Mercury-methylating bacteria are associated with copepods: A proof-of-principle survey in the Baltic Sea.
ABSTRACT: Methylmercury (MeHg) is a potent neurotoxin that biomagnifies in marine food webs. Inorganic mercury (Hg) methylation is conducted by heterotrophic bacteria inhabiting sediment or settling detritus, but endogenous methylation by the gut microbiome of animals in the lower food webs is another possible source. We examined the occurrence of the bacterial gene (hgcA), required for Hg methylation, in the guts of dominant zooplankters in the Northern Baltic Sea. A qPCR assay targeting the hgcA sequence in three main clades (Deltaproteobacteria, Firmicutes and Archaea) was used in the field-collected specimens of copepods (Acartia bifilosa, Eurytemora affinis, Pseudocalanus acuspes and Limnocalanus macrurus) and cladocerans (Bosmina coregoni maritima and Cercopagis pengoi). All copepods were found to carry hgcA genes in their gut microbiome, whereas no amplification was recorded in the cladocerans. In the copepods, hgcA genes belonging to only Deltaproteobacteria and Firmicutes were detected. These findings suggest a possibility that endogenous Hg methylation occurs in zooplankton and may contribute to seasonal, spatial and vertical MeHg variability in the water column and food webs. Additional molecular and metagenomics studies are needed to identify bacteria carrying hgcA genes and improve their quantification in microbiota.
Project description:Knowledge of the diversity of mercury (Hg)-methylating microbes in the environment is limited due to a lack of available molecular biomarkers. Here, we developed novel degenerate PCR primers for a key Hg-methylating gene (hgcA) and amplified successfully the targeted genes from 48 paddy soil samples along an Hg concentration gradient in the Wanshan Hg mining area of China. A significant positive correlation was observed between hgcA gene abundance and methylmercury (MeHg) concentrations, suggesting that microbes containing the genes contribute to Hg methylation in the sampled soils. Canonical correspondence analysis (CCA) showed that the hgcA gene diversity in microbial community structures from paddy soils was high and was influenced by the contents of total Hg, SO4(2-), NH4(+), and organic matter. Phylogenetic analysis showed that hgcA microbes in the sampled soils likely were related to Deltaproteobacteria, Firmicutes, Chloroflexi, Euryarchaeota, and two unclassified groups. This is a novel report of hgcA diversity in paddy habitats, and results here suggest a link between Hg-methylating microbes and MeHg contamination in situ, which would be useful for monitoring and mediating MeHg synthesis in soils.
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: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:The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.
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: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:The formation of the potent neurotoxic methylmercury (MeHg) is a microbially mediated process that has raised much concern because MeHg poses threats to wildlife and human health. Since boreal forest soils can be a source of MeHg in aquatic networks, it is crucial to understand the biogeochemical processes involved in the formation of this pollutant. High-throughput sequencing of 16S rRNA and the mercury methyltransferase, hgcA, combined with geochemical characterisation of soils, were used to determine the microbial populations contributing to MeHg formation in forest soils across Sweden. The hgcA sequences obtained were distributed among diverse clades, including Proteobacteria, Firmicutes, and Methanomicrobia, with Deltaproteobacteria, particularly Geobacteraceae, dominating the libraries across all soils examined. Our results also suggest that MeHg formation is also linked to the composition of non-mercury methylating bacterial communities, likely providing growth substrate (e.g. acetate) for the hgcA-carrying microorganisms responsible for the actual methylation process. While previous research focused on mercury methylating microbial communities of wetlands, this study provides some first insights into the diversity of mercury methylating microorganisms in boreal forest soils.
Project description: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 <i>hgcAB</i>, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized <i>in vivo</i>, 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 <i>Desulfovibrio desulfuricans</i> 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 <i>K<sub>m</sub></i> of 3.2?nM and <i>V</i> <sub>max</sub> of 19.7?fmol · min<sup>-1</sup> · mg<sup>-1</sup> 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 <i>K<sub>m</sub></i> 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.<b>IMPORTANCE</b> 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.
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.