Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

Jeffra K. Schaefer; Sara S. Rocks; Wang Zheng; Liyuan Liang; Baohua Gu; François M. M. Morel

doi:10.1073/pnas.1105781108

New Research In

Contributed by François M. M. Morel, April 12, 2011 (sent for review January 22, 2011)

Abstract

The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron- and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments.

Monomethyl mercury (CH₃Hg⁺, methylmercury, MeHg) is a potent neurotoxic compound (1). It is biomagnified in the food webs of aquatic systems, reaching high concentrations in carnivorous fish, thus posing a risk to human health (2). Understanding the mechanism of inorganic Hg methylation and the parameters that control the extent of methylation in the environment is thus essential for relating patterns of Hg pollution to human exposure. The production of MeHg has been linked to obligate anaerobic bacteria in the δ-Proteobacteria, including iron- and sulfate-reducing bacteria (FeRB and SRB) that live in soil and sediments (3 –6). Although mechanisms of Hg(II) methylation by methylating enzymes have been proposed for some time (7, 8), the mechanism of Hg(II) uptake by the bacteria has remained obscure. The dominant view is that cellular uptake occurs by passive diffusion of neutral Hg(II) complexes, particularly sulfide complexes, through external membranes, leading to accidental methylation of some of the intracellular Hg(II) (9). However, this view is based on indirect data and modeling, as the precipitation of metal sulfides in the medium and the extensive Hg binding to the surface of the organisms (10 –12) have made it difficult to directly measure Hg(II) uptake in methylating bacteria.

In previous work (13), we demonstrated that the cysteine complex of Hg(II) was available to the FeRB Geobacter sulfurreducens PCA and that Hg(II) was likely transported into the cell via an unknown facilitated transport mechanism. Here we examine the energy dependence and specificity of Hg(II) uptake and methylation by both G. sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 in the presence of various thiol compounds.

Results

Partitioning of Hg to Cells.

In 4-h exposure experiments, a variable fraction of Hg(II) complexed to various thiols became associated with cells of G. sulfurreducens at a variety of thiol concentrations (particulate Hg > 0.22 μm): only 10% in the presence of glutathione (GSH) in excess of 10 μM, 60% to 85% in the presence of cysteine depending on the concentration, and intermediate percentages in the presence of penicillamine (PEN) and 2-mercaptopropionate (2MPA; Fig. 1A). This is consistent with our earlier experiments (13), in which 60% of the Hg became associated with the cells in the presence of 10 μM cysteine. In similar experiments, with D. desulfuricans, the bulk of the mercury became associated with the cells under practically all conditions, despite the fact that the experiments were conducted at a much lower cell concentration: 0.3 × 10¹¹ cells/L for D. desulfuricans versus 1.1 × 10¹¹ cells/L for G. sulfurreducens (Fig. 1B). Only at concentrations of GSH and 2MPA of 50 μM did more than 5% of Hg remain in solution. In these experiments at high Hg concentrations (40 nM) and in the absence of growth substrates, less than 1% of the dissolved Hg was present as MeHg.

Fig. 1.

The fraction of total Hg associated with cells of G. sulfurreducens (A) and D. desulfuricans (B) after 4 h in washed cell assays containing 40 nM Hg(II) and 0.5 to 50 μM of the appropriate thiol compound: cysteine (■), 2MPA (○), PEN (▲), or GSH (X). The cell densities were 1.1 and 0.3 × 10¹¹ cells/L in A and B, respectively.

Uptake and Methylation of Hg(II).

The very large fractions of Hg associated with cells in Fig. 1 are unlikely to represent true cellular uptake. As shown in our previous study with G. sulfurreducens, most of the particulate Hg can be removed by a washing protocol that includes exposure to a GSH/HAsc solution (GSH and ascorbic acid; Materials and Methods). The extent of “true cellular uptake” measured in this way is relatively small compared with the total particulate Hg, and is commensurate with the extent of Hg methylation. Small differences in the efficiency of the wash can cause large errors in the measurement of Hg uptake. As a result, uptake experiments are most successful in the presence of a complexing agent that limits the extent of Hg adsorption on the cells, coupled to a washing protocol that removes nearly all Hg adsorbed to the cell surface. For G. sulfurreducens, we decreased the background concentration of particulate Hg (nonzero intercept of uptake curves) and improved the reproducibility of the uptake measurements by adding a second 5-min exposure to the GSH/HAsc solution in our wash protocol. Fig. 2 A and B illustrate the systematic uptake data obtained for live and heat-treated cells and the consistency of the uptake and methylation data obtained simultaneously. For D. desulfuricans, which has a much higher affinity for Hg(II) (Fig. 1), we obtained systematic uptake data in the presence of 100 μM GSH (Fig. 2 C and D), but not in the presence of weaker Hg complexing agents, such as cysteine. In addition to the control provided by pasteurized cells, the consistency of the experiments reported later provide further validation of our experimental protocol.

Fig. 2.

The effect of heat pasteurization on the uptake (A and C) and methylation (B and D) of Hg(II). (A and B) G. sulfurreducens was exposed to a temperature of 50 °C for 0.5 (○), 1.0 (▽), or 2.0 h (□) before inoculation into assays containing 10 nM Hg(II) and 10 μM cysteine. (C and D) D. desulfuricans was exposed to 55 °C for 3.0 h (□) before inoculation to assays containing 10 nM Hg(II) and 100 μM GSH. Control assays (closed symbols) were exposed to a temperature of 29 °C for the same time period. The cell densities were 1.1 cells/L (A and B) and 0.8 × 10¹¹ cells/L (C and D).

In a previous study (13) with G. sulfurreducens, we observed that all the MeHg produced by the cells was present in the external medium, demonstrating efficient export of synthesized MeHg. This finding is not unique to G. sulfurreducens, but is also observed in the SRB, D. desulfuricans, in which the MeHg content of the cells is consistently at or below detection (≤0.07 × 10⁻²¹ cells/L) and the dissolved (<0.22 μm) MeHg concentration is equal to the total MeHg concentration (Fig. S1). Thus, the cellular Hg concentration is a balance between the rates of Hg(II) uptake and MeHg export; in our experiments, after 10 to 20 min, these two rates become approximately equal and the cellular Hg concentration approximately constant.

Active Uptake of Hg(II).

The potential involvement of an active transport mechanism for the uptake of Hg(II) in G. sulfurreducens was investigated by measuring Hg(II) uptake rates in the presence of cysteine, with and without addition of exogenous energy sources, acetate and fumarate. Cells incubated in the absence of electron donor and acceptor for 2 h (i.e., “starved” cells) were unable to take up or methylate Hg(II) (Fig. 3), similar to heat-inactivated cells (Fig. 2 A and B) and assays conducted on ice (13). Upon addition of acetate and fumarate to starved cells, Hg(II) uptake resumed after approximately 15 min, demonstrating that the process is energy-dependent. As expected, starved cells, which did not take up Hg(II), failed to produce MeHg. Although the addition of energy substrates rapidly fostered Hg(II) uptake, it resulted in only a modest extent of methylation over the 40 min of the experiment, producing only approximately 10% of the MeHg typically measured in such experiments with nonstarved cells (Fig. 3B, dashed line). A similar experiment with the SRB, D. desulfuricans ND132, failed to show a significant difference in Hg(II) uptake or methylation between assays with or without added carbon, indicating that starvation may not yet have been achieved during the 4-h preincubation period of this experiment. In our hands, the growth of G. sulfurreducens, but not that of D. desulfuricans, responds readily to a decrease in energy substrates.

Fig. 3.

(A) Hg(II) uptake and (B) methylation in 2-h starved (no electron donor or acceptor added) and nonstarved cells (dashed line) of G. sulfurreducens in assays containing 11 nM Hg(II) and 10 μM cysteine. At time 0, fumarate and acetate (1 mM each) were added to assays indicated by “+C” (■), whereas no carbon was added to assays indicated by “-C” (○). The cell densities were 0.8 and 1.1 × 10¹¹ cells/L in starved and control (dashed line) assays, respectively.

A more direct demonstration of the energy requirement for the uptake of Hg(II) in G. sulfurreducens and D. desulfuricans, was given by the effect of the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (14). As shown in Fig. 4 A and B, the presence of CCCP greatly inhibited Hg(II) uptake in both G. sulfurreducens (in the presence of cysteine) and D. desulfuricans ND132 (in the presence of GSH). Both organisms displayed similar rates of Hg(II) uptake in control assays with similar delays of approximately 10 min observed before the onset of Hg(II) methylation, as discussed later. As expected, the decrease in Hg(II) uptake caused by CCCP resulted in a large decrease in Hg(II) methylation. In other experiments, we observed complete inhibition of MeHg formation by CCCP, even if the effect on Hg uptake was not always clear because of low uptake rates under some growth conditions (Fig. S2). These data show that Hg(II) uptake is energy dependent, either through an electrogenic or ATP-driven mechanism. Examples of carrier-type protein families capable of heavy metal transport in Proteobacteria include those fueled by ATPases (ABC and P₁-type), as well as by proton or chemiosmotic gradients (e.g., NRAMP and ZIP) (15, 16). We tested if an amino acid transporter might be involved in the uptake of Hg(II) in G. sulfurreducens. Millimolar concentrations of neutral amino acids serine, glycine, methionine, valine, leucine, and aspartate, which are known to inhibit the uptake of Hg–cysteine complexes in mammalian cells (17), had no effect on Hg(II) methylation by G. sulfurreducens in the presence of 10 μM cysteine (Fig. S3). The only amino acid that significantly inhibited Hg methylation was cysteine at millimolar concentrations, a phenomenon observed previously (13) and hypothesized to be caused by formation of a biologically unavailable Hg(Cys)₃ complex. The compound 2,6-diaminopimelic acid, a structural analogue of cystine and an inhibitor of low-affinity cystine transporters (18), also had no effect on Hg(II) uptake or methylation (Fig. S3). These results make it doubtful that an amino acid transporter is involved in the uptake of Hg–thiol complexes in our model FeRB.

Fig. 4.

The effect of 20 μM CCCP on the uptake (solid lines) and methylation (dashed lines) of Hg(II) in washed cell suspensions of G. sulfurreducens (A) and D. desulfuricans (B) containing 10 nM Hg(II) and 10 μM cysteine (A) or 100 μM GSH (B). Cells were preincubated with 20 μM CCCP (in ethanol; ○, ●) or 0.2% ethanol only (control cells; □, ■) for 1 h at 29 °C before the start of the assay. The cell densities were 1.0 cells/L (A) and 0.9 × 10¹¹ cells/L (B). The final control assay (time >30 min) was lost in B.

Substrate Specificity of Hg(II) Uptake.

As shown previously (13), G. sulfurreducens is able to take up Hg(II) when complexed to cysteine but not when complexed to GSH. Hg(II) uptake is also seen in the presence of 2MPA, but not in the presence of the thiol analogue PEN (Fig. 5A). This difference in uptake is notable in view of the similar extent of Hg binding to cells observed in the presence of 2MPA and PEN, intermediate between that observed with cysteine and GSH (Fig. 1). We observed Hg(II) methylation only and always when Hg(II) was taken up by G. sulfurreducens. In the presence of cysteine, the uptake of Hg(II) was immediate, followed by a delay of approximately 10 min before the Hg(II) was methylated (Fig. 5B) and exported out of the cell (13). This delay, which was observed in most experiments performed with our improved flash cooling protocol (Materials and Methods), may represent the time-dependent and/or concentration-dependent activation of the methylation process.

Fig. 5.

The effect of ligand structure on (A) Hg uptake and (B) methylation in G. sulfurreducens assays containing 10 nM Hg(II) and 10 μM thiol. Individual thiols tested were cysteine (cys, ■), 2MPA (○), PEN (▲), and GSH (X). Chemical structures for all are provided in Table S1. The cell density in all assays was 1.0 × 10¹¹ cells/L. Similar results were obtained with replicate experiments.

We examined the ability of a variety of Hg–thiol complexes to support Hg methylation in G. sulfurreducens. Small thiols that have local Hg-bonding environments similar to cysteine, with no branching off the thiol carbon, yielded similar rates of Hg methylation (Fig. 6A and Table S1). In contrast, Hg complexes with thiols lacking free hydrogens proximal to the –SH group, were not methylated, with the notable exception of 2MPA. Hg complexes with larger thiols do not support Hg methylation in G. sulfurreducens even when they possess an unsubstituted thiol carbon within the cysteine moiety, as is the case of GSH. These results suggest that the overall size and flexibility of the binding molecule are important for determining the availability of the Hg complex to G. sulfurreducens. This may be caused, for example, by the inability of larger molecules to enter the periplasm and the slow rate of exchange of Hg(II) between rigid compounds and molecules involved in uptake. In this respect, it is notable that uptake of Hg(II) in the presence of 2MPA, the only compound with a single methyl branch on the thiol carbon that supported uptake and methylation, always occurred after a lag of approximately 10 min compared with uptake in the presence of cysteine (Fig. 5A).

Fig. 6.

The effect of Hg-binding ligand on the Hg methylation rate by G. sulfurreducens (A) and D. desulfuricans (B) in washed cell assays containing 10 μM ligand and 5 nM (A) or 8 nM (B) Hg(II). Conditions include assays without any added thiol (buffer), with sulfide, or with one of the following thiols: cysteamine (cysNH2), thioglycolate (TGA), 3-nitrobenzoic mercaptan (3NBM), 2MPA, D-L-PEN (pen), GSH, or N-acetylcysteine (NAC). Methylation rates were determined by measuring total MeHg concentrations after 2 h (A) or 1 h (B). The Cl⁻ concentrations in all assays were 1.6 and 220 mM in GsAB and DdAB, respectively. Chemical structures for all are provided in Table S1. Bars represent averages of two to 35 replicates (typically n = 3) and error bars represent ±1 SD. It should be noted that ND132 was grown in the presence of 0.5 g/L yeast extract in place of 25 μM sulfate (in other experiments), which may explain the high methylation rates shown.

As seen in the dramatic difference in Hg uptake and methylation between the two organisms in the presence of GSH (Figs. 2, 5A, and 6), D. desulfuricans ND132 is able to take up Hg(II) bound to stronger complexing agents than G. sulfurreducens. In addition to GSH, PEN and cysteine promoted Hg methylation in D. desulfuricans ND132 (Fig. 6B). As is the case for adsorption to the cell surface, the transport system of the SRB has a higher affinity for Hg(II) than that of the FeRB. One curious finding was the much higher methylation rate of Hg–chloride complexes in D. desulfuricans than in G. sulfurreducens: approximately 10% of the added Hg(II) in the first versus less than 1% to 3% in the second (Fig. 6, “buffer”). These results are inconsistent with uptake by passive diffusion, as calculations of Hg speciation in the two assay buffers (without thiols) predict more than 99% of the Hg(II) to form neutral HgClOH, HgCl₂, and Hg(OH)₂ complexes in G. sulfurreducens assays, as opposed to only 15% in the estuarine-based assay buffer in D. desulfuricans assays, in which HgCl₄²⁻ and HgCl₃⁻ are calculated to be the dominant Hg(II) species.

Discussion

Our results reveal unexpected properties of the Hg(II) uptake system in methylating bacteria: (i) high rates of Hg(II) uptake and methylation are observed in the presence of some complexing thiols in both FeRB and SRB model organisms; (ii) this uptake is mediated via an active transport mechanism; (iii) uptake is highly dependent on the characteristics of the complexing thiols, with some promoting high rates of uptake and others inhibiting it; and (iv) the SRB D. desulfuricans ND132 has a higher affinity for Hg(II) than the FeRB G. sulfurreducens.

The energy dependence and specificity of the underlying transport system bring up the questions of its physiological function and of the underlying mechanism. The most likely physiological function of the uptake system is for the acquisition of amino acid or trace metals. It seems unlikely that the Hg–cysteine complex is taken up via an amino acid transporter. The uptake system is certainly not specific for amino acids, as chirality has no effect (Table S1), and the presence of amino acid functional groups (e.g., 3NBM, cysteamine, and 2MPA) is unnecessary (Figs. 5 and 6). We also found no effect of the addition of diaminopimelic acid or other amino acids on the methylation of Hg(II)–cysteine complexes. It is much more likely that the function of the transport system, the kinetics of which we observed, is for the uptake of trace metals. In an environment in which essential metals may be present at nanomolar concentrations and readily form complexes with sulfide and thiols, a transport system able to use metal–thiol complexes as substrates may well be necessary. It is possible that such an uptake system would not be highly selective and lead to accidental uptake of Hg(II); alternatively, the transport system could be specific for Hg(II). In either case, the tight stoichiometric coupling observed between Hg(II) uptake and MeHg export avoids the buildup of high Hg concentration inside the cells. Methylation and export could thus serve to reduce Hg(II) toxicity even though MeHg may be as toxic to microorganisms as Hg(II) (19). Such a situation is known for arsenic detoxification, whereby the reduction of As(V) to the more toxic As(III) is tightly coupled to its export (20). Much as in the case of the mer-mediated Hg(II) resistance operon (21), Hg(II) uptake, intracellular Hg methylation, and MeHg export might all be part of a bacterial Hg detoxification system; or, instead, Hg methylation and MeHg export could avoid the buildup and toxicity of Hg accidentally taken up by a transport system for essential metals. There is some evidence that Hg methylation may be part of a detoxification system in the methylating fungus, Neurospora crassa: mutants of N. crassa with increased Hg(II) tolerance are correlated with increased rates of Hg(II) methylation (22). In bacteria, increased resistance to MeHg in methylating organisms have been shown by Baldi et al. (23), but no difference in Hg(II) toxicity was observed between methylating and nonmethylating strains of SRBs (12). The Hg(II) toxicity results were obtained at high concentrations, and it is possible that methylation is a detoxification process in methylating organisms growing at very low concentrations of Hg(II). This system would be constitutive, as we have found no evidence that methylation is up-regulated upon Hg exposure. An analysis of genome and sequence databases showed that the mer operon is absent from obligately anaerobic organisms (24), leaving open the possibility of a different resistance strategy in these bacteria.

Our kinetic data provide some insight into the possible uptake mechanism. Both the absence of an effect of chirality on the uptake of Hg complexes and the remarkable differences seen in the presence of closely related thiol compounds point to exchange with a transport ligand as a key step in Hg(II) uptake. In view of what is known about metal uptake in Gram-negative bacteria (16), we hypothesize that this ligand exchange occurs in the periplasmic space. The uptake of Hg(II) (or of the intended metal substrate) might take place in three steps, as is the case for many Fe(III) complexes (25): (i) transport across the outer membrane (ii), exchange with a soluble metal binding protein in the periplasm, and (iii) transport across the inner membrane. The ability of D. desulfuricans to take up and methylate Hg(II) bound to some thiols that effectively inhibit uptake in G. sulfurreducens presumably results from different chemical characteristics of their transport systems. For example, the outer membrane of the SRB may be less selective for thiol compounds of different sizes than that of the FeRB, its periplasmic ligands may have a higher affinity for Hg(II), or they may allow faster metal exchange kinetics. We have systematically measured the great bulk of the MeHg in the external medium (Fig. S1) (13), so that the organomercurial compound must somehow be exported. However, our data provide no insight into the mechanism of MeHg export, a topic of obvious great interest.

Regardless of the physiological function of Hg(II) uptake and methylation and the underlying cellular and biochemical mechanisms, our results point to the chemistry of the external milieu as a key control on Hg(II) methylation in the environment. Previous work has focused chiefly on the question of Hg(II) complexation by chloride, sulfide, and humic substances (9, 26, 27). Our results show that complexation with thiol compounds is a likely determinant of the rate of methylation by both FeRBs and SRBs. A better understanding of the factors that control Hg(II) methylation in nature likely depends on new information on the nature, concentration, and cycling of thiol compounds in suboxic and anoxic environments.

Materials and Methods

Bacterial Cultures and Media.

G. sulfurreducens PCA (American Type Culture Collection 51573) was grown on 30 mM fumarate and 10 mM acetate in a defined Mops-buffered medium, pH 6.80 (13). D. desulfuricans ND132 (gift from C. Gilmour, Smithsonian Environmental Research Center, Edgewater, MD) was grown on 25 mM pyruvate and 30 mM fumarate in a low-sulfate medium modified from a previously described medium (28) containing (pH 7.3) 10 mM Mops, 1.5 mM KH₂PO₄, 4.7 mM NH₄Cl, 6.7 mM KCl, 3.2 mM MgCl₂, 1.4 mM CaCl₂, 257 mM NaCl, 25 μM Na₂SO₄, 23 nM Na₂SeO₃, 24 nM Na₂WO₄, 3.6 μM FeCl₂, 1 mg/L resazurin, 10 mL/L Wolfe vitamins (29), and 1 mL/L of a sulfate-free SL-7 trace metal solution adapted from one previously described (30). When necessary, titanium citrate (50 μM) was added to the growth medium for ND132 to chemically reduce the medium before inoculation. All media were boiled, allowed to cool while bubbling with O₂-free N₂ gas, dispensed into acid-cleaned serum bottles, sealed with rubber stoppers, and autoclaved.

Hg uptake and methylation assays were prepared under strict anoxic conditions in 20 mL acid-cleaned serum bottles with 9 mL assay buffer containing 1 mM of each electron acceptor/donor pair in which the cells were grown (fumarate and either pyruvate or acetate), unless otherwise noted. The assay buffer, GsAB, for G. sulfurreducens contained 10 mM Mops, 0.1 mM NH₄Cl, 1.3 mM KCl, 0.15 mM MgSO₄, 5 mM NaH₂PO₄, 0.17 mM NaCl, 1 mM acetate, 1 mM fumarate, and 1 mg/L resazurin (13). The assay buffer, Dd-AB, for D. desulfuricans ND132 was free of sulfate and contained 10 mM Mops, 0.1 mM NH₄Cl, 0.5 mM KCl, 0.4 mM MgCl₂, 0.2 mM KH₂PO₄, 170 mM NaCl, 1 mM pyruvate, 1 mM fumarate, and 1 mg/L resazurin.

Hg Sorption on Cells.

Hg partitioning experiments were conducted with washed cell assays containing 0.5 to 50 μM thiols. Before the addition of washed cells, media were allowed to preequilibrate for at least 2 h in glass vials containing 4 mL assay buffer, thiol, and 40 nM Hg(II). The assay buffer was similar to that used for Hg uptake experiments (GsAB or DdAB), except all C-sources (acetate, pyruvate, and fumarate) and Ti-citrate were omitted. The vials were agitated gently in the dark under anaerobic conditions for 4 h. Subsamples (3 mL) were then filtered through 0.2-μm syringe filters, and the filtrate collected and analyzed for dissolved Hg (Hg_diss) and MeHg (MeHg_diss) concentrations. The residual unfiltered solutions were digested in 1% BrCl overnight for total Hg analysis.

Hg Uptake and Methylation Experiments.

All Hg uptake and methylation assays containing 9 mL of GsAB or DdAB were preequilibrated with Hg(II) and thiol at assay temperature of 29 °C for more than 1 h before the addition of washed cells. During this preconditioning period, the cells were harvested by centrifugation at midexponential growth phase, washed at least one or two times, and resuspended in assay buffer. Exposure of the cells to air was minimized by the use of an anaerobic glove box with N₂:H₂ (90:10) atmosphere and the use of N₂-flushed needles and syringes throughout the course of the experiment. Washed cells were resuspended to a final OD₆₆₀ of 0.18 to 0.28 with assay buffer. Each Hg uptake and methylation assay was started by the addition of cells. Typically, no reducing agent was added to G. sulfurreducens assays, whereas 50 μM titanium citrate was added to all D. desulfuricans ND132 assays immediately before inoculation. An exception was the G. sulfurreducens assays with and without carbon, in which 50 μM titanium citrate was added to chemically reduce the medium because cells lacking carbon were unable to sufficiently reduce the medium alone (Fig. 3). Vials that remained oxic after 5 min (resazurin indicator) were not analyzed further. Individual Hg methylation assays were stopped at distinct time periods (usually ≤2 h) by freezing at −20 °C until analysis.

For uptake studies, individual samples were killed at each time point (0.5–60 min) and analyzed for cellular Hg content collected on washed filters, with total Hg (Hg_T) and total MeHg in unfiltered samples. Initial Hg(II) concentrations reported in each figure represent actual measured values in BrCl-digested subsamples taken from each assay vial during the experiment. After subsampling each individual vial for the various Hg species, the vial was immediately submerged in an ice bath to stop methylation until the vial could be frozen. Cellular Hg contents (Hg_cell) were measured by filtering cells (2 or 5 mL for D. desufuricans or G. sulfurreducens, respectively) through 0.2-μm polycarbonate filters (25 mm). Hg adsorbed to the cell surface was washed off the filters as described previously (13), except that a second GSH/HAsc wash was performed. Washed filters were digested overnight in 5 mL MilliQ water and 0.5% BrCl in acid-cleaned Teflon vials. Subsamples collected (1 mL) for total Hg analysis were digested overnight with 0.5% BrCl in acid-cleaned 5-mL glass amber vials.

The wash strategy for the Hg uptake experiments was tested by using heat-treated cells to minimize changes in cell membrane structure and denaturation of proteins while still rendering the cells metabolically inactive. Heat-treatment methods used were 0.5, 1, and 2 h at 50 °C for G. sulfurreducens and 3 h at 55 °C for ND132, as a higher temperature was necessary to render cells inactive and stop Hg methylation. In Hg uptake experiments containing the protonophore, CCCP (20 μM final) was added from a 10-mM stock solution prepared in 100% ethanol (0.2% ethanol, final). Washed cells were preincubated with 20 μM CCCP or 0.2% ethanol (control cells) for 1 h at 29 °C before the start of the experiment.

Hg and MeHg Analyses.

BrCl-digested Hg samples were first reduced with hydroxylamine HCl to react any excess BrCl. The Hg was reduced to Hg(0) with SnCl₂ in glass bubblers purged with N₂ gas in which the Hg(0) was trapped onto gold-coated beads (31). MeHg samples were distilled, derivatized with tetraethylborate, and purged onto Tenax traps (32, 33). When they had been trapped onto gold or Tenax, all Hg compounds were released by thermal desorption, separated by isothermal gas chromatography, pyrolysed to Hg(0), and detected by atomic fluorescence on a Tekran 2500 Hg analyzer (31).

Solutions and Chemicals.

Concentrated solutions of MeHgCl and HgNO₃ were purchased from Brooks Rand Labs and diluted as appropriate for experiments. All thiols were purchased from Sigma-Aldrich within 1 y (typically <3 mo). All thiol, sulfide, and diluted Hg(II) solutions were prepared fresh each day in N₂-flushed serum vials using MilliQ water previously boiled and cooled under O₂-free N₂.

Speciation Calculations.

All Hg(II) speciation calculations were performed for assay buffer (lacking thiols) using MINEQL+ 4.6 and stability constants included within the software. The Cl⁻ concentration in assay buffers GsAB and DdAB were 1.6 and 220 mM, respectively.

Acknowledgments

We thank C. Cobb-Adams for the analysis of Hg and MeHg at Princeton University. We also thank J. Jay and T. Barkay for helpful comments in review of this manuscript. This research was supported in part by the Office of Biological and Environmental Research, Office of Science, US Department of Energy, as part of the Mercury Science Focus Area Program at Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, for the US Department of Energy under contract DE-AC05-00OR22725.

Footnotes

↵¹To whom correspondence should be addressed. E-mail: morel{at}princeton.edu.

Author contributions: J.K.S. and F.M.M.M. designed research; J.K.S., S.S.R., and W.Z. performed research; J.K.S., S.S.R., L.L., B.G., and F.M.M.M. analyzed data; and J.K.S. and F.M.M.M. wrote the paper.
The authors declare no conflict of interest.
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2. Gilmour C,
3. Heyes A,
4. Mason RP,
5. Miller C
(2003) in Biogeochemistry of Environmentally Important Trace Elements, ACS Symposium Series, no. 835, Geochemical and biological controls over methylmercury production and degradation in aquatic ecosystems, eds Chai Y, Braids OC (American Chemical Society, Washington, DC), pp 262–297.
↵
1. Muyzer G,
2. Stams AJM
(2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6:441–454.
OpenUrl CrossRef PubMed
↵
1. Naz N,
2. Young HK,
3. Ahmed N,
4. Gadd GM
(2005) Cadmium accumulation and DNA homology with metal resistance genes in sulfate-reducing bacteria. Appl Environ Microbiol 71:4610–4618.
OpenUrl Abstract/FREE Full Text
↵
1. Henry EA
(1992) The role of sulfate-reducing bacteria in environmental mercury methylation. PhD dissertation (Harvard Univ, Cambridge, MA).
↵
1. Schaefer JK,
2. Morel FMM
(2009) High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens. Nat Geosci 2:123–126.
OpenUrl CrossRef
↵
1. Warthmann R,
2. Cypionka H
(1990) Sulfate transport in Desulfobulbus propionicus and Desulfococcus multivorans. Arch Microbiol 154:144–149.
OpenUrl CrossRef
↵
1. Nies DH
(1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51:730–750.
OpenUrl CrossRef PubMed
↵
1. Waldron KJ,
2. Robinson NJ
(2009) How do bacterial cells ensure that metalloproteins get the correct metal? Nat Rev Microbiol 7:25–35.
OpenUrl CrossRef PubMed
↵
1. Bridges CC,
2. Zalups RK
(2005) Molecular and ionic mimicry and the transport of toxic metals. Toxicol Appl Pharmacol 204:274–308.
OpenUrl CrossRef PubMed
↵
1. Berger EA,
2. Heppel LA
(1972) A binding protein involved in the transport of cystine and diaminopimelic acid in Escherichia coli. J Biol Chem 247:7684–7694.
OpenUrl Abstract/FREE Full Text
↵
1. Jonas RB,
2. Gilmour CC,
3. Stoner DL,
4. Weir MM,
5. Tuttle JH
(1984) Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities. Appl Environ Microbiol 47:1005–1011.
OpenUrl Abstract/FREE Full Text
↵
1. Silver S,
2. Phung LT
(1996) Bacterial heavy metal resistance: New surprises. Annu Rev Microbiol 50:753–789.
OpenUrl CrossRef PubMed
↵
1. Barkay T,
2. Miller SM,
3. Summers AO
(2003) Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 27:355–384.
OpenUrl CrossRef PubMed
↵
1. Landner L
(1971) Biochemical model for the biological methylation of mercury suggested from methylation studies in vivo with Neurospora crassa. Nature 230:452–454.
OpenUrl CrossRef PubMed
↵
1. Baldi F,
2. Pepi M,
3. Filippelli M
(1993) Methylmercury resistance in Desulfovibrio desulfuricans strains in relation to methylmercury degradation. Appl Environ Microbiol 59:2479–2485.
OpenUrl Abstract/FREE Full Text
↵
1. Barkay T,
2. Kritee K,
3. Boyd E,
4. Geesey G
(2010) A thermophilic bacterial origin and subsequent constraints by redox, light and salinity on the evolution of the microbial mercuric reductase. Environ Microbiol 12:2904–2917.
OpenUrl PubMed
↵
1. Ferguson AD,
2. Deisenhofer J
(2004) Metal import through microbial membranes. Cell 116:15–24.
OpenUrl CrossRef PubMed
↵
1. Barkay T,
2. Gillman M,
3. Turner RR
(1997) Effects of dissolved organic carbon and salinity on bioavailability of mercury. Appl Environ Microbiol 63:4267–4271.
OpenUrl Abstract/FREE Full Text
↵
1. Golding GR,
2. et al.
(2002) Evidence for facilitated uptake of Hg(II) by Vibrio anguillarum and Escherichia coli under anaerobic and aerobic conditions. Limnol Oceanogr 47:967–975.
OpenUrl CrossRef
↵
1. Ekstrom EB,
2. Morel FMM,
3. Benoit JM
(2003) Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria. Appl Environ Microbiol 69:5414–5422.
OpenUrl Abstract/FREE Full Text
↵
1. Balch WE,
2. Fox GE,
3. Magrum LJ,
4. Woese CR,
5. Wolfe RS
(1979) Methanogens: Reevaluation of a unique biological group. Microbiol Rev 43:260–296.
OpenUrl FREE Full Text
↵
1. Widdel F,
2. Pfennig N
(1981) Sporulation and further nutritional characteristics of Desulfotomaculum acetoxidans. Arch Microbiol 129:401–402.
OpenUrl CrossRef PubMed
↵
1. Bloom NS,
2. Fitzgerald WF
(1988) Determination of volatile mercury species at the picogram level by low-temperature gas chromatography with cold-vapour atomic fluorescence detection. Anal Chim Acta 208:151–161.
OpenUrl CrossRef
↵
1. Horvat M,
2. Liang L,
3. Bloom NS
(1993) Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples: Part II. Water. Anal Chim Acta 282:153–168.
OpenUrl CrossRef
↵
1. Olson ML,
2. Cleckner LB,
3. Hurley JP,
4. Krabbenhoft DP,
5. Heelan TW
(1997) Resolution of matrix effects on analysis of total and methyl mercury in aqueous samples from the Florida Everglades. Fresenius J Anal Chem 358:392–396.
OpenUrl CrossRef

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Proceedings of the National Academy of Sciences: 108 (21)

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Article Classifications

Biological Sciences
Environmental Sciences

[1] ↵
Clarkson TW
(1997) The toxicology of mercury. Crit Rev Clin Lab Sci 34:369–403.
OpenUrl PubMed

[2] Clarkson TW

[3] ↵
Clarkson TW,
Magos L
(2006) The toxicology of mercury and its chemical compounds. Crit Rev Toxicol 36:609–662.
OpenUrl CrossRef PubMed

[4] Clarkson TW,

[5] Magos L

[6] ↵
Compeau GC,
Bartha R
(1985) Sulfate-reducing bacteria: Principal methylators of mercury in anoxic estuarine sediment. Appl Environ Microbiol 50:498–502.
OpenUrl Abstract/FREE Full Text

[7] Compeau GC,

[8] Bartha R

[9] ↵
Fleming EJ,
Mack EE,
Green PG,
Nelson DC
(2006) Mercury methylation from unexpected sources: Molybdate-inhibited freshwater sediments and an iron-reducing bacterium. Appl Environ Microbiol 72:457–464.
OpenUrl Abstract/FREE Full Text

[10] Fleming EJ,

[11] Mack EE,

[12] Green PG,

[13] Nelson DC

[14] ↵
Gilmour CC,
Henry EA,
Mitchell R
(1992) Sulfate stimulation of mercury methylation in freshwater sediments. Environ Sci Technol 26:2281–2287.
OpenUrl

[15] Gilmour CC,

[16] Henry EA,

[17] Mitchell R

[18] ↵
Kerin EJ,
et al.
(2006) Mercury methylation by dissimilatory iron-reducing bacteria. Appl Environ Microbiol 72:7919–7921.
OpenUrl Abstract/FREE Full Text

[19] Kerin EJ,

[20] et al.

[21] ↵
Choi S-C,
Chase T Jr.,
Bartha R
(1994) Enzymatic catalysis of mercury methylation by Desulfovibrio desulfuricans LS. Appl Environ Microbiol 60:1342–1346.
OpenUrl Abstract/FREE Full Text

[22] Choi S-C,

[23] Chase T Jr.,

[24] Bartha R

[25] ↵
Wood JM,
Kennedy FS,
Rosen CG
(1968) Synthesis of methyl-mercury compounds by extracts of a methanogenic bacterium. Nature 220:173–174.
OpenUrl CrossRef PubMed

[26] Wood JM,

[27] Kennedy FS,

[28] Rosen CG

[29] ↵
Chai Y,
Braids OC
Benoit J,
Gilmour C,
Heyes A,
Mason RP,
Miller C
(2003) in Biogeochemistry of Environmentally Important Trace Elements, ACS Symposium Series, no. 835, Geochemical and biological controls over methylmercury production and degradation in aquatic ecosystems, eds Chai Y, Braids OC (American Chemical Society, Washington, DC), pp 262–297.

[30] Chai Y,

[31] Braids OC

[32] Benoit J,

[33] Gilmour C,

[34] Heyes A,

[35] Mason RP,

[36] Miller C

[37] ↵
Muyzer G,
Stams AJM
(2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6:441–454.
OpenUrl CrossRef PubMed

[38] Muyzer G,

[39] Stams AJM

[40] ↵
Naz N,
Young HK,
Ahmed N,
Gadd GM
(2005) Cadmium accumulation and DNA homology with metal resistance genes in sulfate-reducing bacteria. Appl Environ Microbiol 71:4610–4618.
OpenUrl Abstract/FREE Full Text

[41] Naz N,

[42] Young HK,

[43] Ahmed N,

[44] Gadd GM

[45] ↵
Henry EA
(1992) The role of sulfate-reducing bacteria in environmental mercury methylation. PhD dissertation (Harvard Univ, Cambridge, MA).

[46] Henry EA

[47] ↵
Schaefer JK,
Morel FMM
(2009) High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens. Nat Geosci 2:123–126.
OpenUrl CrossRef

[48] Schaefer JK,

[49] Morel FMM

[50] ↵
Warthmann R,
Cypionka H
(1990) Sulfate transport in Desulfobulbus propionicus and Desulfococcus multivorans. Arch Microbiol 154:144–149.
OpenUrl CrossRef

[51] Warthmann R,

[52] Cypionka H

[53] ↵
Nies DH
(1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51:730–750.
OpenUrl CrossRef PubMed

[54] Nies DH

[55] ↵
Waldron KJ,
Robinson NJ
(2009) How do bacterial cells ensure that metalloproteins get the correct metal? Nat Rev Microbiol 7:25–35.
OpenUrl CrossRef PubMed

[56] Waldron KJ,

[57] Robinson NJ

[58] ↵
Bridges CC,
Zalups RK
(2005) Molecular and ionic mimicry and the transport of toxic metals. Toxicol Appl Pharmacol 204:274–308.
OpenUrl CrossRef PubMed

[59] Bridges CC,

[60] Zalups RK

[61] ↵
Berger EA,
Heppel LA
(1972) A binding protein involved in the transport of cystine and diaminopimelic acid in Escherichia coli. J Biol Chem 247:7684–7694.
OpenUrl Abstract/FREE Full Text

[62] Berger EA,

[63] Heppel LA

[64] ↵
Jonas RB,
Gilmour CC,
Stoner DL,
Weir MM,
Tuttle JH
(1984) Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities. Appl Environ Microbiol 47:1005–1011.
OpenUrl Abstract/FREE Full Text

[65] Jonas RB,

[66] Gilmour CC,

[67] Stoner DL,

[68] Weir MM,

[69] Tuttle JH

[70] ↵
Silver S,
Phung LT
(1996) Bacterial heavy metal resistance: New surprises. Annu Rev Microbiol 50:753–789.
OpenUrl CrossRef PubMed

[71] Silver S,

[72] Phung LT

[73] ↵
Barkay T,
Miller SM,
Summers AO
(2003) Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 27:355–384.
OpenUrl CrossRef PubMed

[74] Barkay T,

[75] Miller SM,

[76] Summers AO

[77] ↵
Landner L
(1971) Biochemical model for the biological methylation of mercury suggested from methylation studies in vivo with Neurospora crassa. Nature 230:452–454.
OpenUrl CrossRef PubMed

[78] Landner L

[79] ↵
Baldi F,
Pepi M,
Filippelli M
(1993) Methylmercury resistance in Desulfovibrio desulfuricans strains in relation to methylmercury degradation. Appl Environ Microbiol 59:2479–2485.
OpenUrl Abstract/FREE Full Text

[80] Baldi F,

[81] Pepi M,

[82] Filippelli M

[83] ↵
Barkay T,
Kritee K,
Boyd E,
Geesey G
(2010) A thermophilic bacterial origin and subsequent constraints by redox, light and salinity on the evolution of the microbial mercuric reductase. Environ Microbiol 12:2904–2917.
OpenUrl PubMed

[84] Barkay T,

[85] Kritee K,

[86] Boyd E,

[87] Geesey G

[88] ↵
Ferguson AD,
Deisenhofer J
(2004) Metal import through microbial membranes. Cell 116:15–24.
OpenUrl CrossRef PubMed

[89] Ferguson AD,

[90] Deisenhofer J

[91] ↵
Barkay T,
Gillman M,
Turner RR
(1997) Effects of dissolved organic carbon and salinity on bioavailability of mercury. Appl Environ Microbiol 63:4267–4271.
OpenUrl Abstract/FREE Full Text

[92] Barkay T,

[93] Gillman M,

[94] Turner RR

[95] ↵
Golding GR,
et al.
(2002) Evidence for facilitated uptake of Hg(II) by Vibrio anguillarum and Escherichia coli under anaerobic and aerobic conditions. Limnol Oceanogr 47:967–975.
OpenUrl CrossRef

[96] Golding GR,

[97] et al.

[98] ↵
Ekstrom EB,
Morel FMM,
Benoit JM
(2003) Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria. Appl Environ Microbiol 69:5414–5422.
OpenUrl Abstract/FREE Full Text

[99] Ekstrom EB,

[100] Morel FMM,

[101] Benoit JM

[102] ↵
Balch WE,
Fox GE,
Magrum LJ,
Woese CR,
Wolfe RS
(1979) Methanogens: Reevaluation of a unique biological group. Microbiol Rev 43:260–296.
OpenUrl FREE Full Text

[103] Balch WE,

[104] Fox GE,

[105] Magrum LJ,

[106] Woese CR,

[107] Wolfe RS

[108] ↵
Widdel F,
Pfennig N
(1981) Sporulation and further nutritional characteristics of Desulfotomaculum acetoxidans. Arch Microbiol 129:401–402.
OpenUrl CrossRef PubMed

[109] Widdel F,

[110] Pfennig N

[111] ↵
Bloom NS,
Fitzgerald WF
(1988) Determination of volatile mercury species at the picogram level by low-temperature gas chromatography with cold-vapour atomic fluorescence detection. Anal Chim Acta 208:151–161.
OpenUrl CrossRef

[112] Bloom NS,

[113] Fitzgerald WF

[114] ↵
Horvat M,
Liang L,
Bloom NS
(1993) Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples: Part II. Water. Anal Chim Acta 282:153–168.
OpenUrl CrossRef

[115] Horvat M,

[116] Liang L,

[117] Bloom NS

[118] ↵
Olson ML,
Cleckner LB,
Hurley JP,
Krabbenhoft DP,
Heelan TW
(1997) Resolution of matrix effects on analysis of total and methyl mercury in aqueous samples from the Florida Everglades. Fresenius J Anal Chem 358:392–396.
OpenUrl CrossRef

[119] Olson ML,

[120] Cleckner LB,

[121] Hurley JP,

[122] Krabbenhoft DP,

[123] Heelan TW

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New Research In

Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

Abstract

Results

Partitioning of Hg to Cells.

Uptake and Methylation of Hg(II).

Active Uptake of Hg(II).

Substrate Specificity of Hg(II) Uptake.

Discussion

Materials and Methods

Bacterial Cultures and Media.

Hg Sorption on Cells.

Hg Uptake and Methylation Experiments.

Hg and MeHg Analyses.

Solutions and Chemicals.

Speciation Calculations.

Acknowledgments

Footnotes

References

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Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

Abstract

Results

Partitioning of Hg to Cells.

Uptake and Methylation of Hg(II).

Active Uptake of Hg(II).

Substrate Specificity of Hg(II) Uptake.

Discussion

Materials and Methods

Bacterial Cultures and Media.

Hg Sorption on Cells.

Hg Uptake and Methylation Experiments.

Hg and MeHg Analyses.

Solutions and Chemicals.

Speciation Calculations.

Acknowledgments

Footnotes

References

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