Potential for Mercury Reduction by Microbes in the High Arctic

The contamination of polar regions due to the global distribution of anthropogenic pollutants is of great concern because it leads to the bioaccumulation of toxic substances, methylmercury among them, in Arctic food chains. Here we present the first evidence that microbes in the high Arctic possess and express diverse merA genes, which specify the reduction of ionic mercury [Hg(II)] to the volatile elemental form [Hg(0)]. The sampled microbial biomass, collected from microbial mats in a coastal lagoon and from the surface of marine macroalgae, was comprised of bacteria that were most closely related to psychrophiles that had previously been described in polar environments. We used a kinetic redox model, taking into consideration photoredox reactions as well as mer-mediated reduction, to assess if the potential for Hg(II) reduction by Arctic microbes can affect the toxicity and environmental mobility of mercury in the high Arctic. Results suggested that mer-mediated Hg(II) reduction could account for most of the Hg(0) that is produced in high Arctic waters. At the surface, with only 5% metabolically active cells, up to 68% of the mercury pool was resolved by the model as biogenic Hg(0). At a greater depth, because of incident light attenuation, the significance of photoredox transformations declined and merA-mediated activity could account for up to 90% of Hg(0) production. These findings highlight the importance of microbial redox transformations in the biogeochemical cycling, and thus the toxicity and mobility, of mercury in polar regions.     

A thermophilic bacterial origin and subsequent constraints by redox,light and salinity on the evolution of the microbial mercuric reductase

Mercuric reductase (MerA) is central to the mercury (Hg) resistance (mer) system, catalyzing the reduction of ionic Hg to volatile Hg(0). A total of 213 merA homologues were identified in sequence databases, the majority of which belonged to microbial lineages that occupy oxic environments. merA was absent among phototrophs and in lineages that inhabit anoxic environments. Phylogenetic reconstructions of MerA indicate that (i) merA originated in a thermophilic bacterium following the divergence of the Archaea and Bacteria with a subsequent acquisition in Archaea via horizontal gene transfer (HGT), (ii) HGT of merA was rare across phylum boundaries and (iii) MerA from marine bacteria formed distinct and strongly supported lineages. Collectively, these observations suggest that a combination of redox, light and salinity conditions constrain MerA to microbial lineages that occupy environments where the most oxidized and toxic form of Hg, Hg(II), predominates. Further, the taxon‐specific distribution of MerA with and without a 70 amino acid N‐terminal extension may reflect intracellular levels of thiols. In conclusion, MerA likely evolved following the widespread oxygenation of the biosphere in a thermal environment and its subsequent evolution has been modulated by the interactions of Hg with the intra‐ and extracellular environment of the organism.     

Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9°N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 μM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45°C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65°C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.     

The relationships of Hg(II) volatilization from a freshwater pond to the abundance ofmer genes in the gene pool of the indigenous microbial community

The role of biological activities in the reduction and volatilization of Hg(II) from a polluted pond was investigated. Elemental mercury was evolved from pond water immediately following spiking with²⁰³Hg(NO₃)₂, whereas an acclimation period of 36 hours was required in control samples collected from a nearby, unpolluted river before onset of volatilization. Genes encoding the bacterial mercuric reductase enzyme (mer genes) were abundant in DNA fractions extracted from biomass of the pond microbial community, but not in samples extracted from control communities. Thus, evolution of Hg⁰ was probably due to activities mediated by the bacterial mercuric reductase. Of four characterizedmer operons, the system encoded by transposon 501 (mer(Tn501)) dominated and likely contributed to the majority of the observed Hg(II) volatilization. Thus,mer-mediated reduction and volatilization could be used to reduce Hg(II) concentrations in polluted waters, in turn decreasing rates of methylmercury formation by limiting substrate availability.     

The effect of aqueous speciation and cellular ligand binding on the biotransformation and bioavailability of methylmercury in mercury-resistant bacteria

Mercury resistant bacteria play a critical role in mercury biogeochemical cycling in that they convert methylmercury (MeHg) and inorganic mercury to elemental mercury, Hg(0). To date there are very few studies on the effects of speciation and bioavailability of MeHg in these organisms, and even fewer studies on the role that binding to cellular ligands plays on MeHg uptake. The objective of this study was to investigate the effects of thiol complexation on the uptake of MeHg by measuring the intracellular demethylation-reduction (transformation) of MeHg to Hg(0) in Hg-resistant bacteria. Short-term intracellular transformation of MeHg was quantified by monitoring the loss of volatile Hg(0) generated during incubations of bacteria containing the complete mer operon (including genes from putative mercury transporters) exposed to MeHg in minimal media compared to negative controls with non-mer or heat-killed cells. The results indicate that the complexes MeHgOH, MeHg-cysteine, and MeHg-glutathione are all bioavailable in these bacteria, and without the mer operon there is very little biological degradation of MeHg. In both Pseudomonas stutzeri and Escherichia coli, there was a pool of MeHg that was not transformed to elemental Hg(0), which was likely rendered unavailable to Mer enzymes by non-specific binding to cellular ligands. Since the rates of MeHg accumulation and transformation varied more between the two species of bacteria examined than among MeHg complexes, microbial bioavailability, and therefore microbial demethylation, of MeHg in aquatic systems likely depends more on the species of microorganism than on the types and relative concentrations of thiols or other MeHg ligands present.     

Mercury and methylmercury detoxification potential by sponge-associated bacteria

Ionic and organic forms of mercury (Hg) are powerful cytotoxic and neurotoxic agents in both humans and wild life. The aim of this study was to analyze the resistance profile and potential detoxification of inorganic and organic forms of Hg of bacteria isolated from marine sponges on the coast of Rio de Janeiro, Brazil. Out of the 1,236 colony forming units associated with eleven species of marine sponges, 100 morphologically different bacterial strains were analyzed in this study. Of these, 21 strains were resistant to Hg, 14 of which were classified as highly resistant because they grew despite exposure to 100 µM HgCl₂. Fifteen resistant strains reduced Hg and presented merA in their genomes. The remaining six strains produced biosurfactants, suggesting that they may tolerate Hg by sequestration. Eleven strains grew in the presence of methylmercury. Our results suggest a potential for mercury detoxification by marine sponge-associated resistant bacteria, either through reduction or sequestration, as well as the possibility of bioremediation of toxic waste containing mercury.     

Distribution of DNA Sequences Encoding Narrow- and Broad-Spectrum Mercury Resistance

The distribution of DNA sequences homologous with three mer genes was determined in unselected and mercury-resistant water and sediment isolates. The maximum proportions of unselected bacterial isolates containing DNA hybridizing with the 358merA, 358merB, and 501merR probes, derived from gram-negative organisms, were 93.8, 21, and 100%, respectively. Up to 53.3% of mercury chloride-resistant isolates and 54% of methylmercury hydroxide-resistant isolates did not contain DNA homologous with 358merA or 358merB, respectively. Hybridizations performed at high and low stringencies demonstrated that divergence of the merA gene accounted for many of the mercury-resistant but probe-negative isolates. Sixteen mercury-resistant Bacillus spp. isolated from the least contaminated site all contained DNA homologous with 258merA, originally from a gram-positive organism, but only four hybridized weakly with 358merA. The results demonstrate the wide distribution of mercury resistance genes but, because of the diversity of genetic determinants, highlight the importance of using multiple detection techniques and gene probes derived from a variety of origins for such studies.     

Nucleotide sequence and expression of the organomercurial-resistance determinants from a Pseudomonas K-62 plasmid pMR26

pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials. DNA sequence of this broad-spectrum resistant mer operon was determined. The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR, merT, merP, merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons. The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons. The remaining ORF named merE, located between merA and merB, had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance. Induction experiments and maxicell analyses of the mer-polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials.© 1997 Elsevier Science B.V. All rights reserved.     

Tn1 generated mutants in the mercuric ion reductase of the Inc P plasmid, R702

Summary Physiological, biochemical and genetic aspects of resistance to inorganic mercury compounds were examined in a group of mercury sensitive derivatives generated in the Inc P plasmid, R702, by Tn1 insertion. Strains carrying each of these insertion mutations had no detectable mercuric ion reductase, were more sensitive to mercuric ion than a plasmidless strain, and exhibited inducible uptake of Hg²⁺. These characteristics indicate that the mutants are altered in the Hg(II) reductase. This hypothesis was supported by complementation and recombination analysis with known point and deletion mutations in the mer operon of the Inc FII plasmid, R100. Such experiments showed that the eight insertions studied had occurred in four distinct regions of the Hg(II) reductase structural gene (merA). Complementation data also demonstrated that the regulatory protein determined by the R702 plasmid has no effect on the expression of the micro-constitutive Hg(II) reductase activity expressed by merR mutants of R100.     

Interactions between Snow Chemistry,Mercury Inputs and Microbial Population Dynamics in an Arctic Snowpack

We investigated the interactions between snowpack chemistry, mercury (Hg) contamination and microbial community structure and function in Arctic snow. Snowpack chemistry (inorganic and organic ions) including mercury (Hg) speciation was studied in samples collected during a two-month field study in a high Arctic site, Svalbard, Norway (79°N). Shifts in microbial community structure were determined by using a 16S rRNA gene phylogenetic microarray. We linked snowpack and meltwater chemistry to changes in microbial community structure by using co-inertia analyses (CIA) and explored changes in community function due to Hg contamination by q-PCR quantification of Hg-resistance genes in metagenomic samples. Based on the CIA, chemical and microbial data were linked (p = 0.006) with bioavailable Hg (BioHg) and methylmercury (MeHg) contributing significantly to the ordination of samples. Mercury was shown to influence community function with increases in merA gene copy numbers at low BioHg levels. Our results show that snowpacks can be considered as dynamic habitats with microbial and chemical components responding rapidly to environmental changes.     

Horizontal Transfer of Mercury Resistance Genes in Environmental Bacterial Populations

The results of studying the horizontal transfer of mercury resistance determinants in environmental bacterial populations are reviewed. Identical or highly homologous mercury resistance (mer) operons and transposons were found in bacteria of different taxonomic groups from geographically distant regions. Recombinant mer operons and transposons were revealed. The data suggest high frequencies of horizontal transfer and of recombination for mercury resistance determinants. The mechanisms of horizontal gene transfer were elucidated in Gram-negative and Gram-positive bacteria. New transposons were found and analyzed.     

Potential Application in Mercury Bioremediation of a Marine Sponge-Isolated Bacillus cereus strain Pj1

Sponges are sessile marine invertebrates that can live for many years in the same location, and therefore, they have the capability to accumulate anthropogenic pollutants such as metals over a long period. Almost all marine sponges harbor a large number of microorganisms within their tissues. The Bacillus cereus strain Pj1 was isolated from a marine sponge, Polymastia janeirensis, and was found to be resistant to 100 μM HgCl₂ and to 10 μM methylmercury (MeHg). Pj1 was also highly resistant to other metals, including CdCl₂ and Pb(NO₃)₂, alone or in combination. The mer operon was located on the bacterial chromosome, and the volatilization test indicated that the B. cereus Pj1 was able to reduce Hg²⁺–Hg⁰. Cold vapor atomic absorption spectrometry demonstrated that Pj1 volatilized 80 % of the total MeHg that it was exposed to and produced elemental Hg when incubated with 1.5 μM MeHg. Pj1 also demonstrated sensitivity to all antibiotics tested. In addition, Pj1 demonstrated a potential for biosurfactant production, presenting an emulsification activity better than synthetic surfactants. The results of this study indicate that B. cereus Pj1 is a strain that can potentially be applied in the bioremediation of HgCl₂ and MeHg contamination in aquatic environments.     

Mercury resistance in <Emphasis Type="Italic">Sporosarcina</Emphasis> sp. G3

Mercuric reductase (MerA) enzyme plays an important role in biogeochemical cycling and detoxification of Hg and recently, has also been shown to be useful in clean up of Hg-contaminated effluents. Present study describes isolation of a heavy metal-resistant isolate of Sporosarcina, which could tolerate up to 40, 525, 210, 2900 and 370 μM of Cd, Co, Zn, Cr and Hg respectively. It was found to reduce and detoxify redox-active metals like Cr and Hg. The chromate reductase and MerA activities in the crude cell extract of the culture were 1.5 and 0.044 units/mg protein respectively. The study also describes designing of a new set of highly degenerate primers based on a dataset of 23 Firmicute merA genes. As the primers encompass the known diversity of merA genes within the phylum Firmicutes, they can be very useful for functional diversity analysis. They were successfully used to amplify a 787 bp merA fragment from the current isolate. A 1174 bp merA fragment was further cloned by designing an additional downstream primer. It was found to show 92% similarity to the putative merA gene from Bacillus cereus AH820. To the best of our knowledge, this is the first report of mercury resistance and merA gene sequence from Sporosarcina.     

Rapidly increasing methyl mercury in endangered ivory gull (Pagophila eburnea) feathers over a 130 year record

Mercury (Hg) is increasing in marine food webs, especially at high latitudes. The bioaccumulation and biomagnification of methyl mercury (MeHg) has serious effects on wildlife, and is most evident in apex predators. The MeHg body burden in birds is the balance of ingestion and excretion, and MeHg in feathers is an effective indicator of overall MeHg burden. Ivory gulls (Pagophila eburnea), which consume ice-associated prey and scavenge marine mammal carcasses, have the highest egg Hg concentrations of any Arctic bird, and the species has declined by more than 80% since the 1980s in Canada. We used feathers from museum specimens from the Canadian Arctic and western Greenland to assess whether exposure to MeHg by ivory gulls increased from 1877 to 2007. Based on constant feather stable-isotope (δ¹³C, δ¹⁵N) values, there was no significant change in ivory gulls'' diet over this period, but feather MeHg concentrations increased 45× (from 0.09 to 4.11 µg g⁻¹ in adults). This dramatic change in the absence of a dietary shift is clear evidence of the impact of anthropogenic Hg on this high-latitude threatened species. Bioavailable Hg is expected to increase in the Arctic, raising concern for continued population declines in high-latitude species that are far from sources of environmental contaminants.     

Environmental Conditions Constrain the Distribution and Diversity of Archaeal <Emphasis Type="Italic">merA</Emphasis> in Yellowstone National Park,Wyoming, U.S.A.

The distribution and phylogeny of extant protein-encoding genes recovered from geochemically diverse environments can provide insight into the physical and chemical parameters that led to the origin and which constrained the evolution of a functional process. Mercuric reductase (MerA) plays an integral role in mercury (Hg) biogeochemistry by catalyzing the transformation of Hg(II) to Hg(0). Putative merA sequences were amplified from DNA extracts of microbial communities associated with mats and sulfur precipitates from physicochemically diverse Hg-containing springs in Yellowstone National Park, Wyoming, using four PCR primer sets that were designed to capture the known diversity of merA. The recovery of novel and deeply rooted MerA lineages from these habitats supports previous evidence that indicates merA originated in a thermophilic environment. Generalized linear models indicate that the distribution of putative archaeal merA lineages was constrained by a combination of pH, dissolved organic carbon, dissolved total mercury and sulfide. The models failed to identify statistically well supported trends for the distribution of putative bacterial merA lineages as a function of these or other measured environmental variables, suggesting that these lineages were either influenced by environmental parameters not considered in the present study, or the bacterial primer sets were designed to target too broad of a class of genes which may have responded differently to environmental stimuli. The widespread occurrence of merA in the geothermal environments implies a prominent role for Hg detoxification in these environments. Moreover, the differences in the distribution of the merA genes amplified with the four merA primer sets suggests that the organisms putatively engaged in this activity have evolved to occupy different ecological niches within the geothermal gradient.     

Dietary Exposure of the Red-Crowned Crane (Grus japonensis) to Total and Methyl Mercury in Zhalong Wetland,Northeastern China

To determine the dietary exposure of the migratory red-crowned crane to mercury (Hg), this study analyzed the concentrations of total mercury (T-Hg) and methyl mercury (MeHg) in its prey, i.e., reeds and three aquatic animal families (Perccottus glenni Dybowski, Cybister japonicus Sharp, and Viviparidae) in northeastern China. Results indicated that the Hg concentration in Zhalong Wetland was elevated through the food chain, and the prey of the red-crowned crane contained measurable levels of T-Hg and MeHg. In prey tissues, MeHg was the main form of the Hg element and accounted for 61 % of total Hg concentration in Viviparidae, 58 % in C. japonicus Sharp, and 85 % in P. glenni Dybowski. The highest T-Hg and MeHg concentrations ranged from 1.66 to 3.89 ppm and from 1.12 to 2.67 ppm, respectively, and they were detected in the feathers of the red-crowned cranes. The lowest T-Hg concentration was determined in the excretions of wild red-crowned cranes at 0.21 ppm; furthermore, the content of MeHg was below the detection limit. In Zhalong Wetland, the level of dietary exposure of the population of red-crowned cranes to Hg is below the threshold of Hg toxicity. Moreover, eggshells are suitable indicators of Hg risk levels to the red-crowned crane.