An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature.
ABSTRACT: 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.
Project description:Neurotoxic methylmercury (MeHg) is produced by anaerobic <i>Bacteria</i> and <i>Archaea</i> possessing the genes <i>hgcAB</i>, 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 <i>hgcAB<sup>+</sup></i> 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 <i>hgcAB<sup>+</sup></i> 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 (<i>Deltaproteobacteria</i>, <i>Firmicutes</i>, and methanogenic <i>Archaea</i>) was measured with clade-specific degenerate <i>hgcA</i> quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of <i>Proteobacteria</i>, <i>Firmicutes</i>, <i>Bacteroidetes</i>, and <i>Actinobacteria</i> Clade-specific qPCR identified <i>hgcA<sup>+</sup></i><i>Deltaproteobacteria</i> and <i>Archaea</i> in all sites but failed to detect <i>hgcA</i><sup>+</sup><i>Firmicutes</i> Cellobiose shifted the communities in all samples to ?90% non-<i>hgcAB</i>-containing <i>Firmicutes</i> (mainly <i>Bacillus</i> spp. and <i>Clostridium</i> spp.). These results suggest that either expression of <i>hgcAB</i> 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.<b>IMPORTANCE</b> 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 <i>Firmicutes</i> This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.
Project description: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.
Project description: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.
Project description:Methylmercury (MeHg) is a bioaccumulative neurotoxin that is produced by certain anaerobic microorganisms, but the abundance and importance of different methylating populations in the environment is not well understood. We combined mercury geochemistry, hgcA gene cloning, rRNA methods, and metagenomics to compare microbial communities associated with MeHg production in two sulfate-impacted lakes on Minnesota's Mesabi Iron Range. The two lakes represent regional endmembers among sulfate-impacted sites in terms of their dissolved sulfide concentrations and MeHg production potential. rRNA amplicon sequencing indicates that sediments and anoxic bottom waters from both lakes contained diverse communities with multiple clades of sulfate reducing Deltaproteobacteria and Clostridia. In hgcA gene clone libraries, however, hgcA sequences were from taxa associated with methanogenesis and iron reduction in addition to sulfate reduction, and the most abundant clones were from unknown groups. We therefore applied metagenomics to identify the unknown populations in the lakes with the capability to methylate mercury, and reconstructed 27 genomic bins with hgcA. Some of the most abundant potential methylating populations were from phyla that are not typically associated with MeHg production, including a relative of the Aminicenantes (formerly candidate phylum OP8) and members of the Kiritimatiellaeota (PVC superphylum) and Spirochaetes that, together, were more than 50% of the potential methylators in some samples. These populations do not have genes for sulfate reduction, and likely degrade organic compounds by fermentation or other anaerobic processes. Our results indicate that previously unrecognized populations with hgcAB are abundant and may be important for MeHg production in some freshwater ecosystems.
Project description:Methylmercury is a potent bioaccumulating neurotoxin that is produced by specific microorganisms that methylate inorganic mercury. Methylmercury production in diverse anaerobic bacteria and archaea was recently linked to the hgcAB genes. However, the full phylogenetic and metabolic diversity of mercury-methylating microorganisms has not been fully unraveled due to the limited number of cultured experimentally verified methylators and the limitations of primer-based molecular methods. Here, we describe the phylogenetic diversity and metabolic flexibility of putative mercury-methylating microorganisms by hgcAB identification in publicly available isolate genomes and metagenome-assembled genomes (MAGs) as well as novel freshwater MAGs. We demonstrate that putative mercury methylators are much more phylogenetically diverse than previously known and that hgcAB distribution among genomes is most likely due to several independent horizontal gene transfer events. The microorganisms we identified possess diverse metabolic capabilities spanning carbon fixation, sulfate reduction, nitrogen fixation, and metal resistance pathways. We identified 111 putative mercury methylators in a set of previously published permafrost metatranscriptomes and demonstrated that different methylating taxa may contribute to hgcA expression at different depths. Overall, we provide a framework for illuminating the microbial basis of mercury methylation using genome-resolved metagenomics and metatranscriptomics to identify putative methylators based upon hgcAB presence and describe their putative functions in the environment.IMPORTANCE Accurately assessing the production of bioaccumulative neurotoxic methylmercury by characterizing the phylogenetic diversity, metabolic functions, and activity of methylators in the environment is crucial for understanding constraints on the mercury cycle. Much of our understanding of methylmercury production is based on cultured anaerobic microorganisms within the Deltaproteobacteria, Firmicutes, and Euryarchaeota. Advances in next-generation sequencing technologies have enabled large-scale cultivation-independent surveys of diverse and poorly characterized microorganisms from numerous ecosystems. We used genome-resolved metagenomics and metatranscriptomics to highlight the vast phylogenetic and metabolic diversity of putative mercury methylators and their depth-discrete activities in thawing permafrost. This work underscores the importance of using genome-resolved metagenomics to survey specific putative methylating populations of a given mercury-impacted ecosystem.
Project description: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.
Project description: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.
Project description:Microbial mercury (Hg) methylation in sediments can result in bioaccumulation of the neurotoxin methylmercury (MMHg) in aquatic food webs. Recently, the discovery of the gene hgcA, required for Hg methylation, revealed that the diversity of Hg methylators is much broader than previously thought. However, little is known about the identity of Hg-methylating microbial organisms and the environmental factors controlling their activity and distribution in lakes. Here, we combined high-throughput sequencing of 16S rRNA and hgcA genes with the chemical characterization of sediments impacted by a waste water treatment plant that releases significant amounts of organic matter and iron. Our results highlight that the ferruginous geochemical conditions prevailing at 1-2?cm depth are conducive to MMHg formation and that the Hg-methylating guild is composed of iron and sulfur-transforming bacteria, syntrophs, and methanogens. Deltaproteobacteria, notably Geobacteraceae, dominated the hgcA carrying communities, while sulfate reducers constituted only a minor component, despite being considered the main Hg methylators in many anoxic aquatic environments. Because iron is widely applied in waste water treatment, the importance of Geobacteraceae for Hg methylation and the complexity of Hg-methylating communities reported here are likely to occur worldwide in sediments impacted by waste water treatment plant discharges and in iron-rich sediments in general.
Project description:Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.
Project description:Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.