EXPLORATION OF THE PHYSIOLOGY OF DESULFOVIBRIO SP. ND132 Δ(HGCA HGCB) MUTANT BY WHOLE GENOME RANDOM TRANSPOSON MUTAGENESIS AND MASSIVELY PARALLEL SEQUENCING
Methylmercury (MeHg) is a neurotoxin generated by a subset of anaerobic bacteria. The overall process of methylation has been intensely investigated but until recently little has been understood about the molecular mechanism. Two genes, hgcA and hgcB, encoding for a corrinoid protein and 2[4Fe-4S] ferrodoxin respectively, have been shown to be essential for production of MeHg. Deletions of these genes in the model methylating organism Desulfovibrio desulfuricans ND132, both single gene deletions and in combination, show complete ablation of MeHg production. Currently, no data implicate HgcA and HgcB as part of a mercury detoxification system and, thus, a putative native function for these proteins remains in question. A newly described protocol, rapid transposon liquid enrichment sequencing (TnLE-Seq), allows for the rapid generation of mutant transposon libraries in the wild-type and mutant strains. DNA from each mutant pool will be prepared for next generation sequencing to determine the location of transposition and abundance of each mutant recovered. Identifying genes differing in fitness levels between the two strains may lead to physiological functions that are altered in the mutant. To date, no data have been provided on the overall response of methylating bacteria to mercury. Utilizing TnLE-Seq, we will also stress both wild-type and double deletion strains in order to observe the global response of the cell to toxic mercury levels.
CHANGING PATHWAYS OF MERCURY METHYLATION ALONG TROPHIC GRADIENTS IN NORTHERN WETLANDS
Methyl mercury (MeHg) can be produced by diverse microbes besides sulfate (SO42-) and iron-reducing bacteria, including syntrophs, methanogens, and fermenters. Many freshwater wetlands, especially those in high norther regions, are deficient in electron acceptors that support the traditional respiratory pathways of methylation. However, MeHg tends to accumulate to high levels in these wetlands. To investigate methylation pathways and to connect these with surface vegetation and microbial communities, experiments were conducted using peats from close to 50 sites in Alaska collected during the summers of 2014-2016. The sites were clustered using multiple factor analysis based on 1) pH and temperature; 2) CH4, CO2, and volatile fatty acids (VFA) production rates; 3) vascular plant composition, and; 4) non-vascular plant composition. pHs varied from <4 to >6. Mercury (Hg) methylation activity in laboratory incubations was determined using the short-lived radioisotope 197Hg(II) (½ life 2.67 days).
In the low pH Sphagnum dominated clusters, methylation rates were less than 0.1% day-1, while the high pH clusters dominated by Carex spp. and active methanogenesis, exhibited Hg methylation rates as high as 10% day-1. In intermediate sites, rich in Sphagnum with less Carex, a gradient in syntrophy and Hg methylation paths was observed. Molecular data revealed a gradient in methanogenic pathways, and an increased importance of fermentation as pH decreased. Incubation data also revealed a sequential loss in the use of specific VFA along the gradient from minerotrophy to ombrotrophy (pH decrease), signifying a gradient in syntrophy pathways. Hg methylation rates in conjunction with the use of microbial inhibitors and stimulators exhibited wide differences in effects along the trophic gradient, e.g., the methanogenic inhibitor BES lead to an inhibition of Hg methylation in minerotrophic sites, but stimulated methylation in intermediates sites, whereas inhibitors had no effect in highly ombrotrophic bogs.
Methanogens were the primary methylators in minerotrophic sites, whereas syntrophs were important methylators in intermediate sites. However, the type of syntrophy responsible for methylation likely varied along the trophic gradient. Primary fermenters were responsible for methylation in the most ombrotrophic sites. As high latitudes warm and decomposition rates and vegetation cover evolve, pathways of Hg methylation should change greatly with an increase in the role of respiratory processes like methanogenesis and probably SO42- and iron reduction, and a decrease in Hg methylation by primary and secondary fermenters (syntrophs).
BIOLOGICAL MERCURY METHYLATION IN A CHRONOSEQUENCE OF WETLANDS: WHAT METHYLATORS DOMINATE THE METHYLATION ACROSS THE AGE GRADIENT?
Earlier pure cultures studies showed that MeHg formation is mainly performed by microorganisms that metabolically rely on anaerobic respiration of sulfate and iron. Additionally, methylating SRB can still methylate Hg in the absence of sulfate via either fermentative growth or syntrophic interactions with methanogens. Yet, the role of syntrophy or fermentation in methylation has not been thoroughly examined in environmental samples. We made use of a unique natural geochemical gradient in a chronosequence of wetlands, where the young mires are more nutrient rich with circumneutral pH and feature an abundance of electron acceptors such as sulfate, while older mires hold much less nutrients, sulfate is scarce and pH is lower. Samples collected from three of these wetlands, representing a gradient in ages, were used for microcosm incubation experiments with specific metabolic resources, inhibitors and Hg isotope tracers. The experimental incubations were used to test the hypothesis that the pathway of Hg methylation varies as a function of wetland age and trophic status. The expectation would be that syntrophy dominates in old and intermediate wetlands, while respiratory pathway is more prominent in young, more nutrient-rich mires. The Hg methylation/demethylation rate constants (Km and Kd) were determined and by means of genomic analyses of incubated samples, microbial populations hosting and expressing the hgcA gene involved in the formation of MeHg could be determined. This was used to establish a relationship between the capacity for net Hg methylation and microbial community composition, with the aim to revealing the pathways of MeHg formation in the three wetlands. Our incubation results showed that microorganisms in young mires had the highest activity because of the highest CO2 and CH4 productions, then followed by intermediate and old ones. The addition of molybdate which is a specific inhibitor of SRB, and sulfate both inhibited CH4 productions but with a decrease and an increase in CO2 productions, respectively. The addition of volatile fatty acids (VFAs) such as lactate, propionate and butyrate, which are important substrates for syntrophic bacteria, enhanced the productions of CO2 and CH4. All of these findings may imply that syntrophic coupling of SRB and methanogens plays an important role on anaerobic respiration of organic matter in wetlands, subsequently impact the formation of MeHg.
Acknowledgements: This work was supported by the Wenner-Gren Foundations to Stefan Bertilsson and Haiyan Hu, the Sino-Swedish Mercury Management Research Framework (SMaReF: VR2013-6978) and National Natural Science Foundation of China (No.41573078 and 41303098).
MERCURY UPTAKE AND BIOAVAILABILITY IN A GRAM-POSITIVE FIRMICUTE, DESULFITOBACTERIUM METALLIREDUCENS
The transport of inorganic Hg into the cell is a key step in the production of methylmercury in anaerobic organisms containing HgcAB. The ability to methylate is distributed across diverse phyla; however, intracellular uptake has only been studied in the δ-Proteobacteria and γ-Proteobacteria. In this study an iron-reducing firmicute, Desulfitobacterium metallireducens, was examined for intracellular Hg(II) transport to determine if the bioavailability and uptake of Hg(II) in this bacterium is similar to that observed in the proteobacteria. Washed cell suspensions of D. metallireducens accumulated Hg(II) when exposed to either Hg-cysteine or HgCl2 complexes, but did not support uptake in the presence of Hg-binding ligands such as sulfide, penicillamine, and glutathione relative to heat-killed controls. Uptake rates in the presence of Hg-cysteine complexes were linear for more than 4 hours; however, no methylmercury production was observed during the 20 h incubation. In contrast, similar experiments in the presence of Hg-cysteine with the Hg-methylating proteobacterium, Geobacter sulffurreducens, showed rapid Hg(II) uptake, methylation, and export of the newly produced methylmercury such that almost 100% of the added Hg(II) was methylated in ~4 h. These differences between these two iron-reducing strains are not due to changes in Hg-speciation, but rather due to enzymatic differences in the Hg methylating proteins, HgcAB, and/or other physiological differences in membrane structure, transporters, or the cytosol. Differences in the putative transporters responsible for Hg(II) uptake were observed between this firmicute and the proteobacteria. Unlike the proteobacteria studied (e.g. G. sulfurreducens), the protonophore, CCCP, failed to inhibit Hg(II) uptake in the firmicute, D. metallireducens. Furthermore, Zn(II) also had minimal or no effect on the cellular accumulation of Hg(II) when complexed to either chloride or cysteine. These specific results indicate that the model of active Hg(II) uptake through Zn transporters observed in proteobacteria may not be the primary mechanism of Hg(II) transport in D. metallireducens. Instead, Hg uptake in this firmicute appears to be a passive process likely mediated by facillitated diffusion through pores or channels in the membrane. These apparent differences in uptake and bioavailability of Hg(II) across phyla are critical to understanding the diverse microbial populations in the environment which are responsible for the production and accumulation of methylmercury.
INSIGHTS INTO THE BIOMOLECULAR MECHANISM OF MICROBIAL MERCURY METHYLATION
Mercury emissions as a result of silver mining, energy production and industrial use have led to a fourfold increase in mercury concentrations near the surface of oceans over the past 500 years. Bioaccumulation of highly toxic methylmercury (MeHg) in the food web is a significant public health concern. MeHg typically accounts for at least 90% of the mercury in fish. Inorganic mercury deposited in soils and sediments is transformed to MeHg by the metabolic activity of anaerobic bacteria and archaea. A two-gene cluster (hgcAB) is required for mercury methylation, and homologs of hgcA and hgcB have been identified in the genomes of more than 150 bacteria and archaea to date. The two genes are predicted to encode a corrinoid-dependent protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, consistent with roles as a methyl carrier and electron shuttle, respectively. However, the molecular structures of HgcA and HgcB and the specific mechanism of mercury methylation have not been determined. Furthermore, there is only limited knowledge about metabolic pathways linked to mercury methylation. This work aims to identify molecular interactions of HgcA and HgcB to elucidate the biochemistry of mercury methylation. Desulfovibrio desulfuricans ND132 strains were engineered for tandem affinity purification of HgcA and HgcB with 3xFLAG/TEV/StrepII tags in order to overcome limitations resulting from low abundance of the native proteins in cells. ND132 strains expressing tagged HgcA or HgcB retain mercury methylation activity at >50% of the wild-type strain. Tandem affinity purification, immunoblotting, crosslinking, and pull-down assays in combination with mass spectrometry are being used to identify other cellular proteins that interact with HgcA and HgcB. The results are expected to reveal protein-protein interactions, which will help delineate the roles of HgcA and HgcB in the context of microbial metabolism. Furthermore, identification of multiprotein complexes may enable molecular characterization that will provide insights into the mechanism of mercury methylation. A comprehensive understanding of the various geochemical and biochemical factors culminating in the production of MeHg will facilitate the development of effective strategies to reduce exposure to this pervasive neurotoxin.
ABIOTIC AND BIOTIC PATHWAYS FOR DIMETHYLMERCURY PRODUCTION IN AQUATIC SYSTEMS
The biogeochemistry of Hg in the oceans and the bioaccumulation of MMeHg in its food webs are of particular interest given that marine fish are the major source of exposure of people to MMeHg. A distinct characteristic of the Hg biogeochemical cycle in the ocean is the occurrence of dimethylmercury (DMeHg), a highly toxic and volatile form of Hg. Reported concentrations of DMeHg in marine waters range from 0.01-0.4 pM and DMeHg has been found to constitute up to 80% of the methylated Hg pool (MMeHg + DMeHg). The underlying formation pathways of DMeHg in the ocean however remains unknown. For the Arctic system, where a singifcant fraction of the methylated pool is present as DMeHg, incubation experiments have shown the methyation rate for the conversion of MMeHg to DMeHg to be one to two orders of mangitue higher than the methylation rate for inorganic Hg to DMeHg. Modelling work by Soerensen et al. futher suggest in situ formation of DMeHg in sea water from MMeHg, rather than from inorganic Hg. Suggested pathways of DMeHg formation from MMeHg under environmentally-relevant conditions include reaction of MMeHg with hydrogen sulfide, selenoaminoacids and methylcobalamin. Formation of DMeHg from MMeHg reacted with L-cysteine has also been suggested, however supporting experimental data is lacking. Up to 90% of the MMeHg in marine waters, and over 99% of the MMeHg in sediments and inside cells, is adsorbed to reduced sulfur groups on mineral surfaces or bound to thiol groups within organic matter. Thus, surface mediated processes leading to DMeHg production could be of great significance. Here, we experimentally show that the methylation of MMeHg to DMeHg can be mediated by different sulfide minerals, of different thermodynamic stability, as well as by organic dithiol compounds. We also show that the reaction can take place on mineral surfaces in either artificial sea water and/or when algal cell components are present, suggesting that MMeHg could be methylated intracellularly by iron-sulfur clusters on protein surfaces. We have also studied the formation of DMeHg from inorganic Hg and MMeHg in pure cultures of bacterial strains previously known to methylate inorganic Hg to MMeHg to examine bacterial production of DMeHg. We will contrast the importance of these various pathways based on our results and information about the potential for these reactions to occur in the marine environment.
MICROBIAL METHYLATION OF MERCURY IN PADDY SOILS AND ITS MAJOR DRIVING FACTORS
Mercury (Hg) has been considered to be a global pollutant because it can be transported over long distances. The Hg in environments could be microbially transformed into highly toxic methylmercury (MeHg), which would threaten human health and ecosystem safety. The MeHg in paddy soil is mainly from Hg microbial methylation, however, the underlying mechanisms of Hg methylation in the soil still remains poorly understood. This study is integrating chemical analysis and microbial ecology to explore the MeHg biosynthesis and characterize the community of Hg microbial methylators in paddy soils. Results show that MeHg concentration in soils was significantly correlated to Hg methylating gene (hgcA) abundance, which mainly distributed into sulfate-reducing microorganisms and iron-reducing microorganisms. In addition, geochemical factors such as soil contents of organic carbon, NH4+, SO42-, and Hg also influence the MeHg biosynthesis. Dynamics of MeHg formation and key microbial groups involved in Hg methylation will be monitored based on a series of mesocosm experiments, and the results will help to unravel the relative contribution of these Hg microbial methylators to MeHg production and the key geochemical factors influencing Hg methylation in the paddy soils. Finally, data from both field studies and incubation experiments will be incorporated to develop biogeochemical models for clarifying the microbial pathways and mechanisms of Hg methylation in paddy soils, which will be helpful to mitigate or manage soil MeHg pollution.
USING MOLECULAR PROBES TO DETECT HGCAB FROM ENRICHED PERIPHYTON BIOFILM SAMPLES FROM THE EAST FORK POPLAR CREEK MERCURY CONTAMINATED SYSTEM
Methylmercury (MeHg) is a harmful neurotoxin that bioaccumulates, thus posing risks to consumers and the environment. While a great deal of mercury (Hg) research has been conducted in aquatic environments, most work has taken place in lakes, reservoirs, and wetlands, with rivers and streams less well studied. The majority of research has also focused on macroscopic MeHg contributors like water and sediment as opposed to microscopic biofilms. Periphyton biofilms in the Hg contaminated East Fork Poplar Creek (EFPC) are a net source of MeHg in the system. Periphyton is a complex mixture of algae, microbes, and detritus that is attached to submerged surfaces in most aquatic ecosystems. These complex assemblages are ubiquitous throughout the creek and contain redox gradients that support Hg-methylating microbial activity even though the bulk water is well oxygenated. These biofilms include metal and sulfate-reducing Deltaproteobacteria and Firmicutes as well as methanogenic Archaea. Our initial attempt to utilize our hgcAB molecular probes on genomic DNA isolated from periphyton samples have been met with challenges. Direct analysis of periphyton was inconclusive, and likely due to the interference of abundant algal DNA in the gDNA extractions.
To increase PCR assay resolution and identify Hg-methylators, periphyton grown on silica disks deployed at upstream (closer to historic point of contamination) and downstream locations (~17km apart) of the EFPC were used to inoculate anaerobic enrichments. Three separate growth conditions for sulfate-reducing, Fe(III)-reducing, and methanogenic bacteria were established to promote growth of potential Hg-methylators and select against algae and other eukaryotes. PCR for hgcAB with gDNA isolated from these enrichments yielded conclusive evidence of hgcAB presence with a decrease in previously observed mispriming from untreated samples. Continuing studies will explore ways to process samples before extraction to better separate Bacterial and Archaeal organisms and DNA from eukaryotes. This accomplishment will allow for direct interrogation of the periphyton to identify Hg-methylators but also determine their native relative abundances. The latter is critical since the methylation extent and potential differs widely amongst Hg-methylating species.
Finally, the enrichments are being used to obtain site representative isolates from the EFPC which will be characterized and used to construct synthetic communities. This approach will allow for the observation of microbial interactions between Hg-methylators. Such interactions which may alter Hg-methylation and demethylation rates, extents, and potentials observed in single cultures, thus providing new and critical information to inform site management and restoration policies.
THE EFFECT OF MARINE DISSOLVED ORGANIC MATTER ON THE FORMATION AND FATE OF Β-HGS NANOPARTICLES
The methylation of mercury is known to depend on the chemical forms of mercury present in the environment and the bacterial methylating activity. In sulfidic sediments, under conditions of supersaturation with respect to metacinnabar, mercury precipitates as β-HgS(s) nanoparticles. Nano-sized particles have enhanced mobility, reactivity, and bioavailability relative to their micro-sized counterparts. Few studies have looked at the formation of β-HgS(s) nanoparticles precipitated in presence of marine DOM. In this work, we use dynamic light scattering (DLS), UV-Vis spectroscopy, and transmission electron microscopy (TEM) to investigate the formation and fate of β-HgS(s) nanoparticles (β-HgS(s)nano). They were formed from solutions containing inorganic mercury (HgII), dissolved sulfide, and marine DOM extracted from: Eastern Long Island Sound, Western Long Island Sound and at the shelf break of the North Atlantic Ocean, as well as with low molecular weight thiols. All the DOM used led to the formation of stable β-HgS(s)nano however, DOM extracted from the shelf break was less effective at inhibiting growth of β-HgS(s)nano relative to coastal DOM. In addition, multiple thiol groups on DOM likely cause inter-staple crosslinking of the β-HgS(s)nano leading to aggregation of particles, increasing their size as measured by DLS. Electron microscopy and calculations based on UV-Vis spectra confirmed that the β-HgS(s)nano however had similar radii, indicating that aggregation was occurring. We also showed that under anoxic conditions, β-HgS(s)nano can remain stable for weeks but will aggregate rapidly on exposure to air and light, suggesting limited persistence of the β-HgS(s)nano in surface waters. Furthermore, we showed that only at very high HgII:DOM ratios (>40 µmole/mg C), much higher than environmental levels, is the DOM concentration insufficient to effectively passivate the surface of β-HgS(s)nano. Our results also suggest that functional groups other than thiols could be involved in the passivation of β-HgS(s)nano by DOM. We will discuss the implications of the results from this work towards understanding the cycling of mercury and the presence of β-HgS(s)nano in sulfidic systems and, as β-HgS(s)nano are bioavailable to methylating bacteria, the implications to mercury methylation in natural ecosystems.
DISTRIBUTION OF MERCURY-METHYLATING MICROBES ALONG SPATIAL AND TEMPORAL REDOX GRADIENTS IN A FRESHWATER LAKE
Methylmercury (MeHg) is the bioaccumulative, neurotoxic form of mercury (Hg). It is taken up by algae and biomagnifies to dangerously high levels up the food chain. MeHg in freshwater lakes is either received from riverine runoff or is produced in situ from inorganic Hg(II) by anaerobic bacteria and archea that contain the hgcAB gene cluster. These microorganisms are tremendously diverse both metabolically and phylogenetically, and produce MeHg at different rates. In freshwater lakes, MeHg production can occur both in anoxic lake sediments and the anoxic water column, suggesting that these microorganisms inhabit both niches. In the water column of stratified lakes, changing redox conditions due to the onset of anoxia establishes a temporal and spatial gradient of electron acceptors and nutrients. The impact of these gradients on the distribution and identity of hgcAB-containing organisms, and thus on methylation potential, is not well understood.
Here, we examined the distribution of hgcAB-containing microorganisms relative to these gradients in water chemistry in a dimictic, eutrophic lake with high levels of MeHg. We collected depth-discrete DNA samples from Lake Mendota approximately every two weeks over the course of the summer, starting at the onset of anoxia and concluding at fall turnover. We also collected samples for water chemistry analysis to establish redox conditions and nutrient status at each of our sample sites. We performed PCR using primers for the hgcAB genes to determine which sites had organisms that contained hgcAB, then sequenced hgcAB amplicons from each of these sites for an overview of the diversity of hgcAB-containing organisms. Samples from representative sites were used to generate clone libraries for Sanger sequencing to further probe the diversity and to identify the organisms. These samples will be used for metagenomic sequencing to characterize the metabolic context for Hg methylation, as well as the community structure around methylation. Understanding how these varying conditions affect the identity of the methylating organisms will lead to a greater understanding of how MeHg production is influenced by the water chemistry of a lake, where and why methylation hotspots occur, and what role the methylating machinery plays in the physiology of methylating organisms.
SORPTION OF MERCURY TO AGED IRON SULFIDES AND THE IMPLICATIONS FOR HG BIOAVAILABILITY
The potential for inorganic mercury (Hg) to be converted to methylmercury depends on its bioavailability to microorganisms, which is in part controlled by the chemical speciation of Hg. In anaerobic settings, sulfides play an important role in controlling Hg speciation with numerous studies focusing on the formation of HgS and Hg-organic matter interactions and their effects on Hg bioavailability. Likewise, sulfide speciation can be dominated by ferrous iron sulfide (FeS), which can sorb or coprecipitate with Hg. The objective of this study was to determine if the aging state of FeS alters the reactivity and bioavailability of sorbed and coprecipitated Hg species. Synthesized FeS particles (with and without Hg) were aged under anaerobic conditions for multiple time frames spanning 1 hour to 1 month. Divalent Hg was subsequently sorbed to the non-Hg FeS for 1 day. The results indicated that more than 99% of the Hg sorbed to FeS (33 ug Hg per g FeS), regardless of the FeS aging state but in the Hg-Fe-S system aging affected Hg uptake (56% (1 hr) to 76% (1 month)). The recalcitrance of the Hg was assessed by exposing the particles to a solution of dissolved glutathione (a thiolate-based Hg chelator). In the sorbed Hg-FeS system, more Hg desorbed from the 1 month-old FeS than from the 1-h old FeS. For the Hg-Fe-S coprecipitate system, the desorption potential of Hg decreased with Hg-Fe-S aging time. Analysis of Hg speciation by X-ray absorption near edge spectroscopy (XANES) revealed qualitative differences between sorbed and coprecipitated Hg-FeS forms indicating a possible difference in binding mechanisms. However, the spectral quality was insufficient in elucidating quantitative information on the Hg phases present. In future work, these various forms of synthesized Hg-FeS will be exposed to cultures of methylating bacteria in order to compare bioavailability and Hg desorption potential from FeS particles.
SOIL CHEMISTRY IMPACTS ON hgcAB DISTRIBUTION AND MERCURY METHYLATION RATES IN WETLAND SOILS
The discovery of the genes responsible for microbial methylmercury production, hgcA and hgcB, has led to the identification of novel mercury (Hg) methylators with diverse metabolisms including iron and sulfate -reducing bacteria, syntrophs, and methanogens. Although these microorganisms are known to be widespread in nature, little is known about how soil chemistry shapes the distribution and abundance of Hg-methylating microorganisms. The objectives of this work were to 1) determine the abundance and distribution of hgcAB+ organisms in mid-Atlantic marshes and 2) to evaluate their distribution in relation to biogeochemical parameters in the marsh soils, including Hg methylation rates. The overall goal of this research is to assess the relative importance of site geochemistry and microbial community structure on Hg-methylation.
Soil cores were obtained from several marsh sites in the mid-Atlantic with salinities ranging from 2 to 13 parts per thousand. The sites were selected to contain different dominant vegetation types, including: no vegetation, Typha latifolia and angustifolia, Phragmites australis, and Spartina patens. The soil organic content varied from 1% to 73%, and porewater iron and sulfate concentrations varied according to both salinity and vegetation type. Microbial activity, as assessed by CO2 and methane production, varied widely across sites, with methane production being highest in the lowest salinity marsh site and CO2 production highest in the site dominated by Spartina patens. CO2 production correlated with soil MeHg concentration, and methylation rates were highest in the low-salinity marsh samples. hgcA was found in all samples tested, and methylation rates correlated with hgcA copy number for deltas and methanogens for samples with a core depth of 0-4 cm. Work is ongoing to expand the data set for hgcA abundance across all the sample sites and core depths.
IONIC STRENGTH CONTROLS ANAEROBIC HG UPTAKE
In the absence of dissolved organic matter, chloride and sulphide are the dominant complexing agents affecting mercury (Hg) speciation in the environment. Bioreporters are good tools in evaluating bioavailable species of Hg. However, signal production for current Hg bioreporters requires oxygen, namely for lux and gfp gene encoded proteins, resulting in a paucity of data in anaerobic bioavailability of Hg species where it is methylated to toxic CH3Hg+. We developed and optimized a whole-cell Hg bioreporter using Escherichia coli 5 capable of functioning in aerobic and anaerobic conditions by transforming a vector containing the gene fusion between the regulatory circuitry of the mer-operon and a flavin mononucleotide-based fluorescent protein. The bioreporter exhibited no physiological limitations with respect to signal production over a chloride gradient ranging from 0 to 0.55 M. The bioreporter had a detection limit of 1 nM Hg(II) however we used 5nM Hg(II) as the optimal working concentration for Hg speciation assays. We report that HgCl4-2 is highly bioavailable under anoxic conditions in contrast to what was observed under oxic conditions where Hg was not bioavailable. HgCl3- was not bioavailable regardless of the presence of oxygen which we attributed to either coordination polymers consisting of [HgCl3-]x chains on the cell surface or molecular mimicry of HgCl4-2 onto anaerobically expressed periplasmic binding proteins specific for divalent polyatomic anions shuttling Hg to the inner membrane. The unexpected bioavailability of HgCl4-2 indicates the possibility of a novel mechanism for Hg uptake in marine environments.
METHYLMERCURY FORMATION AND MOBILIZATION IN TROPICAL SOILS FROM A RAINFOREST WATERSHED (FRENCH GUIANA)
Small scale gold-mining activities are both releasing anthropogenic amalgamated Hg and remobilizing endogenous Hg naturally accumulated in tropical forest soils. On the other hand, the main sources of methylmercury (MeHg) in tropical rainforest environments are still under debate. In this way, we investigated mercury (Hg) methylation pathways and microbial diversity in French Guiana rainforest soils from a small scale watershed containing significant concentration of endogenous Hg during early and late wet season (Febr June 2013). The experiments were realized along a typical soil gradient from upper oxisols down to hydromorphic soils. All these soils were strongly different in term of redox conditions, the hydromorphic soils being much more under reducing conditions, especially during the late wet season. Selected samples from different types of soils were investigated for potential methylation and demethylation rates during in situ experiments after incubations with isotopic tracers in the presence or absence of potential stimulators (sulfate, reducible iron or propionate). Soils and incubation batches bacterial communities diversity was characterized by NGS methods.
The microbial communities structure was extremely different between communities from upper oxisols, intermediate ultisols and hydromorphic soils periodically flooded with water. In most samples, community composition exhibit high proportions of Deltaproteobacteria dominated by syntrophic bacteria and Geobacteraceae, while Sulfate reducers were mostly found in reduced hydromorphic soils. Microbial diversity was compared with mercury transformation potentials (i.e. methylation and demethylation) in order to identify the main Hg methylators in tropical forest soils, especially taking into account sulfate reducers, iron reducers and syntrophs that were found at significant abundance. While the Hg methylation in oxisols remains negligible, significant methylation extent has been measured (up to ~1%.day-1) in hydromorphic soils during the late wet season. Such larger methylation extent was strongly enhanced by iron or sulfate amendment which suggests the main implication of both sulfate and iron reducing bacteria. The seasonal hydromorphic alteration in the lower section of the watershed (ie hydromorphic soils) provides geochemical and microbial conditions in soils favorable for in situ Hg methylation. During rain events, MeHg was also found to be actively remobilized into soil porewater and superficial flood stream. Tropical forest soils are thus able to generate significant amount of MeHg which can be further released to downstream hydrosystems, suggesting important implications for the environmental impact of Hg in such environments.
ADSORPTION OF METHYLMERCURY ONTO GEOBACTER BEMIDIJENSIS BEM
Anaerobic bacteria play an important role in the production and degradation of methylmercury (MeHg). The adsorption of MeHg onto anaerobic bacterial cells can affect the release of MeHg into aquatic environments as well as the uptake of MeHg for demethylation. The anaerobic bacterium Geobacter bemidijensis Bem has the unique ability to both produce and degrade methylmercury. To date, the binding of MeHg onto Hg-methylating and MeHg degrading bacteria remains poorly understood. In this study, we quantified the adsorption of MeHg onto G. bemidijensis Bem and applied X-ray absorption spectroscopy to elucidate the mechanism of MeHg binding on the bacterial cell wall. We hypothesized that MeHg adsorption on bacterial surfaces occurs via complexation with thiol functional groups in the cell wall. To test this hypothesis, fluorescent thiol-specific probes were used to determine the concentration of thiol functional groups on the bacterial surface. Methylmercury adsorption experiments were conducted over a range of MeHg concentrations and adsorption isotherms were used to quantify MeHg binding constants. The local coordination environment of methylmercury adsorbed onto G. bemidijensis Bem was then examined using extended X-ray absorption fine structure (EXAFS) spectroscopy. The results showed that MeHg adsorption onto G. bemidijensis Bem was rapid and equilibrium was reached in less than 1 hour. At low MeHg concentrations, up to 95% of MeHg was adsorbed to the bacterial cells. Blocking of the thiol functional groups with fluorescent thiol-specific probes significantly decreased the extent of MeHg binding. Results from the EXAFS analysis will be discussed in the presentation. Our study indicates that MeHg binds strongly onto G. bemidijensis Bem and that complexation reactions with cell wall thiol functional groups can affect the release and uptake of MeHg in anaerobic bacteria.
EXPLORING ENVIRONMENTS FOR THE PRESENCE OF MICROBIAL MERCURY METHYLATORS USING HGCAB MOLECULAR PROBES
The genes hgcA and hgcB have been shown to be responsible for microbial mercury (Hg) methylation. Recently, we developed a cost-effective PCR-based assay utilizing degenerate primers to assess the presence of hgcAB in genomic DNA (gDNA) from 30 pure cultures with the intention of application in the field. The goal for the present work was to survey environmental samples, including multiple soil/sediment/periphyton types from diverse geographical locations, for the detection and quantification of hgcAB from both known and novel microorganisms.
Here we present a selection of those environments which were examined, including; 1) sediment and periphyton collected from the Hg-contaminated East Fork Poplar Creek as well as the background Hinds Creek in Oak Ridge, Tennessee, each differing in relative methyl mercury (MeHg) and total mercury (HgT) concentrations, 2) sediment from microcosm experiments originating from these locations amended with carbon sources, 3) soil cores from the Marcell Experimental Forest in Minnesota, 4) organic carbon-rich permafrost cores from the Seward peninsula in Alaska, part of the Next-Generation Ecosystem Experiments (NGEE-Arctic), and 5) salt-marsh soils from the Smithsonian Environmental Research Center (SERC) on the shores of the Chesapeake Bay sub-estuary in Maryland. The hgcAB gene pair was present across all locations and the obtained PCR products amplified were observed at the expected size. Within each set of samples, hgcAB was only detected within the anaerobic zone. For each unique environment, gDNA isolation required optimization in order to account for differences in sediment texture, organic carbon content and pH, to name a few. Most importantly, our degenerate primers worked efficiently to amplify hgcAB and sequencing data revealed these genes were captured from uncultured and novel microorganisms among the samples as well as those known to be present at a particular location.
EFFECTS OF NATURAL ORGANIC MATTER ON MICROBIAL METHYLATION OF MERCURY (HG) UNDER ANAEROBIC CONDITIONS
Naturally dissolved organic matter (DOM) is known to affect mercury (Hg) redox reactions and microbial methylation in the environment. Several studies have shown that DOM may enhance Hg methylation, particularly under sulfidic conditions, whereas others found that DOM inhibits Hg methylation due to strong complexation between Hg and DOM molecules. In this study, we systematically investigated and compared the effects of DOM in Hg methylation by an iron-reducing bacterium Geobacter Sulfurreducens PCA and a sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 under anaerobic conditions. The experiment was performed with washed cells in laboratory incubations in a phosphate buffered saline either in the presence or absence of a DOM isolate extracted from a mercury-contaminated creek water. Additionally, we compared DOM effects on Hg methylation in the presence or absence of 20 µM cysteine, which is known to enhance Hg methylation. Our results demonstrate that DOM effects on microbial methylation are bacterial strain-specific, time- and DOM-concentration dependent. Addition of DOM (at 0.1–5 mg/L DOC (dissolved organic carbon)) greatly inhibited Hg methylation by Geobacter Sulfurreducens PCA but somewhat enhanced methylation by Desulfovibrio desulfuricans ND132. However, addition of cysteine with DOM not only alleviated the inhibitory effects of DOM but enhanced Hg methylation by Geobacter Sulfurreducens PCA (at 0.1-2.5 mg/L DOC). These observations suggest that the effects of DOM and small thiol compounds such as cysteine on Hg methylation vary greatly, likely due to their influences on Hg bioavailability through complexation, and this effect should be considered when evaluating Hg methylation potential in the natural environment.
CONTRASTING RESPONSES OF METHYLMERCURY ACCUMULATION IN RICE AND WHEAT TO BIOCHAR AMENDMENT
There is growing concern about methylmercury (MeHg) accumulation in crops and thus enhanced dietary exposure to MeHg. Here, we explored the possibility of reducing grain MeHg levels by biochar amendment, and the underlying mechanisms. Two-year pot (i.e., rice-wheat-rice cultivation in biochar amended soils) and batch experiments (i.e., incubation of amended soils under laboratory conditions) were carried out, to investigate MeHg dynamics (i.e., MeHg production, partitioning and phytoavailability in soils, and MeHg uptake by rice or wheat) under biochar amendment (14% of soil mass). We demonstrate for the first time that biochar amendment could evidently increase net MeHg production in soils during rice (36-303% higher, compared to control) or wheat (52-292%) cultivation, probably explained by the release of dissolvable sulfate from biochar and thus enhanced microbial production of MeHg (e.g., by sulfate-reducing bacteria, indicated by copies of dsrAB genes). Interestingly, contrasting responses of MeHg accumulation in rice and wheat to biochar amendment were observed: biochar amendment resulted in reduced MeHg levels in rice (4992% lower, compared to control), but enhanced MeHg concentrations in wheat (48-84% higher). The differences were attributed to the changes in sulfur speciation in soils during rice (generally anoxic) or wheat (oxic) cultivation, examined by XANES, and thus different binding strength between MeHg and biochar amended soils. These observations together with mechanistic explanations improve understanding of MeHg dynamics in soil-plant systems, and support the possibility of reducing MeHg phytoaccumulation under biochar amendment.