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.

Uptake Mechanisms of a Novel, Activated Carbon-Based Equilibrium Passive Sampler for Estimating Porewater Methylmercury.

We describe the validation of a novel polymeric equilibrium passive sampler comprised of agarose gel with embedded activated carbon particles (ag+AC), to estimate aqueous monomethylmercury (MeHg) concentrations. Sampler behavior was tested using a combination of idealized media and realistic sediment microcosms. Isotherm bottle experiments with ag+AC polymers were conducted to constrain partitioning to these materials by various environmentally relevant species of MeHg bound to dissolved organic matter (MeHgDOM) across a range of sizes and character. Log of partitioning coefficients for passive samplers (Kps ) ranged from 1.98 ± 0.09 for MeHg bound to Suwannee River humic acid to 3.15 ± 0.05 for MeHg complexed with Upper Mississippi River natural organic matter. Reversible equilibrium exchange of environmentally relevant MeHg species was demonstrated through a series of dual isotope-labeled exchange experiments. Isotopically labeled MeHgDOM species approached equilibrium in the samplers over 14 days, while mass balance was maintained, providing strong evidence that the ag+AC polymer material is capable of equilibrium measurements of environmentally relevant MeHg species within a reasonable deployment time frame. Samplers deployed across the sediment-water interface of sediment microcosms estimated both overlying water and porewater MeHg concentrations within a factor of 2 to 4 of measured values, based on the average measured Kps values for species of MeHg bound to natural organic matter in the isotherm experiments. Taken together, our results indicate that ag+AC polymers, used as equilibrium samplers, can provide accurate MeHg estimations across many site chemistries, with a simple back-calculation based on a standardized Kps. Environ Toxicol Chem 2022;41:2052-2064. © 2022 SETAC.

Experimental evidence for recovery of mercury-contaminated fish populations.

Anthropogenic releases of mercury (Hg)1-3 are a human health issue4 because the potent toxicant methylmercury (MeHg), formed primarily by microbial methylation of inorganic Hg in aquatic ecosystems, bioaccumulates to high concentrations in fish consumed by humans5,6. Predicting the efficacy of Hg pollution controls on fish MeHg concentrations is complex because many factors influence the production and bioaccumulation of MeHg7-9. Here we conducted a 15-year whole-ecosystem, single-factor experiment to determine the magnitude and timing of reductions in fish MeHg concentrations following reductions in Hg additions to a boreal lake and its watershed. During the seven-year addition phase, we applied enriched Hg isotopes to increase local Hg wet deposition rates fivefold. The Hg isotopes became increasingly incorporated into the food web as MeHg, predominantly from additions to the lake because most of those in the watershed remained there. Thereafter, isotopic additions were stopped, resulting in an approximately 100% reduction in Hg loading to the lake. The concentration of labelled MeHg quickly decreased by up to 91% in lower trophic level organisms, initiating rapid decreases of 38-76% of MeHg concentration in large-bodied fish populations in eight years. Although Hg loading from watersheds may not decline in step with lowering deposition rates, this experiment clearly demonstrates that any reduction in Hg loadings to lakes, whether from direct deposition or runoff, will have immediate benefits to fish consumers.

Complete Genome Sequence of Desulfobulbus oligotrophicus Prop6, an Anaerobic Deltabacterota Strain That Lacks Mercury Methylation Capability.

Desulfobulbus oligotrophicus Prop6 is a sulfate-reducing, propionate-oxidizing Deltabacterota (formerly Deltaproteobacteria) strain from sewage sludge. Desulfobulbus species are found in anoxic environments, in animal microbiota, and some produce the neurotoxin methylmercury. The 3.1-Mbp D. oligotrophicus genome sequence enables studies of diverse environmental adaptations and the evolutionary genomics of mercury methylation mechanisms.

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.

The assessment and remediation of mercury contaminated sites: A review of current approaches.

Remediation of mercury (Hg) contaminated sites has long relied on traditional approaches, such as removal and containment/capping. Here we review contemporary practices in the assessment and remediation of industrial-scale Hg contaminated sites and discuss recent advances. Significant improvements have been made in site assessment, including the use of XRF to rapidly identify the spatial extent of contamination, Hg stable isotope fractionation to identify sources and transformation processes, and solid-phase characterization (XAFS) to evaluate Hg forms. The understanding of Hg bioavailability for methylation has been improved by methods such as sequential chemical extractions and porewater measurements, including the use of diffuse gradient in thin-film (DGT) samplers. These approaches have shown varying success in identifying bioavailable Hg fractions and further study and field applications are needed. The downstream accumulation of methylmercury (MeHg) in biota is a concern at many contaminated sites. Identifying the variables limiting/controlling MeHg production-such as bioavailable inorganic Hg, organic carbon, and/or terminal electron acceptors (e.g. sulfate, iron) is critical. Mercury can be released from contaminated sites to the air and water, both of which are influenced by meteorological and hydrological conditions. Mercury mobilized from contaminated sites is predominantly bound to particles, highly correlated with total sediment solids (TSS), and elevated during stormflow. Remediation techniques to address Hg contamination can include the removal or containment of Hg contaminated materials, the application of amendments to reduce mobility and bioavailability, landscape/waterbody manipulations to reduce MeHg production, and food web manipulations through stocking or extirpation to reduce MeHg accumulated in desired species. These approaches often rely on knowledge of the Hg forms/speciation at the site, and utilize physical, chemical, thermal and biological methods to achieve remediation goals. Overall, the complexity of Hg cycling allows many different opportunities to reduce/mitigate impacts, which creates flexibility in determining suitable and logistically feasible remedies.

Development of a Novel Equilibrium Passive Sampling Device for Methylmercury in Sediment and Soil Porewaters.

We explored the concept of equilibrium passive sampling for methylmercury (MeHg) using the strategy developed for hydrophobic organic chemicals. Passive sampling should allow prediction of the concentration of the chemically labile fraction of MeHg in sediment porewaters based on equilibrium partitioning into the sampler, without modeling diffusion rates through the sampler material. Our goals were to identify sampler materials with the potential to mimic MeHg partitioning into animals and sediments and provide reversible sorption in a time frame appropriate for in situ samplers. Candidate materials tested included a range of polymers embedded with suitable sorbents for MeHg. The most promising were activated carbon (AC) embedded in agarose, thiol-self-assembled monolayers on mesoporous supports embedded in agarose, and cysteine-functionalized polyethylene terephthalate, which yielded log sampler-water partition coefficients of 2.8 to 5 for MeHgOH and MeHg complexed with dissolved organic matter (Suwannee River humic acid). Sampler equilibration time in sediments was approximately 1 to 2 wk. Investigation of the MeHg accumulation mechanism by AC embedded in agarose suggested that sampling was kinetically influenced by MeHg interactions with AC particles and not limited by diffusion through the gel for this material. Also, AC exhibited relatively rapid desorption of Hg and MeHg, indicating that this sorbent is capable of reversible, equilibrium measurements. In sediment:water microcosms, porewater concentrations made with isotherm-calibrated passive samplers agreed within a factor of 2 (unamended sediment) or 4 (AC-amended sediment) with directly measured concentrations. The present study demonstrates a potential new approach to passive sampling of MeHg. Environ Toxicol Chem 2020;39:323-334. © 2019 SETAC.

Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations.

Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03-14 mg kg-1 dry wt. soil) and MeHg (0.05-27 µg kg-1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB+ species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.

Impact of dissolved organic matter on mercury and methylmercury sorption to activated carbon in soils: implications for remediation.

Activated carbon (AC) amendments have shown promise in reducing inorganic mercury (Hg(ii) complexes, "Hg") and methylmercury (MeHg) risk in contaminated soils. However, the effectiveness of AC in Hg and MeHg immobilization has varied among studies, suggesting that site biogeochemistry might dictate efficacy. In this study, we examined the effect of dissolved organic matter (DOM) on MeHg and Hg sorption to AC. We evaluated the impact of Suwannee River Humic Acid (SRHA) on sorption to AC directly using an isotherm approach and in a soil/AC mixture using slurry microcosms. Aqueous sorption coefficients to AC (log KAC) for Hg-SRHA and MeHg-SRHA complexes were one to two orders of magnitude lower (Hg-SRHA = 4.53, MeHgSRHA = 4.35) than those for chloride complexes (HgCl2 = 6.55, MeHgCl = 4.90) and more closely resembled the log KAC of SRHA (3.64). In anoxic, sulfidic soil slurries, the KAC for sulfide species appeared stronger than for chloride or SRHA species for both Hg and MeHg. AC significantly reduced porewater concentrations of both ambient MeHg and a fresh Me199Hg spike, and the addition of up to 60 mg L-1 SRHA did not reduce sorption to AC. The AC also reduced ambient Hg and 201Hg porewater concentrations, but as SRHA concentration increased, the magnitude of solid phase sorption decreased. Speciation modeling revealed that SRHA may have impacted Hg distribution to the solid phase by reducing HgS precipitation. This study highlights the need for site-specific evaluation of AC efficacy and the value in developing biogeochemical models of AC performance for Hg control.

Pseudodesulfovibrio mercurii sp. nov., a mercury-methylating bacterium isolated from sediment.

The sulfate-reducing, mercury-methylating strain ND132T was isolated from the brackish anaerobic bottom sediments of Chesapeake Bay, USA. Capable of high levels of mercury (Hg) methylation, ND132T has been widely used as a model strain to study the process and to determine the genetic basis of Hg methylation. Originally called Desulfovibrio desulfuricans ND132T on the basis of an early partial 16S rRNA sequence, the strain has never been formally described. Phylogenetic and physiological traits place this strain within the genus Pseudodesulfovibrio, in the recently reclassified phylum Desulfobacterota (formerly Deltaproteobacteria). ND132T is most closely related to Pseudodesulfovibrio hydrargyri BerOc1T and Pseudodesulfovibrio indicus J2T. Analysis of average nucleotide identity (ANI) of whole-genome sequences showed roughly 88 % ANI between P. hydrargyri BerOc1T and ND132T, and 84 % similarity between ND132T and P. indicus J2T. These cut-off scores <95 %, along with a multi-gene phylogenetic analysis of members of the family Desulfovibrionacea, and differences in physiology indicate that all three strains represent separate species. The Gram-stain-negative cells are vibrio-shaped, motile and not sporulated. ND132T is a salt-tolerant mesophile with optimal growth in the laboratory at 32 °C, 2 % salinity, and pH 7.8. The DNA G+C content of the genomic DNA is 65.2 %. It is an incomplete oxidizer of short chain fatty acids, using lactate, pyruvate and fumarate with sulfate or sulfite as the terminal electron acceptors. ND132T can respire fumarate using pyruvate as an electron donor. The major fatty acids are iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0, iso-C17 : 1ω9c and anteiso-C17 : 0. We propose the classification of strain ND132T (DSM 110689, ATCC TSD-224) as the type strain Pseudodesulfovibrio mercurii sp. nov.

Persistent reductions in the bioavailability of PCBs at a tidally inundated Phragmites australis marsh amended with activated carbon.

In situ amendment of sediments with highly sorbent materials like activated carbon (AC) is an increasingly viable strategy to reduce the bioavailability of persistent, sediment-associated contaminants to benthic communities. Because in situ sediment remediation is an emerging strategy, much remains to be learned about the field conditions under which amendments can be effective, the resilience of amendment materials toward extreme weather conditions, and the optimal design of engineered applications. We report the results of a multiyear, pilot-scale field investigation designed to measure the persistence and efficacy of AC amendments to reduce the bioavailability of polychlorinated biphenyls (PCBs) in an intertidal Phragmites marsh. The amendments tested were granular AC (GAC), GAC with a layer of sand, and a pelletized fine AC. Key metrics presented include vertically resolved black carbon concentrations in sediment and PCB concentrations in sediment, porewater, and several invertebrate species. The results demonstrate that all 3 amendments withstood Hurricane Sandy and remained in place for the duration of the study, successfully reducing porewater PCB concentrations by 34 to 97%. Reductions in invertebrate bioaccumulation were observed in all amendment scenarios, with pelletized fine AC producing the most pronounced effect. The present findings support the use of engineered AC amendments in intertidal marshes and can be used to inform amendment design, delivery, and monitoring at other contaminated sediment sites. Environ Toxicol Chem 2018;37:2496-2505. © 2018 SETAC.

Robust Mercury Methylation across Diverse Methanogenic Archaea.

Methylmercury (MeHg) production was compared among nine cultured methanogenic archaea that contain hgcAB, a gene pair that codes for mercury (Hg) methylation. The methanogens tested produced MeHg at inherently different rates, even when normalized to growth rate and Hg availability. Eight of the nine tested were capable of MeHg production greater than that of spent- and uninoculated-medium controls during batch culture growth. Methanococcoides methylutens, an hgcAB+ strain with a fused gene pair, was unable to produce more MeHg than controls. Maximal conversion of Hg to MeHg through a full batch culture growth cycle for each species (except M. methylutens) ranged from 2 to >50% of the added Hg(II) or between 0.2 and 17 pmol of MeHg/mg of protein. Three of the species produced >10% MeHg. The ability to produce MeHg was confirmed in several hgcAB+ methanogens that had not previously been tested (Methanocella paludicola SANAE, Methanocorpusculum bavaricum, Methanofollis liminatans GKZPZ, and Methanosphaerula palustris E1-9c). Maximal methylation was observed at low sulfide concentrations (<100 µM) and in the presence of 0.5 to 5 mM cysteine. For M. hollandica, the addition of up to 5 mM cysteine enhanced MeHg production and cell growth in a concentration-dependent manner. As observed for bacterial Hg methylators, sulfide inhibited MeHg production. An initial evaluation of sulfide and thiol impacts on bioavailability showed methanogens responding to Hg complexation in the same way as do Deltaproteobacteria The mercury methylation rates of several methanogens rival those of the better-studied Hg-methylating sulfate- and iron-reducing DeltaproteobacteriaIMPORTANCEArchaea, specifically methanogenic organisms, play a role in mercury methylation in nature, but their global importance to MeHg production and the subsequent risk to ecosystems are not known. Methanogenesis has been linked to Hg methylation in several natural habitats where methylmercury production incurs risk to people and ecosystems, including rice paddies and permafrost. In this study, we confirm that most methanogens carrying the hgcAB gene pair are capable of Hg methylation. We found that methylation rates vary inherently among hgcAB+ methanogens but that several species are capable of MeHg production at rates that rival those of the better-know Hg-methylating sulfate- and iron-reducing bacteria. Methanogens may need to be considered equally with sulfate and iron reducers in evaluations of MeHg production in nature.

Challenges and opportunities for managing aquatic mercury pollution in altered landscapes.

The environmental cycling of mercury (Hg) can be affected by natural and anthropogenic perturbations. Of particular concern is how these disruptions increase mobilization of Hg from sites and alter the formation of monomethylmercury (MeHg), a bioaccumulative form of Hg for humans and wildlife. The scientific community has made significant advances in recent years in understanding the processes contributing to the risk of MeHg in the environment. The objective of this paper is to synthesize the scientific understanding of how Hg cycling in the aquatic environment is influenced by landscape perturbations at the local scale, perturbations that include watershed loadings, deforestation, reservoir and wetland creation, rice production, urbanization, mining and industrial point source pollution, and remediation. We focus on the major challenges associated with each type of alteration, as well as management opportunities that could lessen both MeHg levels in biota and exposure to humans. For example, our understanding of approximate response times to changes in Hg inputs from various sources or landscape alterations could lead to policies that prioritize the avoidance of certain activities in the most vulnerable systems and sequestration of Hg in deep soil and sediment pools. The remediation of Hg pollution from historical mining and other industries is shifting towards in situ technologies that could be less disruptive and less costly than conventional approaches. Contemporary artisanal gold mining has well-documented impacts with respect to Hg; however, significant social and political challenges remain in implementing effective policies to minimize Hg use. Much remains to be learned as we strive towards the meaningful application of our understanding for stakeholders, including communities living near Hg-polluted sites, environmental policy makers, and scientists and engineers tasked with developing watershed management solutions. Site-specific assessments of MeHg exposure risk will require new methods to predict the impacts of anthropogenic perturbations and an understanding of the complexity of Hg cycling at the local scale.

Carbon Amendments Alter Microbial Community Structure and Net Mercury Methylation Potential in Sediments.

Neurotoxic methylmercury (MeHg) is produced by anaerobic Bacteria and Archaea possessing the genes hgcAB, but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution of hgcAB+ microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence of hgcAB+ organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators (Deltaproteobacteria, Firmicutes, and methanogenic Archaea) was measured with clade-specific degenerate hgcA quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria Clade-specific qPCR identified hgcA+Deltaproteobacteria and Archaea in all sites but failed to detect hgcA+Firmicutes Cellobiose shifted the communities in all samples to ∼90% non-hgcAB-containing Firmicutes (mainly Bacillus spp. and Clostridium spp.). These results suggest that either expression of hgcAB is downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment.IMPORTANCE Methylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylating Firmicutes This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.

Sulfurization of Dissolved Organic Matter Increases Hg-Sulfide-Dissolved Organic Matter Bioavailability to a Hg-Methylating Bacterium.

Reactions of dissolved organic matter (DOM) with aqueous sulfide (termed sulfurization) in anoxic environments can substantially increase DOM's reduced sulfur functional group content. Sulfurization may affect DOM-trace metal interactions, including complexation and metal-containing particle precipitation, aggregation, and dissolution. Using a diverse suite of DOM samples, we found that susceptibility to additional sulfur incorporation via reaction with aqueous sulfide increased with increasing DOM aromatic-, carbonyl-, and carboxyl-C content. The role of DOM sulfurization in enhancing Hg bioavailability for microbial methylation was evaluated under conditions typical of Hg methylation environments (µM sulfide concentrations and low Hg-to-DOM molar ratios). Under the conditions of predicted metacinnabar supersaturation, microbial Hg methylation increased with increasing DOM sulfurization, likely reflecting either effective inhibition of metacinnabar growth and aggregation or the formation of Hg(II)-DOM thiol complexes with high bioavailability. Remarkably, Hg methylation efficiencies with the most sulfurized DOM samples were similar (>85% of total Hg methylated) to that observed in the presence of l-cysteine, a ligand facilitating rapid Hg(II) biouptake and methylation. This suggests that complexes of Hg(II) with DOM thiols have similar bioavailability to Hg(II) complexes with low-molecular-weight thiols. Overall, our results are a demonstration of the importance of DOM sulfurization to trace metal and metalloid (especially mercury) fate in the environment. DOM sulfurization likely represents another link between anthropogenic sulfate enrichment and MeHg production in the environment.

Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.

Global prevalence and distribution of genes and microorganisms involved in mercury methylation.

Mercury (Hg) methylation produces the neurotoxic, highly bioaccumulative methylmercury (MeHg). The highly conserved nature of the recently identified Hg methylation genes hgcAB provides a foundation for broadly evaluating spatial and niche-specific patterns of microbial Hg methylation potential in nature. We queried hgcAB diversity and distribution in >3500 publicly available microbial metagenomes, encompassing a broad range of environments and generating a new global view of Hg methylation potential. The hgcAB genes were found in nearly all anaerobic (but not aerobic) environments, including oxygenated layers of the open ocean. Critically, hgcAB was effectively absent in ~1500 human and mammalian microbiomes, suggesting a low risk of endogenous MeHg production. New potential methylation habitats were identified, including invertebrate digestive tracts, thawing permafrost soils, coastal "dead zones," soils, sediments, and extreme environments, suggesting multiple routes for MeHg entry into food webs. Several new taxonomic groups capable of methylating Hg emerged, including lineages having no cultured representatives. Phylogenetic analysis points to an evolutionary relationship between hgcA and genes encoding corrinoid iron-sulfur proteins functioning in the ancient Wood-Ljungdahl carbon fixation pathway, suggesting that methanogenic Archaea may have been the first to perform these biotransformations.

Activated carbon mitigates mercury and methylmercury bioavailability in contaminated sediments.

There are few available in situ remediation options for Hg contaminated sediments, short of capping. Here we present the first tests of activated carbon and other sorbents as potential in situ amendments for remediation of mercury and methylmercury (MeHg), using a study design that combined 2 L sediment/water microcosms with 14 day bioaccumulation assays. Our key end points were pore water concentrations, and bioaccumulation of total Hg and MeHg by a deposit-feeding oligochaete Lumbriculus variegatus. Four amendments were tested: an activated carbon (AC); CETCO Organoclay MRM (MRM); Thiol-SAMMS (TS), a thiol-functionalized mesoporous silica; and AMBERSEP GT74, an ion-exchange resin. Amendments were tested in four separate microcosm assays using Hg-contaminated sediments from two freshwater and two estuarine sites. AC and TS amendments, added at 2-7% of the dry weight of sediments significantly reduced both MeHg concentrations in pore waters, relative to unamended controls (by 45-95%) and bioaccumulation of MeHg by Lumbriculus (by between 30 and 90%). Both amendments had only small impacts on microcosm surface water, sediment and pore water chemistry, with the exception of significant reductions in pore water dissolved organic matter. The effectiveness of amendments in reducing bioaccumulation was well-correlated with their effectiveness in increasing sediment:water partitioning, especially of MeHg. Sediments with low native sediment:water MeHg partition coefficients were most effectively treated. Thus, in situ sediment sorbent amendments may be able to reduce the risk of biotic Hg and MeHg uptake in contaminated sediments, and subsequent contamination of food webs.

Mercury methylation by novel microorganisms from new environments.

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.

Draft genome sequences for three mercury-methylating, sulfate-reducing bacteria.

The genetic basis for bacterial mercury methylation has been described recently. For insights into the physiology of mercury-methylating bacteria, we present genome sequences for Desulfococcus multivorans strain DSM 2059, Desulfovibrio alkalitolerans strain DSM 16529, and Desulfovibrio species strain X2.