Shallow Lakes.

Algal blooms bring massive amounts of algal organic matter (AOM) into eutrophic lakes, which influences microbial methylmercury (MeHg) production. However, because of the complexity of AOM and its dynamic changes during algal decomposition, the relationship between AOM and microbial Hg methylators remains poorly understood, which hinders predicting MeHg production and its bioaccumulation in eutrophic shallow lakes. To address that, we explored the impacts of AOM on microbial Hg methylators and MeHg production by characterizing dissolved organic matter with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy and quantifying the microbial Hg methylation gene hgcA. We first reveal that the predominance of methanogens, facilitated by eutrophication-induced carbon input, could drive MeHg production in lake water. Specifically, bioavailable components of AOM (i.e., CHONs such as aromatic proteins and soluble microbial byproduct-like materials) increased the abundances (Archaea-hgcA gene: 438-2240% higher) and activities (net CH4 production: 16.0-44.4% higher) of Archaea (e.g., methanogens). These in turn led to enhanced dissolved MeHg levels (24.3-15,918% higher) for three major eutrophic shallow lakes in China. Nevertheless, our model results indicate that AOM-facilitated MeHg production could be offset by AOM-induced MeHg biodilution under eutrophication. Our study would help reduce uncertainties in predicting MeHg production, providing a basis for mitigating the MeHg risk in eutrophic lakes.

Effects of sulfur-rich biochar amendment on microbial methylation of mercury in rhizosphere paddy soil and methylmercury accumulation in rice

Biochar amendment has the potential to reduce methylmercury (MeHg) uptake by rice grains in soil-rice ecosystem. Considering that sulfur can strongly bind Hg and thus reduce its bioavailability, S-modified biochar has been used to immobilize Hg in soils. However, whether natural S-enriched biochar can further reduce Hg and MeHg phytoavailability remains unknown. Moreover, the rhizosphere is one of the most important microbial hotspots regulating the pollutant dynamics in terrestrial ecosystems. Therefore, it is of greater practical significance to examine the impact of biochar amendment on MeHg production and phytoavailability in the rhizosphere versus nonrhizosphere. Here, by conducting a pot experiment, we evaluated the efficacy of biochar derived from sulfur-enriched oilseed rape straw to reduce MeHg accumulation in rice. The results demonstrated that: (1) biochar-induced enhancement of chloride ion and sulfate levels in the overlying water and pore water facilitate microbial methylation of Hg and thus MeHg production in rhizosphere soil. (2) biochar amendment increased rhizosphere soil sulfur content and humic acid-like substances, strengthening MeHg binding to soil, and thus reducing grain MeHg levels by 47%–75%. Our results highlight the necessity to applying natural sulfur-rich biochar accompanied with exogenous sulfur to further reduce MeHg phytoavailability.

Microbial methylation potential of mercury sulfide particles dictated by surface structure

Environmental contamination by mercury in its organometallic form, methylmercury, remains a major global concern due to its neurotoxicity, environmental persistence and biomagnification through the food chain. Accurate prediction of mercury methylation cannot be achieved based on aqueous speciation alone, and there remains limited mechanistic understanding of microbial methylation of particulate-phase mercury. Here we assess the time-dependent changes in structural properties and methylation potential of nanoparticulate mercury using microscopic and spectroscopic analyses, microcosm bioassays and theoretical calculations. We show that the methylation potential of a mercury sulfide mineral ubiquitous in contaminated soils and sediments (nanoparticulate metacinnabar) is determined by its crystal structure. Methylmercury production increases when more of nano-metacinnabar’s exposed surfaces occur as the (111) facet, due to its large binding affinity to methylating bacteria, likely via the protein transporter responsible for mercury cellular uptake prior to methylation. During nanocrystal growth, the (111) facet diminishes, lessening methylation of nano-metacinnabar. However, natural ligands alleviate this process by preferentially adsorbing to the (111) facet, and consequently hinder natural attenuation of mercury methylation. We show that the methylation potential of nanoparticulate mercury is independent of surface area. Instead, the nano-scale surface structure of nanoparticulate mercury is crucial for understanding the environmental behaviour of mercury and other nutrient or toxic soft elements. The environmental behaviour of mercury and other toxic soft elements is in part dictated by the surface structure of nanoparticulates, according to a combination of microcosm bioassays and theoretical calculations.

Impacts of climate change on methylmercury formation and bioaccumulation in the 21st century ocean

Impacts of climate change on methylmercury formation and bioaccumulation in the 21st century ocean

Natural organic matter facilitates formation and microbial methylation of mercury selenide nanoparticles

Tiemannite nanoparticles available for microbial mercury methylation are formed during the co-precipitation of natural organic matter, divalent mercury and selenium.

An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature.

The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novel hgcAB sequences similar to Lentisphaerae, Bacteroidetes, Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. Gene abundance data also corroborate the important role of two “classic” groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcAB sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature.