Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution

To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH₃Hg⁺) and accumulated more mercury from CH₃Hg⁺-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH₃Hg⁺ to Hg²⁺, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH₃Hg⁺ and Hg²⁺ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg⁰) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.     

Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

Effects of Inorganic Mercury and Methylmercury on the Ionic Currents of Cultured Rat Hippocampal Neurons

1. The effects of inorganic Hg²⁺ and methylmercuric chloride on the ionic currents of cultured hippocampal neurons were studied and compared. We examined the effects of acute exposure to the two forms of mercury on the properties of voltage-activated Ca²⁺ and Na⁺ currents and N-methyl-D-aspartate (NMDA)-induced currents.2. High-voltage activated Ca²⁺ currents (L type) were inhibited by both compounds at low micromolar concentrations in an irreversible manner. Mercuric chloride was five times as potent as methylmercury in blocking L-channels.3. Both compounds caused a transient increase in the low-voltage activated (T-type) currents at low concentrations (1 M) but blocked at higher concentrations and with longer periods of time.4. Inorganic mercury blockade was partially use dependent, but that by methylmercury was not. There was no effect of exposure of either form of mercury on the I–V characteristics of Ca²⁺ currents.5. Na⁺- and NMDA-induced currents were essentially unaffected by either mercury compound, showing only a delayed nonspecific effect at a time of overall damage of the membrane.6. We conclude that both mercury compounds show a relatively selective blockade of Ca²⁺ currents, but inorganic mercury is more potent than methylmercury.     

Seasonal influences on partitioning and transport of total and methylmercury in rivers from contrasting watersheds

Seven Wisconsin rivers with contrasting, relativelyhomogeneous watershed composition were selected toassess the factors controlling mercury transport.Together, these watersheds allow comparisons ofwetland, forest, urban and agricultural land-uses.Each site was sampled nine times between September1993 and September 1994 to establish seasonalsignatures and transport processes of total mercury(Hg_T) and methylmercury (MeHg). Our resultsclearly show that land use and land cover stronglyinfluence mercury transport processes. Under base-flowconditions, unfiltered MeHg yield varies by a factorof sixteen (12–195 mg km^-2 d^-1), andincreases with the fraction of wetland area in thewatershed. Elevated mercury yields during high floware particle-phase associated in agricultural sites,but filtered-phase associated in wetland sites.Methylmercury represented less than 5% of totalmercury mobilized during the spring thaw across allwatersheds. Autumn MeHg yield was generally 11–15%of Hg_T in wetland influenced watersheds, thougha maximum of 51% was observed. In some cases, singlehigh-flow events may dominate the annual export ofmercury from a watershed. For example, one high-flowevent on the agricultural Rattlesnake Creek had thelargest Hg_T and MeHg yield in the study (107 and2.32 mg km^-2 d^-1, respectively). The mass ofmercury transported downstream by this single eventwas an order of magnitude larger than the eight other(non-event) sampling dates combined. These resultsunderscore the importance of watershed characteristicsand seasonal events on the fate of mercury in freshwater rivers.     

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg⁰, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.     

Automated Speciation of Mercury in the Hair of Breastfed Infants Exposed to Ethylmercury from Thimerosal-Containing Vaccines

A simplified thiourea-based chromatography method, originally developed for methyl and inorganic mercury, was adapted to separate methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg^II) in infants' hair. Samples were weighed and leached with an acidic thiourea solution. Leachates were concentrated on a polymeric resin prior to analysis by Hg-thiourea liquid chromatography/cold vapor atomic fluorescence spectrometry. All but one sample showed small amounts of EtHg, and four of the six analyzed samples had proportionally higher Hg^II as a percent of total Hg. Breastfed infants from riverine Amazonian communities are exposed to mercury in breast milk (from high levels of maternal sources that include both fish consumption and dental amalgam) and to EtHg in vaccines (from thimerosal). The method proved sensitive enough to detect and quantify acute EtHg exposure after shots of thimerosal-containing vaccines. Based on work with MeHg and Hg^II, estimated detection limits for this method are 0.050, 0.10, and 0.10 ng g⁻¹ for MeHg, Hg^II, and EtHg, respectively, for a 20-mg sample. Specific limits depend on the amount of sample extracted and the amount of extract injected.     

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn<Emphasis Type="Italic">MERI1</Emphasis>

Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.     

Behavior of mercury in the Patuxent River estuary

An overview of a comprehensive study of the behavior and fate of mercury in the estuarine Patuxent River is presented. Total Hg (Hg_T) and methylmercury (MeHg) exhibited weakly non-conservative behavior in the estuary. Total Hg concentrations ranged from 6 ng L^-1 in the upper reaches of the sub-urbanized tidal freshwater river to <0.5 ng L^-1 in the mesohaline lower estuary. Filterable (0.2 µm) Hg_T ranged from 0.2 to 1.5 ng L^-1. On average, MeHg accounted for <5% of unfiltered Hg_T and <2% of filterable Hg_T. Dissolved gaseous section Hg (DGHg) concentrations were highest (up to 150 pg L^-1) in the summer in the mesohaline, but were not well correlated with primary production or chlorophyll a, demonstrating the complex nature of Hg⁰ formation and cycling in an estuarine environment. Organic matter content appeared to control the Hg_T content of sediments, while MeHg in sediments was positively correlated with Hg_T and organic matter, and negatively correlated with sulfide. MeHg in sediments was low (0.1 to 0.5% of Hg_T). Preliminary findings suggest that net MeHg production within sediments exceeds net accumulation. Although Hg_T in pore waters increased with increasing sulfide, bulk MeHg concentrations decreased. The concentration of MeHg in sediments was not related to the concentration of Hg_T in pore waters. These observations support the hypothesis that sulfide affects the speciation and therefore bioavailability of dissolved and/or solid-phase Hg for methylation. Comparison with other ecosystems, and the negative correlation between pore water sulfide and sediment MeHg, suggest that sulfide limits production and accumulation of MeHg in this system.     

Mercury inputs and outputs at a small lake in northern Minnesota

Storages and cycling of total mercury (Hg_T), methylmercury (MeHg), and Hg⁰ are described for Spring Lake, a small bog lake in the Marcell Experimental Forest in north-central Minnesota. We quantified photoredox transformations, MeHg photolysis, burial to the sediments, and internal and external loadings of Hg_T and MeHg. Atmospheric deposition was the main input of Hg_T; MeHg was supplied by a combination of atmospheric, near-shore wetland, and biotic (methylation) sources. Hg_T outputs were dominated by burial (67%), and Hg⁰ evasion accounted for 26% of Hg_T outputs. The watershed of Spring Lake is small (3.7× lake surface area), and accordingly, bog and upland runoff were minor contributors to both Hg_T and MeHg inputs. Wet deposition was ∼9% of total MeHg input, and other external inputs (runoff, sediment porewater) provided only an additional 7%, indicating that internal production of MeHg was occurring in the lake. Photolysis of MeHg, measured in the field and laboratory, removed ∼3× the lake mass of MeHg (20 mg) annually, and was the dominant sink for MeHg. Residence times of MeHg and Hg_T in the lake were 48 and 61 days, respectively, during the open-water season, compared with only 8 days for the residence time of MeHg on settling particles (seston). Photoreduction of Hg²⁺ to Hg⁰ was greater than the reverse reaction (Hg⁰ photooxidation), and the residence time of Hg⁰ in the photic zone was short (hours). Data from this study show active cycling of all the measured species of mercury (Hg_T, MeHg, and Hg⁰) and the importance of MeHg photolysis and photo-redox processes.     

Mercury-resistant plasmids in bacteria from a mercury and antimony deposit area

Summary Most bacterial cells (Pseudomonas, Acinetobacter) obtained from the soil at the Khaidarkan mercury and antimony mine (Kirghiz USSR) contain R plasmids with mercury (HgCl₂) resistance determinants. The plasmids have a large molecular mass (about 100 MD, though smaller ones also occur), and at least some of them are transmissive. Many of the Hg^r bacteria also display an elevated antimony (SbCl₃) resistance, though this trait was not shown to be plasmid-dependent. There are practically no Hg^r plasmids in bacteria taken from the soil at different distances from the mine: the saturation of bacteria with Hg^r plasmids is maintained by selective pressure only in the area with a high enough toxin concentration.In the same mercury and antimony deposit area Hg^r plasmids were also found in Escherichia coli isolates from the gut of Mus musculus mice and Bufo viridis toads. At least some of the bacterial plasmids obtained from animals also carry antibiotic-resistance determinants (Tc^r, Cm^r, Sm^r). These plasmids are also transmissive. They display internal instability and lose their resistance determinants after a conjugation transfer to other E. coli trains.     

Reduction of Mercury Chloride by Chlorella: Evidence for a Reducing Factor

Evidence is brought for the presence of low molecular weight, heat stable, mercury reducing factors in Chlorella cells. Some of the properties of these factors, which appears to be normal metabolities, are described. These factors are also present in the medium in which the cells grow. The reduced mercury, Hg⁰, is volatilized from the culture medium more rapidly than Hg⁺². The resultant decrease in the Hg concentration appears to be the main reason for the recovery of the cells from Hg stress. No resistance to Hg developed in cells emerging from the stress.     

Spatial distribution and seasonal changes in methylmercury concentrations in shallow lakes

The environmental factors governing the distribution and behaviour of total and methylmercury within mercury contaminated shallow lakes, associated with the River Yare, Norfolk, UK, have been assessed in situ through the use of sediment cores. These were analysed for microbial and chemical changes on both a temporal and spatial scale. The distribution of total mercury proved to be site dependent and related to the hydrology, sediment transport dynamics, the degree of sediment disturbance and distance from the contamination source. The core profiles revealed a subsurface peak in total mercury with maximum concentrations residing at depths of 12 and 36 cm, depending upon location, with enrichment extending down to depths in excess of 88 cm. This vertical distribution was deemed to reflect historical emission in the late 1960s and early 1970s rather than post depositional migration effects. The distribution of methylmercury was distinct from that of its inorganic counterpart since it also displayed temporal variability with highest concentrations occurring in the spring and summer. Maximum concentrations also prevailed in the uppermost 12 cm of sediment, with peak loadings at or just below the sediment/water interface and detectable levels being restricted to the upper 36 cm. The temporal and spatial behaviour of methylmercury appeared to reflect variations in redox potential, the availability of the Hg²⁺ ion and the nature of microbial populations.     

Sensitivity to antimicrobial agents of some mercury-sensitive and mercury-resistant strains of Gram-negative bacteria

The sensitivity and resistance of some Gram-negative mercury (Hg²⁺)-sensitive and-resistant strains to chemotherapeutic agents and to disinfectants and preservatives are described.Escherichia coli andPseudomonas aeruginosa strains harboring plasmid pUB 1351 [pUB 367:Tn 501] andE. coli bearing R100-1 were resistant to inorganic mercury and to various antibiotics, but were not more resistant to organic mercury and other preservatives and disinfectants than plasmidless strains.     

Influence of dietary selenite on the binding characteristics of rat serum proteins to mercurial compounds

The effect of dietary selenite on the binding characteristics of serum proteins was investigated with rats. In the control serum, the maximal binding of phenylmercuric acetate (PMA) and methylmercuric chloride (MMC) to rat serum protein was approx. 18 and 9 nmoles per mg protein, respectively. The binding of Hg²⁺ was biphasic and it did not reach a maximum at the concentrations used.Selenite treatment caused a reduction in binding capacity of serum proteins to Hg²⁺ and PMA, and an increase in the binding affinities. However, there were no such changes for the binding of MMC. Selenite protection from mercury toxicity, therefore, acts not only via a change in tissue distribution and a change in the formation of seleno-proteins but, also, via a change in the binding characteristics to some mercury compounds. In the case of methylmercury, a different mechanism of protection must exist as the modification of tissue distribution, its binding to subcellular and soluble proteins and the binding characteristics remained equivocal.     

Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation

Background

Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds.

Methodology/Principal Findings

To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg²⁺. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg²⁺, 0.12 and CH₃Hg⁺, 0.08. The addition of Hg²⁺ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg²⁺ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg²⁺ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg²⁺ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg²⁺ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h.

Conclusions/Significance

A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.     

Occurrence and transformation of mercury in formerly contaminated soils due to operation of amalgamation techniques and assessment of consequences

Abstract

Soils formerly contaminated with mercury due to the implementation of amalgamation processes at two localities in the Czech Republic were analyzed to assess the impact of such contamination on both the environment and human health. One site is an abandoned gold mine at Lib?ice, where, in the past, gold was extracted from the raw ore by the mercury amalgamation technique. The second site is the environs of a small building in Prague where dental surgery was formerly undertaken. Soils were tested for total mercury (T-Hg), elemental mercury (Hg⁰), methylmercury (MeHg⁺), phenylmercury (PhHg⁺), and gaseous elemental mercury (GEM). The T-Hg concentrations in both localities exceeded many times the maximum permissible limit for soils in the Czech Republic. The most contaminated soils were found around the dental surgery releasing GEM at concentrations of up to 1308?ng m^?3, which can represent a danger mainly for people through inhalation. Soils near the abandoned gold mine release GEM at concentrations of up to 26?ng m^?3, which indicates a long-term burden on the environment and a danger for surrounding farmland.     

Reduced sulphur sources favour HgII reduction during anoxygenic photosynthesis by Heliobacteria

The consumption of rice has become a global food safety issue because rice paddies support the production of high levels of the potent neurotoxin, methylmercury. The production of methylmercury is carried out by chemotrophic anaerobes that rely on a diversity of terminal electron acceptors, namely sulphate. Sulphur can be a limiting nutrient in rice paddies, and sulphate amendments are often used to stimulate crop production, which can increase methylmercury production. Mercury (Hg) redox cycling can affect Hg methylation by controlling the delivery of inorganic Hg substrates to methylators in anoxic habitats. Whereas sulphur is recognized as a key substrate controlling methylmercury production, the controls sulphur exerts on other microbe‐mediated Hg transformations remain poorly understood. To explore the potential coupling between sulphur assimilation and anaerobic Hg^II reduction to Hg⁰, we studied Heliobacillus mobilis, a mesophilic anoxygenic phototroph representative from the Heliobacteriacea family originally isolated from a rice paddy. Here, we tested whether the redox state of the sulphur sources available to H. mobilis would affect its ability to reduce Hg^II. By comparing Hg⁰ production over a redox gradient of sulphur sources, we demonstrate that phototrophic Hg^II reduction is favoured in the presence of reduced sulphur sources such as thiosulphate and cysteine. We also show that cysteine exerts dynamic control on Hg cycling by affecting not only Hg's bioavailability but also its abiotic photoreduction under low light conditions. Specifically, in the absence of cells we show that organic matter (as yeast extract) and cysteine are both required for photoreduction to occur. This study offers insights into how one of the most primitive forms of photosynthesis affects Hg redox transformations and frames Heliobacteria as key players in Hg cycling within paddy soils, forming a basis for management strategies to mitigate Hg accumulation in rice.     

Diel variability of mercury phase and species distributions in the Florida Everglades

Preliminary studies of mercury (Hg) cycling in the Everglades revealed that dissolved gaseous mercury (DGM), total mercury (Hg_T), and reactive mercury (Hg_R) show reproducible, diel trends. Peak water-column DGM concentrations were observed on or about noon, with a 3 to 7 fold increase over night-time concentrations. Production of DGM appears to cease during dark periods, with nearly constant water column concentrations that were at or near saturation with respect to the overlying air. A simple mass balance shows that the flux of Hg to the atmosphere from diel DGM production and evasion represents about 10% of the annual input from atmospheric deposition. Production of DGM is likely the result of an indirect photolysis reaction that involves the production of reductive species and/or reduction by electron transfer. Diel variability in Hg_T and Hg_R appears to be controlled by two factors: inputs from rainfall and photolytic sorption/desorption processes. A possible mechanism involves photolysis of chromophores on the surface of a solid substrate (e.g., the periphyton mat) giving rise to destabilization of sorbed mercury and net desorption during daylight. At night, the sorption reactions predominate and the water-column Hg_T decreases. Methylmercury (MeHg) also showed diel trends in concentration but were not clearly linked to the solar cycle or rainfall at the study site.     

In vitro study of thimerosal reactions in human whole blood and plasma surrogate samples

Because of its bactericidal and fungicidal properties, thimerosal is used as a preservative in drugs and vaccines and is thus deliberately injected into the human body. In aqueous environment, it decomposes into thiosalicylic acid and the ethylmercury cation. This organomercury fragment is a potent neurotoxin and is suspected to have similar toxicity and bioavailability like the methylmercury cation. In this work, human whole blood and physiological simulation solutions were incubated with thimerosal to investigate its behaviour and binding partners in the blood stream. Inductively coupled plasma with optical emission spectrometry (ICP-OES) was used for total mercury determination in different blood fractions, while liquid chromatography (LC) coupled to electrospray ionisation time-of-flight (ESI-TOF) and inductively coupled plasma-mass spectrometry (ICP-MS) provided information on the individual mercury species in plasma surrogate samples. Analogous behaviour of methylmercury and ethylmercury species in human blood was shown and an ethylmercury-glutathione adduct was identified.     

Review of characteristics of mercury speciation and mobility from areas of mercury mining in semi-arid environments

The speciation of mercury—including most phase minerals, secondary phases, gaseous and aqueous species—is very important for evaluating the environmental impact and mobilization of this contaminant. Mining activities produce mercury mine waste, which includes several types of material (mainly mine waste and calcines) with varying mercury content and speciation depending on the ore deposit and processing technology. The main phase minerals are cinnabar, metacinnabar, metallic Hg⁰, corderoite, livingstonite, calomel and schuetteite. The aqueous mobilization of mercury is controlled by complex formation, pH-Eh conditions, the primary phase mineral of mercury, and organic-matter and iron oxyhydroxide content. The possibility of colloidal transport of mercury from mine waste is influenced by the atmospheric emission of metallic Hg⁰ and the leaching of waste by episodic high-intensity precipitations. In these climatic conditions, mercury can be mobilized to pore water, surface water or groundwater by the dissolution of metallic Hg⁰ and cinnabar in acidic conditions, and by the colloidal transport. The presence of Hg-soluble phases (chlorides and oxychlorides) may enhance the mobilization of mercury. In the semi-arid conditions of the Valle del Azogue (SE Spain) the atmospheric emissions of the Hg⁰ present in calcines and mine waste may be significant and the dissolution of Hg⁰ and metal-sulfate salts during episodic runoff events may explain the mobilization of Hg, Fe, Pb, Zn and other heavy metals.