Redox gradient shapes the abundance and diversity of mercury-methylating microorganisms along the water column of the Black Sea.

In the global context of seawater deoxygenation triggered by climate change and anthropogenic activities, changes in redox gradients impacting biogeochemical transformations of pollutants, such as mercury, become more likely. Being the largest anoxic basin worldwide, with high concentrations of the potent neurotoxic methylmercury (MeHg), the Black Sea is an ideal natural laboratory to provide new insights about the link between dissolved oxygen concentration and hgcAB gene-carrying (hgc+) microorganisms involved in the formation of MeHg. We combined geochemical and microbial approaches to assess the effect of vertical redox gradients on abundance, diversity, and metabolic potential of hgc+ microorganisms in the Black Sea water column. The abundance of hgcA genes [congruently estimated by quantitative PCR (qPCR) and metagenomics] correlated with MeHg concentration, both maximal in the upper part of the anoxic water. Besides the predominant Desulfobacterales, hgc+ microorganisms belonged to a unique assemblage of diverse-previously underappreciated-anaerobic fermenters from Anaerolineales, Phycisphaerae (characteristic of the anoxic and sulfidic zone), Kiritimatiellales, and Bacteroidales (characteristic of the suboxic zone). The metabolic versatility of Desulfobacterota differed from strict sulfate reduction in the anoxic water to reduction of various electron acceptors in the suboxic water. Linking microbial activity and contaminant concentration in environmental studies is rare due to the complexity of biological pathways. In this study, we disentangle the role of oxygen in shaping the distribution of Hg-methylating microorganisms consistently with MeHg concentration, and we highlight their taxonomic and metabolic niche partitioning across redox gradients, improving the prediction of the response of marine communities to the expansion of oxygen-deficient zones. IMPORTANCE Methylmercury (MeHg) is a neurotoxin detected at high concentrations in certain marine ecosystems, posing a threat to human health. MeHg production is mainly mediated by hgcAB gene-carrying (hgc+) microorganisms. Oxygen is one of the main factors controlling Hg methylation; however, its effect on the diversity and ecology of hgc+ microorganisms remains unknown. Under the current context of seawater deoxygenation, mercury cycling is expected to be disturbed. Here, we show the strong effect of oxygen gradients on the distribution of potential Hg methylators. In addition, we show for the first time the significant contribution of a unique assemblage of potential fermenters from Anaerolineales, Phycisphaerae, and Kiritimatiellales to Hg methylation, stratified in different redox niches along the Black Sea gradient. Our results considerably expand the known taxonomic diversity and ecological niches prone to the formation of MeHg and contribute to better apprehend the consequences of oxygen depletion in seawater.

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.

"Trojan Horse" Type Internalization Increases the Bioavailability of Mercury Sulfide Nanoparticles and Methylation after Intracellular Dissolution.

Mercury sulfide nanoparticles (HgSNP), as natural metal-containing nanoparticles, are the dominant Hg species in anoxic zones. Although the microbial Hg methylation of HgSNP has been previously reported, the importance of this process in Hg methylation has yet to be clarified due to the lack of knowledge on the internalization and transformation of HgSNP. Here, we investigated the internalization and transformation of HgSNP in microbial methylator Geobacter sulfurreducens PCA through total Hg analysis and different Hg species quantification in medium and cytoplasm. We found that the microbial uptake of HgSNP, via a passive diffusion pathway, was significantly higher than that of the Hg2+-dissolved organic matter (Hg2+-DOM) complex. Internalized HgSNP were dissolved to Hg2+ in cytoplasm with a maximal dissolution of 41%, suggesting a "Trojan horse" mechanism. The intracellular Hg2+ from HgSNP exposure at the initial stage (8 h) was higher than that in Hg2+-DOM group, which led to higher methylation of HgSNP. Furthermore, no differences in methylmercury (MeHg) production from HgSNP were observed between the hgcAB gene knockout (ΔhgcAB) and wild-type strains, suggesting that HgSNP methylation may occur through HgcAB-independent pathways. Considering the possibility of a broad range of hgcAB-lacking microbes serving as methylators for HgSNP and the ubiquity of HgSNP in anoxic environments, this study highlights the importance of HgSNP internalization and methylation in MeHg production and demonstrates the necessity of understanding the assimilation and transformation of nutrient and toxic metal nanoparticles in general.

Distribution and phylogeny of mercury methylation, demethylation, and reduction genes in the Seto Inland Sea of Japan.

Mercury (Hg) adversely affects human and environmental health. To evaluate the mercury (Hg) speciation (methylation, demethylation, and reduction) of microorganisms in coastal seawater, we analyzed the microbial functional gene sets involved in Hg methylation (hgcA and hgcB), demethylation (merB), and reduction (merA) using a metagenomic approach in the eastern and western parts (the Kii and Bungo channels, respectively) of the Seto Inland Sea (SIS) of Japan. We determined the concentration of dissolved total mercury (dTHg) and methylated mercury (dMeHg) in seawater. The metagenomic analysis detected hgcAB, merA, and merB in both channels, whereas the phylogenies of these genes differed between them. A correlation between Hg concentration (both dTHg and dMeHg) and the relative abundance of each gene was not observed. Our data suggests that microbial Hg methylation and demethylation could occur in the SIS and there could be a distinct microbial Hg speciation process between the Kii and Bungo channels.

Anaerobic mercury methylators inhabit sinking particles of oxic water columns.

Increased concentration of mercury, particularly methylmercury, in the environment is a worldwide concern because of its toxicity in severely exposed humans. Although the formation of methylmercury in oxic water columns has been previously suggested, there is no evidence of the presence of microorganisms able to perform this process, using the hgcAB gene pair (hgc+ microorganisms), in such environments. Here we show the prevalence of hgc+ microorganisms in sinking particles of the oxic water column of Lake Geneva (Switzerland and France) and its anoxic bottom sediments. Compared to anoxic sediments, sinking particles found in oxic waters exhibited relatively high proportion of hgc+genes taxonomically assigned to Firmicutes. In contrast hgc+members from Nitrospirae, Chloroflexota and PVC superphylum were prevalent in anoxic sediment while hgc+ Desulfobacterota were found in both environments. Altogether, the description of the diversity of putative mercury methylators in the oxic water column expand our understanding on MeHg formation in aquatic environments and at a global scale.

A consensus protocol for the recovery of mercury methylation genes from metagenomes.

Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

Long-term mercury contamination does not affect the microbial gene potential for C and N cycling in soils but enhances detoxification gene abundance.

Soil microorganisms are key transformers of mercury (Hg), a toxic and widespread pollutant. It remains uncertain, however, how long-term exposure to Hg affects crucial microbial functions, such as litter decomposition and nitrogen cycling. Here, we used a metagenomic approach to investigate the state of soil functions in an agricultural floodplain contaminated with Hg for more than 80 years. We sampled soils along a gradient of Hg contamination (high, moderate, low). Hg concentrations at the highly contaminated site (36 mg kg-1 dry soil on average) were approximately 10 times higher than at the moderately contaminated site (3 mg kg-1 dry soil) and more than 100 times higher than at the site with low contamination (0.25 mg kg-1 dry soil; corresponding to the natural background concentration in Switzerland). The analysis of the CAZy and NCyc databases showed that carbon and nitrogen cycling was not strongly affected with high Hg concentrations, although a significant change in the beta-diversity of the predicted genes was observed. The only functional classes from the CAZy database that were significantly positively overrepresented under higher Hg concentrations were genes involved in pectin degradation, and from the NCyc database dissimilatory nitrate reduction and N-fixation. When comparing between low and high Hg concentrations the genes of the EggNOG functional category of inorganic ion transport and metabolism, two genes encoding Hg transport proteins and one gene involved in heavy metal transport detoxification were among those that were highly significantly overrepresented. A look at genes specifically involved in detoxification of Hg species, such as the mer and hgc genes, showed a significant overrepresentation when Hg contamination was increased. Normalized counts of these genes revealed a dominant role for the phylum Proteobacteria. In particular, most counts for almost all mer genes were found in Betaproteobacteria. In contrast, hgc genes were most abundant in Desulfuromonadales. Overall, we conclude from this metagenomic analysis that long-term exposure to high Hg triggers shifts in the functional beta-diversity of the predicted microbial genes, but we do not see a dramatic change or breakdown in functional capabilities, but rather functional redundancy.

Expression Levels of hgcAB Genes and Mercury Availability Jointly Explain Methylmercury Formation in Stratified Brackish Waters.

Neurotoxic methylmercury (MeHg) is formed by microbial methylation of inorganic divalent Hg (HgII) and constitutes severe environmental and human health risks. The methylation is enabled by hgcA and hgcB genes, but it is not known if the associated molecular-level processes are rate-limiting or enable accurate prediction of MeHg formation in nature. In this study, we investigated the relationships between hgc genes and MeHg across redox-stratified water columns in the brackish Baltic Sea. We showed, for the first time, that hgc transcript abundance and the concentration of dissolved HgII-sulfide species were strong predictors of both the HgII methylation rate and MeHg concentration, implying their roles as principal joint drivers of MeHg formation in these systems. Additionally, we characterized the metabolic capacities of hgc+ microorganisms by reconstructing their genomes from metagenomes (i.e., hgc+ MAGs), which highlighted the versatility of putative HgII methylators in the water column of the Baltic Sea. In establishing relationships between hgc transcripts and the HgII methylation rate, we advance the fundamental understanding of mechanistic principles governing MeHg formation in nature and enable refined predictions of MeHg levels in coastal seas in response to the accelerating spread of oxygen-deficient zones.

Microbial mercury transformations: Molecules, functions and organisms.

Mercury (Hg) methylation, methylmercury (MeHg) demethylation, and inorganic redox transformations of Hg are microbe-mediating processes that determine the fate and cycling of Hg and MeHg in many environments, and by doing so influence the health of humans and wild life. The discovery of the Hg methylation genes, hgcAB, in the last decade together with advances in high throughput and genome sequencing methods, have resulted in an expanded appreciation of the diversity of Hg methylating microbes. This review aims to describe experimentally confirmed and recently discovered hgcAB gene-carrying Hg methylating microbes; phylogenetic and taxonomic analyses are presented. In addition, the current knowledge on transformation mechanisms, the organisms that carry them out, and the impact of environmental parameters on Hg methylation, MeHg demethylation, and inorganic Hg reduction and oxidation is summarized. This knowledge provides a foundation for future action toward mitigating the impact of environmental Hg pollution.

Nitrospina-like Bacteria Are Dominant Potential Mercury Methylators in Both the Oyashio and Kuroshio Regions of the Western North Pacific.

Highly neurotoxic methylmercury (MeHg) accumulates in marine organisms, thereby negatively affecting human and environmental health. Recent studies have revealed that oceanic prokaryotes harboring the hgcAB gene pair are involved in Hg methylation. Presently, little is known about the distribution and phylogeny of these genes in distinct oceanic regions of the western North Pacific. In this study, we used metagenomics to survey the distribution of hgcAB genes in the seawater columns of the subarctic Oyashio region and the subtropical Kuroshio region. The hgcAB genes were detected in the MeHg-rich offshore mesopelagic layers of both the Oyashio region, which is a highly productive area in the western North Pacific, and the Kuroshio region, which has low productivity. Comparative analysis revealed that hgcAB genes belonging to the Nitrospina-like lineage were dominant in the MeHg-rich mesopelagic layers of both regions. These results indicate that Nitrospina-like bacteria are the dominant Hg methylators in the mesopelagic layers throughout the western North Pacific. IMPORTANCE MeHg is highly neurotoxic and accumulates in marine organisms. Thus, understanding MeHg production in seawater is critical for environmental and human health. Recent studies have shown that microorganisms harboring mercury-methylating genes (hgcA and hgcB) are involved in MeHg production in several marine environments. Knowing the distribution and phylogeny of hgcAB genes in seawater columns can facilitate assessment of microbial MeHg production in the ocean. We report that hgcAB genes affiliated with the microaerophilic Nitrospina lineage were detected in the MeHg-rich mesopelagic layers of two hydrologically distinct oceanic regions of the western North Pacific. This finding facilitates understanding of the microbial Hg methylation and accumulation in seawater columns of the western North Pacific.

The role of plastic debris in the biogeochemical cycle of mercury in Lake Erie and San Francisco Bay.

The accumulation of plastic debris that concentrates hydrophobic compounds and microbial communities creates the potential for altered aquatic biogeochemical cycles. This study investigated the role of plastic debris in the biogeochemical cycling of mercury in surface waters of the San Francisco Bay, Sacramento River, Lake Erie, and in coastal seawater. Total mercury and monomethylmercury were measured on plastic debris from all study sites. Plastic-bound microbial communities from Lake Erie and San Francisco Bay contained several lineages of known mercury methylating microbes, however the hgcAB gene cluster was not detected using polymerase chain reaction. These plastic-bound microbial communities also contained species that possess the mer operon, and merA genes were detected using polymerase chain reaction. In coastal seawater incubations, rapid mercury methylation percentages were greater in the presence of microplastics and demethylation percentages decreased as monomethylmercury additions adsorbed to microplastics. These findings suggest that plastic pollution has the potential to alter the biogeochemical cycling of mercury in aquatic ecosystems.

Microbial Diversity and Mercury Methylation Activity in Periphytic Biofilms at a Run-of-River Hydroelectric Dam and Constructed Wetlands.

Periphytic biofilms have the potential to greatly influence the microbial production of the neurotoxicant monomethylmercury in freshwaters although few studies have simultaneously assessed periphyton mercury methylation and demethylation rates and the microbial communities associated with these transformations. We performed a field study on periphyton from a river affected by run-of-river power plants and artificial wetlands in a boreal landscape (Québec, Canada). In situ incubations were performed on three sites using environmental concentrations of isotopically enriched monomethylmercury (MM198Hg) and inorganic mercury (200Hg) for demethylation and methylation rate measurements. Periphytic microbial communities were investigated through 16S rRNA gene analyses and metagenomic screenings for the hgcA gene, involved in mercury methylation. Positive mercury methylation rates ([5.9 ± 3.4] × 10-3 day-1) were observed only in the wetlands, and demethylation rates averaged 1.78 ± 0.21 day-1 for the three studied sites. The 16S rRNA gene analyses revealed Proteobacteria as the most abundant phylum across all sites (36.3% ± 1.4%), from which families associated with mercury methylation were mostly found in the wetland site. Metagenome screening for HgcA identified 24 different hgcA sequences in the constructed wetland site only, associated with 8 known families, where the iron-reducing Geobacteraceae were the most abundant. This work brings new information on mercury methylation in periphyton from habitats of impacted rivers, associating it mostly with putative iron-reducing bacteria.IMPORTANCE Monomethylmercury (MMHg) is a biomagnifiable neurotoxin of global concern with risks to human health mostly associated with fish consumption. Hydroelectric reservoirs are known to be sources of MMHg many years after their impoundment. Little is known, however, on run-of-river dams flooding smaller terrestrial areas, although their numbers are expected to increase considerably worldwide in decades to come. Production of MMHg is associated mostly with anaerobic processes, but Hg methylation has been shown to occur in periphytic biofilms located in oxic zones of the water column. Therefore, in this study, we investigated in situ production of MMHg by periphytic communities in habitats impacted by the construction of a run-of-river dam by combining transformation rate measurements with genomic approaches targeting hgcAB genes, responsible for mercury methylation. These results provide extended knowledge on mercury methylators in river ecosystems impacted by run-of-river dams in temperate habitats.

Deltaproteobacteria and Spirochaetes-Like Bacteria Are Abundant Putative Mercury Methylators in Oxygen-Deficient Water and Marine Particles in the Baltic Sea.

Methylmercury (MeHg), a neurotoxic compound biomagnifying in aquatic food webs, can be a threat to human health via fish consumption. However, the composition and distribution of the microbial communities mediating the methylation of mercury (Hg) to MeHg in marine systems remain largely unknown. In order to fill this knowledge gap, we used the Baltic Sea Reference Metagenome (BARM) dataset to study the abundance and distribution of the genes involved in Hg methylation (the hgcAB gene cluster). We determined the relative abundance of the hgcAB genes and their taxonomic identity in 81 brackish metagenomes that cover spatial, seasonal and redox variability in the Baltic Sea water column. The hgcAB genes were predominantly detected in anoxic water, but some hgcAB genes were also detected in hypoxic and normoxic waters. Phylogenetic analysis identified putative Hg methylators within Deltaproteobacteria, in oxygen-deficient water layers, but also Spirochaetes-like and Kiritimatiellaeota-like bacteria. Higher relative quantities of hgcAB genes were found in metagenomes from marine particles compared to free-living communities in anoxic water, suggesting that such particles are hotspot habitats for Hg methylators in oxygen-depleted seawater. Altogether, our work unveils the diversity of the microorganisms with the potential to mediate MeHg production in the Baltic Sea and pinpoint the important ecological niches for these microorganisms within the marine water column.

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.

Nitrospina-Like Bacteria Are Potential Mercury Methylators in the Mesopelagic Zone in the East China Sea.

In natural environments, the production of neurotoxic and bioaccumulative methylmercury (MeHg) is mediated by microorganisms carrying the genes hgcA and hgcB. However, the contribution of these microorganisms to mercury (Hg) methylation or MeHg accumulation in the ocean is poorly understood. Here we determined the total Hg (THg) and MeHg concentrations in seawater samples and conducted a metagenomic survey of the hgcAB genes and functional modules involved in metabolic pathways in the East China Sea (ECS). In the metagenomic analyses, we used paired-end reads and assembled contigs for hgcAB enumeration and phylogenetic analyses in the seawater column. To evaluate the relative abundance of hgcAB in the metagenomic data, we estimated the abundance of recA (single-copy gene of bacteria) as well and then compared them. Moreover, the profiles of prokaryotic community composition were analyzed by 16S rRNA gene (V4 region) deep-sequencing. In the mesopelagic layers, the hgcA sequences were detected, and there was a positive correlation between hgcA abundance relative to the recA and MeHg concentrations. Thus, the quantification of the hgcA sequences could provide valuable information to evaluate the potential environments of microbial MeHg accumulation in the seawater column. A phylogenetic analysis using the assembled contigs revealed that all of the hgcA sequences in the mesopelagic layers were affiliated with Nitrospina-like sequences. The 16S rRNA gene analysis revealed that Nitrospinae were abundant in the mesopelagic layers. Although the lineages of Deltaproteobacteria, Firmicutes, and Spirochaetes were detected in the seawater column, their hgcAB sequences were not detected in our metagenomes, despite the fact that they are closely related to previously identified Hg methylators. The metabolic pathway analysis revealed that the modules related to sulfur and methane metabolism were prominent in the mesopelagic layers. However, no hgcA sequences affiliated with sulfate-reducing bacteria (SRB) or methanogens were detected in these layers, suggesting that these bacteria could not be strongly involved in the Hg accumulation in the seawater column. Our results indicate that Nitrospina-like bacteria with hgcAB genes could play a critical role in microbial Hg accumulation in the oxygenated mesopelagic layers of the ECS.

Genome-Resolved Metagenomics and Detailed Geochemical Speciation Analyses Yield New Insights into Microbial Mercury Cycling in Geothermal Springs.

Geothermal systems emit substantial amounts of aqueous, gaseous, and methylated mercury, but little is known about microbial influences on mercury speciation. Here, we report results from genome-resolved metagenomics and mercury speciation analysis of acidic warm springs in the Ngawha Geothermal Field (<55°C, pH <4.5), Northland Region, Aotearoa New Zealand. Our aim was to identify the microorganisms genetically equipped for mercury methylation, demethylation, or Hg(II) reduction to volatile Hg(0) in these springs. Dissolved total and methylated mercury concentrations in two adjacent springs with different mercury speciation ranked among the highest reported from natural sources (250 to 16,000 ng liter-1 and 0.5 to 13.9 ng liter-1, respectively). Total solid mercury concentrations in spring sediments ranged from 1,274 to 7,000 µg g-1 In the context of such ultrahigh mercury levels, the geothermal microbiome was unexpectedly diverse and dominated by acidophilic and mesophilic sulfur- and iron-cycling bacteria, mercury- and arsenic-resistant bacteria, and thermophilic and acidophilic archaea. By integrating microbiome structure and metagenomic potential with geochemical constraints, we constructed a conceptual model for biogeochemical mercury cycling in geothermal springs. The model includes abiotic and biotic controls on mercury speciation and illustrates how geothermal mercury cycling may couple to microbial community dynamics and sulfur and iron biogeochemistry.IMPORTANCE Little is currently known about biogeochemical mercury cycling in geothermal systems. The manuscript presents a new conceptual model, supported by genome-resolved metagenomic analysis and detailed geochemical measurements. The model illustrates environmental factors that influence mercury cycling in acidic springs, including transitions between solid (mineral) and aqueous phases of mercury, as well as the interconnections among mercury, sulfur, and iron cycles. This work provides a framework for studying natural geothermal mercury emissions globally. Specifically, our findings have implications for mercury speciation in wastewaters from geothermal power plants and the potential environmental impacts of microbially and abiotically formed mercury species, particularly where they are mobilized in spring waters that mix with surface or groundwaters. Furthermore, in the context of thermophilic origins for microbial mercury volatilization, this report yields new insights into how such processes may have evolved alongside microbial mercury methylation/demethylation and the environmental constraints imposed by the geochemistry and mineralogy of geothermal systems.

Mercury methylation-related microbes and genes in the sediments of the Pearl River Estuary and the South China Sea.

Methylmercury (MeHg) is a toxicant that mainly originates from in situ microbial methylation of inorganic mercury (Hg) in the environment and poses a severe health risk to the public. However, the characteristics of the Hg-methylating microbial community and its relationship with MeHg production in various environments remain to be understood. In the present study, Hg-methylating microbial communities and genes (hgcAB cluster) in the sediments of the Pearl River (PR), Pearl River Estuary (PRE) and South China Sea (SCS) were investigated at a large spatial scale using high-throughput sequencing-based approaches. The results showed that sulfur-reducing bacteria (SRB) and iron-reducing bacteria (IRB) were consistently the dominant microbial strains responsible for the methylation of inorganic Hg in all three regions investigated. The abundance and diversity of Hg-methylating communities and genes were both found to be higher in the PR sediments compared to that in the PRE and SCS sediments, and in good agreement with the spatial distribution of MeHg. Furthermore, a significant correlation was observed between the MeHg concentration and the abundance of both hgcA and hgcB genes in the sediments of the PR, PRE and SCS regions. Overall, the present study suggested that there was the presence of a close link between MeHg and Hg-methylating communities or genes in the ambient aquatic environment, which could be used to reflect the potential of in situ MeHg production.

Periphyton and Flocculent Materials Are Important Ecological Compartments Supporting Abundant and Diverse Mercury Methylator Assemblages in the Florida Everglades.

Mercury (Hg) methylation in the Florida Everglades is of great environmental concern because of its adverse effects on human and wildlife health through biomagnification in aquatic food webs. Periphyton and flocculant materials (floc) overlaying peat soil are important ecological compartments producing methylmercury (MeHg) in this ecosystem. These compartments retain higher concentrations of MeHg than did soil at study sites across nutrient and/or sulfate gradient(s). To better understand what controls Hg methylation in these compartments, the present study explored the structures and abundances of Hg methylators using genes hgcAB as biomarkers. The hgcA sequences indicated that these compartments hosted a high diversity of Hg methylators, including Deltaproteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia, with community compositions that differed between these habitats. The copy numbers of hgcAB quantified by quantitative PCR revealed that floc and soil supported higher numbers of Hg methylators than periphyton in the Everglades ecosystem. The abundance of Hg methylators was strongly positively correlated with concentrations of carbon and nutrients (e.g., phosphorus and nitrogen) according to redundancy analysis. Strong correlations were also observed among numbers of sulfate reducers, methanogens, and the dominant hgcAB-carrying groups, suggesting that hgcAB would spread primarily through the growth of those assemblages. The abundances of Hg methylators were weakly negatively correlated to MeHg concentrations, suggesting that the size of this population would not solely determine the final concentrations of MeHg in the ecological compartments studied. This study extends the knowledge regarding the distribution of diverse potential mercury methylators in different environmental compartments in a wetland of national concern.IMPORTANCE Methylmercury is a potent neurotoxin that impacts the health of humans and wildlife. Most mercury in wetlands such as the Florida Everglades enters as inorganic mercury via atmospheric deposition, some of which is transformed to the more toxic methylmercury through the activities of anaerobic microorganisms. We investigated the numbers and phylogenetic diversity of hgcAB, genes that are linked to mercury methylation, in the soil, floc, and periphyton in areas of the Everglades with different sulfate and nutrient concentrations. Soil harbored relatively high numbers of cells capable of methylating mercury; however, little detectable methylmercury was present in soil. The greatest concentrations of methylmercury were found in floc and periphyton. The dominant methylators in those compartments included methanogens and Syntrophobacteriales This work provides significant insight into the microbial processes that control methylation and form the basis for accumulation through the food chain in this important environment.

Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB.

Methylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pair hgcAB, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized in vivo, the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood. Here, we report the kinetics of Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen sensitive, irreversible, and follows Michaelis-Menten kinetics, with an apparent Km of 3.2 nM and Vmax of 19.7 fmol · min-1 · mg-1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Interestingly, increasing thiol/Hg(II) ratios did not impact Hg methylation rates, which suggests that HgcAB-mediated Hg methylation effectively competes with cellular thiols for Hg(II), consistent with the low apparent Km Supplementation of 5-methyltetrahydrofolate or pyruvate did not enhance MeHg production, while both ATP and a nonhydrolyzable ATP analog decreased Hg methylation rates in cell lysates under the experimental conditions. These studies provide insights into the biomolecular processes associated with Hg methylation in anaerobic bacteria.IMPORTANCE The concentration of Hg in the biosphere has increased dramatically over the last century as a result of industrial activities. The microbial conversion of inorganic Hg to MeHg is a global public health concern due to bioaccumulation and biomagnification of MeHg in food webs. Exposure to neurotoxic MeHg through the consumption of fish represents a significant risk to human health and can result in neuropathies and developmental disorders. Anaerobic microbial communities in sediments and periphyton biofilms have been identified as sources of MeHg in aquatic systems, but the associated biomolecular mechanisms are not fully understood. In the present study, we investigate the biochemical mechanisms and kinetics of MeHg formation by HgcAB in sulfate-reducing bacteria. These findings advance our understanding of microbial MeHg production and may help inform strategies to limit the formation of MeHg in the environment.

Quantitative Proteomic Analysis of Biological Processes and Responses of the Bacterium Desulfovibrio desulfuricans ND132 upon Deletion of Its Mercury Methylation Genes.

Recent studies of microbial mercury (Hg) methylation revealed a key gene pair, hgcAB, which is essential for methylmercury (MeHg) production in the environment. However, many aspects of the mechanism and biological processes underlying Hg methylation, as well as any additional physiological functions of the hgcAB genes, remain unknown. Here, quantitative proteomics are used to identify changes in potential functional processes related to hgcAB gene deletion in the Hg-methylating bacterium Desulfovibrio desulfuricans ND132. Global proteomics analyses indicate that the wild type and ΔhgcAB strains are similar with respect to the whole proteome and the identified number of proteins, but differ significantly in the abundance of specific proteins. The authors observe changes in the abundance of proteins related to the glycolysis pathway and one-carbon metabolism, suggesting that the hgcAB gene pair is linked to carbon metabolism. Unexpectedly, the authors find that the deletion of hgcAB significantly impacts a range of metal transport proteins, specifically membrane efflux pumps such as those associated with heavy metal copper (Cu) export, leading to decreased Cu uptake in the ΔhgcAB mutant. This observation indicates possible linkages between this set of proteins and metal homeostasis in the cell. However, hgcAB gene expression is not induced by Hg, as evidenced by similarly low abundance of HgcA and HgcB proteins in the absence or presence of Hg (500 nm). Taken together, these results suggest an apparent link between HgcAB, one-carbon metabolism, and metal homeostasis, thereby providing insights for further exploration of biochemical mechanisms and biological functions of microbial Hg methylation.