Microbial Biosynthesis of Thiol Compounds: Implications for

Article Cite This: Environ. Sci. Technol. 2019, 53, 8187−8196

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Microbial Biosynthesis of Thiol Compounds: Implications for Speciation, Cellular Uptake, and Methylation of Hg(II) Gbotemi A. Adediran,† Van Liem-Nguyen,†,‡ Yu Song,§ Jeffra K. Schaefer,∥ Ulf Skyllberg,§ and Erik Björn*,† †

Department of Chemistry, Umeå University, SE- 90187 Umeå, Sweden School of Science and Technology, Ö rebro University, SE-70182 Ö rebro, Sweden § Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden ∥ Department of Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, New Jersey 08901, United States

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‡

S Supporting Information *

ABSTRACT: Cellular uptake of inorganic divalent mercury (Hg(II)) is a key step in microbial formation of neurotoxic methylmercury (MeHg), but the mechanisms remain largely unidentified. We show that the iron reducing bacterium Geobacter sulfurreducens produces and exports appreciable amounts of low molecular mass thiol (LMM-RSH) compounds reaching concentrations of about 100 nM in the assay medium. These compounds largely control the chemical speciation and bioavailability of Hg(II) by the formation of Hg(LMM-RS)2 complexes (primarily with cysteine) in assays without added thiols. By characterizing these effects, we show that the thermodynamic stability of Hg(II)-complexes is a principal controlling factor for Hg(II) methylation by this bacterium such that less stable complexes with mixed ligation involving LMM-RSH, OH−, and Cl− are methylated at higher rates than the more stable Hg(LMM-RS)2 complexes. The Hg(II) methylation rate across different Hg(LMM-RS)2 compounds is also influenced by the chemical structure of the complexes. In contrast to the current perception of microbial uptake of Hg, our results adhere to generalized theories for metal biouptake based on metal complexation with cell surface ligands and refine the mechanistic understanding of Hg(II) availability for microbial methylation.

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amino acid or thiol transporters6,15 but possibly heavy metal transporters targeting, e.g., zinc.12 Experimental additions of ∼1−10 μM concentrations of specific LMM-RSH compounds, in particular cysteine (Cys), have been reported to enhance rates of bacterial uptake and methylation of Hg(II) above controls.6,14−20 Some studies have demonstrated a more complex pattern with either enhancements or suppressions depending on bacterial cell line, incubation time, and thiol concentration.19,21 It has generally been assumed that micromolar additions of thiols cause a shift in the chemical speciation of Hg(II)(aq) from HgCl2 and Hg(OH)2, prevailing in the control systems, to a dominance of Hg(LMM-RS)2 complexes, and that the enhanced methylation occurs despite a higher thermodynamic stability of the Hg(LMM-RS)2 complexes. Such a conclusion could be supported if Hg(II) is internalized as intact Hg(LMM-RS)2 complexes via specific transport systems targeting the LMM-RSH compounds, but it is not coherent

INTRODUCTION

Methylation of inorganic mercury (Hg(II)) by phylogenetically diverse microorganisms is a key step in the production and bioaccumulation of neurotoxic methylmercury (MeHg).1,2 The methylation occurs intracellularly3−7 via the activity of the corrinoid-binding protein HgcA which transfers a methyl group to Hg(II) and the ferredoxin HgcB which reduces the cobalamin in HgcA for a new round of synthesis.7,8 The ability to produce MeHg is constitutive and not responsive to Hg(II)5 and does not correlate with hgcA and hgcB expression levels.9 Instead, cellular uptake of Hg(II) and the biotic and abiotic factors controlling this process appear to be key steps for the rate and thus amount of MeHg formed.1,5,6 The molecular mechanisms of uptake, however, remain unclear. While uptake by passive diffusion is plausible for some types of Hg(II) species,10,11 recent studies emphasize metabolically active transport of Hg(II) across membranes.6,12,13 It was also shown that the presence of some low molecular mass thiol (LMM-RSH) compounds, in particular cysteine (Cys), enhances Hg(II) uptake and methylation, while other LMMRSH compounds can suppress these processes.6,14 The mechanisms of Hg(II) uptake does not seem to involve © 2019 American Chemical Society

Received: Revised: Accepted: Published: 8187

March 11, 2019 June 19, 2019 June 20, 2019 June 20, 2019 DOI: 10.1021/acs.est.9b01502 Environ. Sci. Technol. 2019, 53, 8187−8196

Article

Environmental Science & Technology

centrifugation under anaerobic condition, washed at least three times, and resuspended in an assay buffer. For all determinations of LMM-RSH compounds and Hg(II) methylation experiments, assays were inoculated with 1 mL aliquots of washed cell suspensions to a final assay with ∼108 cells ml−1, a cell density typical of pure culture methylation experiments with G. sulfurreducens.6,12,14 The basic conditions of the Hg(II) methylation assay followed the procedure by Schaefer et al.6 In all experiments, pH was 6.85−6.90 and the assay medium contained 1.6 mM Cl−. All treatments were replicated at least 3 times. The experiment nos. 1−4 were conducted under different chemical formulations. (1) Extracellular and cell-associated concentrations of LMM-RSH compounds were determined after 6 and 48 h incubation with G. sulfurreducens in assay medium. The LMM-RSH compound cysteine (Cys) was further quantified in assay samples with and without addition of 1 mM fumarate to live bacteria and in assays with heat-killed (at 70 °C for 2.5 h) bacteria. (2) Methylation of Hg(II) was studied in 6 h incubation assays over a broad range of Hg(II) additions (5−200 nM in the form of HgCl2 yielding 1.5−72 nM of dissolved Hg(II) in the medium and the remaining fraction being cell associated Hg(II) or produced MeHg) to stepwise complex and saturate the LMM-RSH compounds synthesized by G. sulfurreducens. This approach allowed us to relate Hg(II) methylation to thermodynamic binding stability by comparing methylation rate in the presence of the stronger Hg(LMM-RS)2 complexes to those with lower thermodynamic stability involving mixed coordination of Hg(II) to RS−, RO−, OH−, and Cl− functional groups. (3) The methylation rate of 20−25 nM Hg(II) was determined at moderate additions (50−500 nM) of the reduced sulfur compounds cysteine (Cys), cysteamine (CystN), N-acetylcysteine (NACCys), and sulfide (HS−). Hg(II) was equilibrated with each of the reduced sulfur compounds for at least 2 h in the assay medium before addition of bacteria and Hg(II) methylation was assayed 6 h after the addition of cells. (4) The effect of a large excess of Cys on Hg(II) methylation was determined as previously described except that 5 nM of Hg(II) was equilibrated with 10 μM Cys for 2 h before bacteria addition. The Hg(II) methylation was monitored hourly over a 4 h incubation period. Measurements and Modeling of LMM-RSH Compounds and Hg Species in G. sulfurreducens Assays. The concentrations of specific LMM-RSH compounds were determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) after derivatization with 4-(hydroxymercuri) benzoate (PHMB) and online preconcentration by solid-phase extraction as described by Liem-Nguyen et al.35 Measurements were done without prereduction of organic disulfides (RSSR) and thus only the thiol form (RSH) was quantified for each LMM-RSH compound. The compounds were determined in extracellular and cellular fractions of the G. sulfurreducens assay culture. Extracellular concentrations of LMM-RSH compounds were determined in the assay culture samples after filtration through 0.2 μm syringe filters, and cellular concentrations were determined by subtracting extracellular concentrations from whole assay concentrations that were determined after subjecting unfiltered assay culture to physical cell lysing by sonication.36 Sulfide was measured in the assay medium by absorption spectrometry after preconcentration by purging 100

with generalized and established theoretical frameworks for biouptake of metals. These generalized concepts (including the free ion activity model,22,23 the biotic ligand model24−26 and surface complexation models27−29) rely on a chemical bond formed between the metal-ion and a biotic ligand at the cell surface. They therefore postulate a higher bioavailability for thermodynamically less stable metal complexes.24,30,31 At further increased Cys concentrations of >100 μM, Hg(II) uptake and methylation are suppressed.14,19 This has been explained in terms of a shift in the composition of Hg(II)-Cys speciation from a stoichiometry of Hg(Cys)2 toward three- and four-coordinated Hg(Cys)3− and Hg(Cys)42− complexes at higher Cys concentrations, with a presumed lower bioavailability. Following additions of 1000 μM of Cys, formation of αHgS(s) and β-HgS(s) was observed at cell surfaces of G. sulfurreducens,32 which could be another mechanism contributing to decreased Hg(II) availability at very high Cys concentrations. Furthermore, the structural geometry of Hg(II)(aq) complexes has been recognized as important for uptake and methylation in G. sulfurreducens and D. desulfuricans.6 It was found that uptake and methylation by G. sulfurreducens was facilitated by Hg(II) complexes formed with small LMM-RSH ligands lacking branching close to the thiol group, as compared to ligands with more “bulky”, branching structure and/or having an overall larger size which may partially involve in a chelation of Hg(II). Thiols may also be metabolically produced and exported by microorganisms,33 yet specific LMM-RSH compounds associated with Hg(II) methylating bacteria strains have not been identified and quantified. The chemical speciation of Hg(II) and MeHg in the presence of metabolically active bacteria is therefore uncertain, in particular for systems without added ligands. Addition of artificially high concentrations of ligands in excess (as discussed above) has been one approach to circumvent the potential impact from biologically in situ produced ligands. Both these circumstances introduce uncertainties in the interpretation of the role of chemical speciation for cellular uptake of Hg(II). In this study, we demonstrate that G. sulfurreducens produces LMM-RSH compounds in vivo and exports them to yield concentrations exceeding 100 nM in the medium, sufficient to control Hg(II) speciation, cellular uptake, and methylation in incubation assays in absence of externally added ligands. By quantifying and taking into account these microbially produced LMMRSH compounds, we carried out experiments at environmentally relevant concentrations of such compounds in the nanomolar range and generated refined speciation models for Hg(II) in assay media. This approach leads to new important insights on the role of chemical speciation of Hg(II), and the function of Cys and other LMM-RSH compounds, for cellular uptake and methylation of Hg(II).

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MATERIALS AND METHODS Bacteria Culture Assay Conditions. Geobacter sulfurreducens PCA (ATCC 51573)34 purchased from DSMZ was grown under N2 atmosphere at 28 °C and pH 6.8 in a defined Hg(II) free growth medium. An assay buffer typical of most Hg(II) methylation experiment with G. sulfurreducens was adopted in this study.14 The assay buffer, GsAB, 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−1 resazurin. Cells were harvested from the growth medium at the midexponential phase by 8188

DOI: 10.1021/acs.est.9b01502 Environ. Sci. Technol. 2019, 53, 8187−8196


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RESULTS Metabolically Active Synthesis and Cellular Export of LMM-RSH compounds by G. sulfurreducens. Eight different LMM-RSH compounds were detected within cells (Figure S1) and in the assay medium (Table 1, Figure S1) in

mL of assay medium into 5 mL of 0.25 N NaOH trapping solution.37,38 The amount of MeHg produced during incubation was determined by thermal desorption gas chromatography− ICPMS after sample digestion with 25% NaOH for 24 h (adapted from Carrasco and Vassileva39) followed by adjustment of pH to ∼4.5 using a 10 M HCl and 2 M CH3COONH4 buffer and subsequent derivatization with NaB(C2H5)4 and purge and trap on Tenax adsorbent.40 Total Hg concentration was measured with cold vapor-ICPMS after 24 h digestion with BrCl following the EPA 1631E method.41 Total Hg and MeHg determined in unfiltered assays after incubation is denoted Hg(T) and MeHg(T), respectively, and apparent dissolved Hg and MeHg determined in 0.2 μm filter-passing solution is denoted Hg(aq) and MeHg(aq), respectively. Total and aqueous inorganic divalent Hg, Hg(II), were calculated as Hg(II)(T) = Hg(T) − MeHg(T) and Hg(II)(aq) = Hg(T)(aq) − MeHg(aq), respectively. Cell-associated Hg(II) and MeHg were calculated as Hg(II)(cell) = Hg(II)(T) − Hg(II)(aq), and MeHg(cell) = MeHg(T) − MeHg(aq), respectively. We adopted the thermodynamic model developed by LiemNguyen et al.42 to calculate the chemical speciation of Hg(II)(aq) and MeHg(aq) in the G. sulfurreducens assay medium using the software WinSGW.43 The complete speciation model is given in Table S1, and the reactions most central for the discussion of results are given below.

Table 1. Average (n = 4; ± Standard Error) Total Concentrations of Low Molecular Mass Thiol Compounds (LMM-RSH) Detected in GsAB Assay Buffer with Metabolically Active G. sulfurreducens (Cell Density ∼108 cell ml−1) at 6 and 48 h after Incubationa thiol concentrations (nM) 6h cysteine (Cys) penicillamine (Pen) N-acetylcysteine (NacCys) glutamylcysteine (GluCys) cysteamine (CystN) monothioglycerol (Glyc) mercaptoacetic acid (Mac) homocysteine (HCys)

(2)

Hg 2 + + Cl− + (LMMRS−)n = HgCl(LMMRS)1 + n ; (3)

Hg 2 + + (LMMRORS−)n = Hg(LMMRORS)2 + n ; log K = 25.0 − 26.5 (ref 46) Hg 2 + + 2OH− = Hg(OH)2 ;

(4)

log K = 21.2 (ref 47) (5)

Hg 2 + + OH− + Cl− = HgOHCl; log K = 18.0 (ref 47) Hg 2 + + 2Cl− = HgCl2;

(6)

log K = 13.8 (ref 47)

4.2A, b 1.7B, b 1.1DE, b 0.60C, a 0.91C, a 0.48CD, a 0.26E, a 0.36E, a

48 h 63 18 12 7.8 8.1 6.0 3.6 2.2

± ± ± ± ± ± ± ±

2.5A, a 1.9B, a 1.2C, a 1.2DE, a 0.55D, a 0.63E, a 0.45F, a 0.38F, a

the presence of metabolically active G. sulfurreducens but without external addition of thiols. The detected thiol compounds were cysteine (Cys), penicillamine (Pen), Nacetylcysteine (NACCys), glutamylcysteine (GluCys), cysteamine (CystN), monothioglycerol (Glyc), mercaptoacetic acid (Mac), and homocysteine (HCys). The total concentration of the LMM-RSH compounds reached up to ∼120 nM in the assay medium (Table 1) at 1.1 × 108 cells ml−1. The cells were harvested at the midexponential phase, washed and transferred to the experimental assay systems such that there were no external thiols present in the medium at t = 0 of the assay. Time-resolved measurements showed that the initial production and export of LMM-RSH compounds was fast, reaching a concentration of >30 nM already after 1 h of incubation (Figure S2). The concentration levels were reproducible for within-day batches (4−26% relative standard deviation) and fairly consistent (within a factor of 2) for between-day batches. The extracellular and/or cellular concentrations changed significantly from 6 to 48 h of incubation for Cys, Pen, NACCys, and GluCys but appeared more constant for CystN, Glyc, Mac, and HCys (Table 1, Figure S1). Cysteine was consistently present at the highest concentrations in the presence of live bacteria with fumarate added (Figure S3). In the absence of fumarate, and in heat-killed cells, the Cys concentration was significantly lower and less variable over time as compared to assays with live cells. Confocal laser scanning imaging of live (green) and membrane-ruptured/ dead cells (red) incubated for 48 h, either without Hg(II) or in the presence of 100 nM Hg(II) addition, showed that over 98% of the cells were alive and without ruptured membrane (Figure S4). We also analyzed GsAB assay without bacteria inoculation, and there was no detectable formation of LMMRSH compounds from degradation of GsAB assay constituents. Together these results demonstrate that Cys (and likely

Hg 2 + + OH− + (LMMRS−)n = HgOH(LMMRS)1 + n ;

log K = 28.5 (ref 45)

± ± ± ± ± ± ± ±

Different capital letters indicate significant (p < 0.05 ANOVA followed by Fisher’s Least Significance Difference post hoc test) differences among the detected thiol compounds at 6 h incubation and among the detected thiol compounds at 48 h incubation. Different lowercase letters indicate significant (p < 0.05) differences between 6 and 48 h of incubation for each individual thiol compound.

(1)

log K = 32.2 (ref 45)

53 16 4.2 7.6 7.0 6.2 3.5 2.1

a

Hg 2 + + 2(LMMRS−)n = Hg(LMMRS)2 2 + 2n ; log K = 36.9 − 40.9 (ref 44)

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The ranges given for log K values of reactions 1 and 4 represent the intervals for the LMM−RSH compounds cysteine (Cys), penicillamine (Pen), N-acetylcysteine (NACCys), glutamylcysteine (GluCys), cysteamine (CystN), monothioglycerol (Glyc), mercaptoacetic acid (Mac), and homocysteine (HCys). The charge n varies for different (LMM−RS−)n compounds depending on the presence of carboxyl and amino functional groups in the molecule and is for simplicity omitted in the text. Further details on the speciation modeling considerations are given in the SI Text 1. 8189


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Figure 1. Average (n = 4; ± standard error) absolute and percentage concentrations of Hg(II) species in the assay medium and total MeHg in the assay after 6 h incubation of G. sulfurreducens plotted against measured total aqueous concentrations of Hg(II) (nM) in the assay medium. (a) Summed concentrations of Hg(LMM-RS)2 species and of other Hg(II)(aq) species, (b) summed concentrations of Hg(II)(aq) complexes formed with two LMM-RSH molecules (∑Hg(LMM-RS)2), with one LMM-RSH molecule (∑Hg(LMM-RORS)), with a mixture of OH−, Cl−, and LMM-RSH molecules (ΣHgOHnCl1−n(LMM-RS)), and with a mixture of OH− and Cl− (ΣHgOHnCl2−n), (c) percentage distribution of the Hg(II)(aq) species in (b), and (d) measured total MeHg concentration (nM).

unidentified outer membrane protein for both periplasmic and outer membrane cytochrome assembly.51,52 Chemical Speciation of Hg(II)(aq) in Assay Medium with Active G. sulfurreducens without External Addition of Thiols. In the absence of aqueous phase RSH ligands, chemical speciation modeling predicts that HgCl2(aq) or Hg(OH)2(aq) are the dominant Hg(II)(aq) complexes in typical assay media used for bacteria incubation experiments.6,12,14,15,19 Rates of cellular Hg(II) uptake, methylation, and reduction in such control/reference systems without externally added RSH compounds have therefore been attributed to this expected chemical composition. In sharp contrast, results from our control systems show that the metabolically active synthesis and cellular export of LMM-RSH compounds by G. sulfurreducens controlled the chemical speciation of Hg(II)(aq) for concentrations at least up to 70 nM Hg(II) (aq) in assay media in the absence of added ligands

also Pen, NACCys, and GluCys) encountered in the assay media were biosynthesized in vivo by G. sulfurreducens through an active metabolism and were not caused by dead disrupted cells. Further, the production and cellular export of LMM-RSH compounds were neither induced nor suppressed by total Hg(II) concentrations up to at least 130 nM (Figure S5). Complete pathways for cysteine (e.g., cysE, cysK/cysM) and homocysteine biosynthesis (e.g., metX, metY)48 were observed in the G. sulfurreducens genome (NCBI Accession NC_002939). Other organisms such as Escherichia coli have been shown to transport cysteine (and other LMM-RSH) from the cytosol to the periplasm via an ATP-driven transporter (CydDC) to supply cysteine necessary for full maturation of periplasmic cytochromes.49,50 Homologues to CydDC (gene loci GSU1215−1216) were identified in the G. sulfurreducens genome, suggesting possible involvement of CydDC and an 8190


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determine kmeth for the different types of Hg(II)(aq) species, we fitted a second-order rate model, containing five methylation rate constants, to this entire experimental data set (only excluding data from experiments with sulfide and 200 and 500 nM Cys additions, as discussed further below). The model included bacteria cell density, (depicted as [cell]), and the concentrations of the individual Hg(II) species Hg(Cys)2, Hg(CystN)2, Hg(NACCys)2, the sum of the mixed-ligation species ΣHg(LMM-RORS) + ΣHgOHnCl1−n(LMM-RS), and the sum of HgOHnCl2−n species:

(Figure 1a−c). Note that all speciation modeling in this work is based on measured Hg(aq) concentrations in the assay media at each experimental condition. Mercury-thiol complexes with 1:2 stoichiometry, i.e., Hg(LMM-RS)2, dominated the speciation of Hg(II)(aq) up to ∼30 nM Hg(II)(aq) with Hg(Cys)2 showing the highest concentrations, followed by Hg(Pen)2, Hg(NACCys)2, and Hg(CystN)2 (Figure S6). The Hg(II)(aq) concentration is typically kept lower than 30 nM in bacteria assay studies on Hg(II) methylation and reduction. When the Hg(II)(aq) concentration is increased into the range 30−100 nM, the speciation is dominated by Hg(II)(aq) complexes with a mixed ligation of the types HgOH(LMMRS), HgCl(LMM-RS), and by Hg-thiol complexes with a 1:1 stoichiometry, Hg(LMM-RORS). Only at conditions with Hg(II)(aq) concentrations ≥100 nM do complexes with OH− and Cl− (HgOHnCl2−n with n = 0, 1, or 2, i.e., Hg(OH)2 + HgCl2 + HgOHCl) dominate the chemical speciation of Hg(II)(aq). Thus, depending on the Hg(II)(aq) concentration, a G. sulfurreducens control assay may have a very variable chemical composition. For example, at 5, 30, and 60 nM of Hg(II)(aq) the complex Hg(Cys)2 constitutes about 80%, 25%, and close to 0% of the Hg(II)(aq) speciation, respectively, in our assay systems. Such large variability in Hg(II) chemical speciation may have substantial implications for the interpretation of experimental results and illustrates the importance of considering microbially produced LMM-RSH compounds. These findings suggest that for additions up to ∼30 nM Hg(II), the chemical speciation of Hg(II)(aq) in control systems of many previous studies with this bacterium likely have been dominated by the LMM thiol complex Hg(Cys)2 rather than by the anticipated inorganic complexes Hg(OH)2 or HgCl2. In this context, it is also important to note that sulfide, another ligand forming stable complexes with Hg, was not detected (LOD 5 nM) in the assay medium of our experiments with metabolically active G. sulfurreducens. Mercury Methylation Rate for the Hg(LMM-RS)2, Hg(LMM-RORS), HgOHnCl1−n(LMM-RS) and HgOHnCl2−n Species. Our experiments represent important advancements of Hg(II) methylation assay studies as they were established under conditions where (i) LMM-RSH compounds produced by the bacteria were taken into account, (ii) the chemical speciation of Hg(II)(aq) was established in systems with and without externally added ligands, and (iii) LMM-RSH additions were kept at nanomolar concentrations relevant for natural systems. In this way, we could (1) determine Hg(II) methylation rate constants (kmeth) for Hg(II) species having lower thermodynamic stabilities than Hg(LMM-RS)2 complexes. This was accomplished by adding Hg(II) (5−200 nM additions yielding 1.5−72 nM of dissolved Hg(II) in the medium and the remaining fraction being cell associated Hg(II) (Figure S7) or produced MeHg) to gradually “titrate” the biosynthesized LMM-RSH ligands by Hg(II) in a controlled manner (experiment no. 2, Figure 1), (2) determine kmeth for individual Hg(LMM-RS)2 species at minimum (50− 500 nM) additions of excess LMM-RSH ligands relevant to natural conditions (experiment no. 3, Figure 2), (3) disentangle how LMM-RSH compounds affect Hg(II) methylation by controlling Hg(II) speciation and by other mechanisms at large excess of LMM-RSH compounds (experiment nos. 3 and 4, Figures 2 and S8). The data set obtained by combining experiment nos. 2 and 3 spanned assay conditions being highly variable in Hg(II)(aq) speciation and methylation rates (Figures 1 and 2). To

Δ[MeHg tot] Δt × [cell]

= k meth1 × [Hg(NACCys)2 ] + k meth2 × [Hg(Cys)2 ] + k meth3 × [Hg(CystN)2 ] + k meth4 × ([Hg(LMM − RORS) + HgOH nCl1 − n(LMM − RS)]) + k meth5 × [HgOH nCl 2 − n]

(8)

Fitting eq 8 to data resulted in a significant multiple linear regression (MLR) model [F (5, 17) = 694, p < 0.001, R2 = 0.995] with Hg(II) species-specific kmeth values (Table 2) Table 2. Mercury(II) Species-Specific Second-Order Methylation Rate Constants (kmeth, × 10−13 L cell−1 h−1, ± standard error) Determined by Fitting a Multiple Linear Regression Model, Eq 8, to Experimental Dataa kmeth (× 10−13 L cell−1 h−1)

Hg(II) species Hg(NACCys)2 Hg(Cys)2 Hg(CystN)2 Hg(LMM-RORS), HgOHnCl1−n (LMM-RS) HgOHnCl2−n

0.49 3.4 7.3 8.8 45

± ± ± ± ±

2.1a 1.5a 5.0ab 0.7b 4c

a

Different lowercase letters indicate significant (p < 0.05 ANOVA followed by Fisher’s least significance difference post hoc test) differences.

increasing in the order Hg(NACCys)2 (relative rate constant normalized to the kmeth for Hg(NACCys)2, k′meth = 1) < Hg(Cys)2 (k′meth = 7) < Hg(CystN)2 (k′meth = 15) < Hg(LMM-RORS), HgOHnCl1−n(LMM-RS) (k′meth = 18) < HgOHnCl2−n (k′meth = 91). These results demonstrate that complexes having lower thermodynamic stabilities, such as the mixed ligation complexes Hg(LMM-RORS), HgOHnCl1−n (LMM-RS), and HgOHnCl2−n enhances the MeHg formation rate compared to Hg(Cys)2 and the other investigated Hg(LMM-RS)2 species having a higher thermodynamic stability. This finding agrees with the fundamental concept of generalized metal uptake models involving biotic ligands and partly challenges the current perspective1,6,14,53 on Hg bioavailability for methylating bacteria. Noteworthy, the amount of MeHg produced increased exponentially, not linearly, with added Hg(II) concentration (Figure 1d) due to the shift to complexes with mixed ligation and higher methylation rates. The MLR model was fitted to a data set generated from independent experiments done on separate cell batches prepared several months apart with variable Hg(II) additions and with bacterially produced LMM-RSH only and with externally added LMM-RSH compounds. The fit of the 8191


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higher rates observed at 200 and 500 nM Cys additions. Further, the speciation modeling showed that the increase in methylation at ≥200 nM Cys occurred despite no significant change in the speciation of Hg(II)(aq), which was largely dominated by Hg(Cys)2 in the controls (∼85% of Hg(II)(aq)) and in all experiments with Cys concentrations in the 50−500 nM range (>98% of Hg(II)(aq)) (Figure 2b). Even at the considerably higher Cys additions of 10 μM, Hg(Cys)2 was the only quantitatively important Hg(II) species and the added Hg(II) approached complete methylation (99% of added 5 nM Hg(II)) within 4 h of incubation (Figure S8). The results in Figures 2 and S8 show that the presence of ≥200 nM Cys enhances methylation by mechanisms not only related to the speciation of Hg(II)(aq) in the assay medium.

model to data obtained at all these conditions demonstrates a high robustness and consistency of the determined kmeth values. Although the species with the highest methylation rates influenced the MLR model results the most, the kmeth values calculated for Hg(NACCys)2, Hg(Cys)2, and Hg(CystN)2 from data obtained in the individual ligand addition experiments alone (Figure 2) were not significantly different (but had lower uncertainties) than the values determined from the complete data set. Using the same G. sulfurreducens strain as in our study, Schaefer and Morel14 determined Hg(II) methylation rates for Hg(Cys)2 at variable concentrations of Cys (10 nM to 1000 μM). At 10 and 100 nM Cys, i.e., comparable to the conditions in our study, the kmeth value for Hg(Cys)2 recalculated from the data reported by Schaefer and Morel agrees remarkably well with our result from eq 8, 2.4 ± 0.4 × 10−13, and 3.4 ± 1.5 × 10−13 L cell−1 h−1, respectively. This agreement further demonstrates a generally high consistency and robustness of MeHg formation in G. sulfurreducens assays. Mercury Methylation of individual Hg(LMM-RS)2 Species at Nanomolar Additions of LMM-RSH Compounds. Although the kmeth values were lower for Hg(LMMRS)2 complexes than for the weaker mixed-ligation Hg(II) complexes, the differences were relatively small when comparing Hg(Cys)2 or Hg(CystN)2 with Hg(LMM-RORS) or HgOHnCl1−n(LMM-RS). To further investigate the methylation of individual Hg(LMM-RS)2 species, and of HgS(s), we added 50−500 nM of Cys, CystN, NACCys, or HS− to G. sulfurreducens assays with 20−25 nM Hg(II). Our results show that differences in thermodynamic stability do not explain the considerable difference in methylation rates among the different types of Hg(LMM-RS)2 complexes (Figure 2), given that log K for reaction 1 is 37.5, 40.3, and 40.6 for Hg(Cys)2, Hg(CystN)2, and Hg(NACCys)2, respectively.44 The net charge of the Hg(LMM-RS)2 complex does not appear to be a controlling factor either since at pH 6.9 Hg(Cys)2 and Hg(NACCys)2 have a net charge of 0 (due to the presence of one −COO− and one −NH3+ or −NH2+− group in each LMM-RSH molecule), and Hg(CystN)2 has a net charge of +2. Instead, differences in the molecular geometry is a likely explanation for these differences in methylation rates of Hg(LMM-RS)2 complexes. The results in Figure 2 are in line with previous observations6 that an unsubstituted thiol carbon and overall small size of the LMMRSH compound favor a high methylation rate of the corresponding Hg(LMM-RS)2 complex. It is striking that moderate structural differences between Cys and NACCys (−NH3+ compared to − NH2+COCH3 at the β-carbon atom from the −SH group) consistently cause substantially higher rate of methylation for Hg(Cys)2 compared to Hg(NACCys)2 in our and in previous studies.6 Indeed, the kmeth of Hg(NACCys)2 was almost as low as the rate observed for βHgS(s) formed at additions of 50−500 nM HS− (Figure 2, Supporting Information). We further found the formation of MeHg to be independent of the concentration of added NACCys and CystN (within measurement uncertainty) in the investigated range (Figure 2). In contrast, there was a distinct Cys concentration-dependent methylation rate which increased by a factor of 3 at 200 and 500 nM Cys, compared to lower Cys concentrations. Importantly, the Hg(II) methylation rates calculated by eq 8 for all assays in Figures 1 and 2 could be fitted to measured rates only by using the lower methylation rate for Hg(Cys)2 observed at 50 and 100 nM Cys addition but not by using the

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DISCUSSION The molecular-level mechanisms for cellular uptake of Hg(II) by methylating microbes remain to be identified. Until now, proposed mechanisms have been based on cellular uptake of Hg(II) species with high thermodynamic stabilities via specialcase processes. These include passive diffusion of neutral HgS0(aq) and Hg(SH)20(aq) complexes,53 active uptake processes favoring the Hg(Cys)2 complex,14 and incompletely defined uptake processes of nanoparticulate forms of HgS.1 A high cellular uptake rate of thermodynamically stable metal complexes, e.g., Hg(LMM-RS) 2 , can be explained by mechanisms where the intact metal complex is internalized.54 Such mechanisms include passive diffusion of lipophilic complexes across the lipid bilayer and accidental, so-called “piggyback”, uptake of metal-anion complexes via anion transporters targeting the metal binding ligand.54 However, Hg(LMM-RS)2 complexes are hydrophilic and internalized via active transport, and their uptake is not mediated by amino acid or thiol anion transporters.6,15 Instead Hg(II) uptake has been suggested to occur via metal transporters in such organisms.6,12 Uptake of intact metal complexes via metal transporters has been reported but are exceptions.55 While it remains possible that Hg(LMM-RS)2 is taken up as intact complexes via metal transporters in bacteria, the large variation in structural size, charge, and chirality of LMM-RSH ligands proven to promote Hg(II) uptake and methylation makes this mechanism an unlikely candidate.6 The generalized theoretical framework for biouptake of metals is based on binding of the metal ion to a functional group at the cell surface.56,57 For metal complexes, such uptake mechanisms include ligand-exchange reactions, either via dissociation of the complex in solution (the free ion activity model22,23 and biotic ligand model24−26) or via the formation of transient ternary complexes with the metal (M) binding to one cell surface ligand (RS) and one aqueous ligand (L), L-MRs (surface complexation models27−29). Irrespective of the ligand-exchange mechanism, the net transfer rate (v) of the metal from solution to the cell surface is governed by the relative affinity of the metal to the aqueous ligand (KML) and to the cell surface ligand (Ks), and on the relative abundances of these ligands ([L] and [RS], respectively) in the system, i.e., v = Ks × [RS]/(KML × [L]).24,25,27,54 The bioavailability thus decreases with increased thermodynamic stability of the aqueous metal complex.24,30,31 Our results, demonstrating a higher kmeth for the less thermodynamically stable mixed ligation Hg(II) complexes than for the Hg(LMM-RS) 2 complexes, are in agreement with this framework, suggesting cellular uptake by G. sulfurreducens via bonding of Hg(II) to 8192


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Figure 2. (a) Fraction MeHg of total Hg(II) and (b) percentage distribution of aqueous Hg(II) species in assay media after 6 h incubation with G. sulfurreducens without added sulfur compounds and with addition of 50−500 nM of sulfide (HS−), N-acetyl cysteine (NACCYs), cysteamine (CystN), or cysteine (Cys). Different letters in (a) indicate significant (p < 0.05 ANOVA followed by Fisher’s least significance difference post hoc test) differences among Hg(II) methylation rates (n = 4) and error bars show standard errors.

Further, relatively small differences in the chemical structures of Hg(NACCys)2, Hg(Cys)2, and Hg(CystN)2 obviously resulted in a kmeth being 1 order of magnitude lower for the former molecule. Similarly small structural differences resulting in markedly different kmeth were previously observed6,14 at the micromolar level additions of LMM-RSH compounds. Our study expands on these results by demonstrating that the effect is pronounced already at environmentally relevant LMM-RSH concentrations in the nanomolar range.42,58 Higher cellular uptake than predicted by the classic biotic ligand model has been observed for several metal complexes,27,29,54,59 and explained by the formation of ternary complexes of the type L-M-Rs although this was not explicitly demonstrated. In principle, uptake of Hg(II) via formation of ternary complexes

cell membrane ligands. In contrast, the previously proposed higher cellular uptake rate of Hg(LMM-RS)2 complexes compared to HgCl2 and Hg(LMM-RORS), as discussed in the Introduction, are not consistent with this theoretical framework. Even if models based on metal binding to biotic ligands can explain most of our data, it is also obvious that there are aspects of microbial uptake and methylation of Hg(II) which cannot be explained by ligand affinity alone and which are Hg(II) species-dependent on a more detailed level than captured by current models for metal biouptake. Notably the kmeth value for the complexes Hg(Cys)2 and Hg(CystN)2 is of the same order of magnitude as for the mixed ligation complexes Hg(LMM-RORS) and HgOHnCl1−n(LMM-RS). 8193


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complexes in the extracellular medium and that other mechanisms controlling later steps in the biochemical pathway of Hg(II) methylation need to be invoked. The fact that Hg(II) methylation is enhanced by increased concentrations of Cys also at environmentally relevant levels (nM) corroborate the potential importance of this process for MeHg production in the environment and further studies of the underlying mechanism(s) are warranted. The range in kmeth value observed for the different types of Hg(II) species considered in our study spans 2 orders of magnitude. Our results thereby show that natural spatial and temporal fluctuations in the chemical speciation of Hg(II) could be of considerable significance for MeHg formation in the environment. There is compelling evidence from bacteria culture experiments and field studies that microbes can produce MeHg following exposure to Hg(II) species with high thermodynamic stability, such as Hg(SH)2, Hg(LMMRS)2, Hg(II) complexes, with thiol groups in natural organic matter (Hg(NOM-RS)2) and nanoparticulate HgS. On the basis of our findings, the fundamental principle when comparing Hg(II) availability for MeHg formation should be that less stable Hg(II) complexes have a higher availability for methylation. The concentrations of mixed ligation Hg(II) species with comparably low thermodynamic stability are less likely to be quantitatively important in natural environments under thermodynamic equilibrium conditions. However, they may be kinetically favored62,63 under certain conditions, and our study shows that if present, such species are important contributors to MeHg formation. Kinetic constraints can be induced by the high spatial and temporal dynamics in formation and release of LMM-RSH in, e.g., soil/sediment porewaters. 58 Further, it has been hypothesized that thermodynamically stable Hg(LMM-RS)2 complexes may undergo Hg(II)−Hg(0) reduction−oxidation cycles,64,65 such that a nonequilibrated pool of Hg(II) is continuously present. The large difference in kmeth for individual Hg(LMM-RS)2 complexes illustrate that the specific composition of LMMRSH compounds included in the “pool” of dissolved natural organic matter likely is an important factor for MeHg formation in the environment. At present such molecular compositions of LMM-RSH compounds are poorly characterized.

could occur for a wide range of dissolved Hg(II) complexes which are sufficiently kinetically labile27 to readily exchange ligands with thiol moieties at the cell surface17 and which can access the specific binding site(s) of the transporter from which Hg(II) is internalized. The log K for different Hg(RS)2 complexes is constrained to a relatively narrow range,44,46 and Hg(RS)2 complexes exchange thiol ligands at appreciable rates.60 On the basis of this, it is expected that Hg(LMM-RS)2 complexes can generally exchange ligands with thiol functional groups at cell surfaces (albeit at lower rate than less stable mixed ligation Hg(II)(aq) complexes). However, steric hindrance may impede the formation of ternary complexes, which has been suggested for metal-chelates, e.g., Zn(II)EDTA (but also for the Zn(Cys)2 complex) in marine phytoplankton.27 Steric hindrance could decrease the rate for ligand-exchange and formation of ternary complexes at certain cell surface sites also for Hg(LMM-RS)2 complexes which are too large and/or for which chelating binding interactions are present, possibly, e.g., Hg(NACCys)2.6 Our results showing considerably lower kmeth value for Hg(NACCys)2 compared to Hg(Cys)2 and Hg(CystN)2 may suggest that Hg(II) ligandexchange reactions with specific sterically constrained sites, as part of a controlled biochemical pathway, is critical for cellular uptake and/or methylation of Hg(II) in G. sulfurreducens and may explain observed differences in methylation of Hg(LMMRS)2 complexes across related taxa (D. desulfuricans and Shewanella oneidensis). Notably, the sulfate reducing bacterium D. desulfuricans is able to methylate Hg(II) at high rates from a wider range of Hg(LMM-RS)2 complexes than G. sulfurreducens.6 The reasons for these differences are not known but may be due to biochemical differences in membrane structure and transporter binding sites impacting ligand exchange reactions at the cell surface. Our results also show that LMM-RSH compounds can affect Hg(II) methylation via additional mechanisms than by controlling the chemical speciation of Hg(II)(aq) in the extracellular medium. The presence of an excess of Cys, but not of CystN or NACCys, caused a substantial increase in Hg(II) methylation already at concentrations of 200−500 nM Cys. Several previous studies have demonstrated high Hg(II) uptake and methylation (rates or amounts) by G. sulfurreducens14 and D. desulfuricans6 and high Hg(II) uptake by the nonmethylating bacteria E. coli17,61 and S. oneidensis16 in the presence of ∼1−10 μM concentrations of Cys. It has further been shown that micromolar concentrations of Cys enhance uptake and methylation of Hg(II) in both whole cells14 and in spheroplasts12 and enhances Hg(II) methylation in cell lysates14 of G. sulfurreducens. The enhanced Hg(II) methylation at micromolar levels of Cys compared to controls and lower Cys addition experiments was interpreted as a concentration-dependent change in the speciation from HgCl2 or Hg(Cys) to the Hg(Cys)2 complex with a presumed higher bioavailability.14,19 These speciation models however under-predicted the competitiveness of Hg(Cys)2 over HgCl2 and Hg(Cys). In contrast, our speciation modeling showed that the methylation increased without changes in Hg(II)(aq) speciation, which was highly dominated by the Hg(Cys)2 complex at all Cys additions in the range of 0−10 μM. The findings in previous studies suggest that Cys promotes both the uptake of Hg(II) by the bacteria, potentially across both the outer and inner membranes, and the intracellular enzymatic formation of MeHg. Our results show that the enhancement is not caused by changes in the speciation of Hg(II)(aq)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.9b01502.

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Summary of extracellular concentrations, time-resolved concentration, average cysteine concentrations per cell, confocal laser scanning images, concentrations of biosynthesized compounds, average MeHg production in G. sulfurreducens assay, and text on Hg(II) distribution (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yu Song: 0000-0002-8610-0525 Ulf Skyllberg: 0000-0001-6939-8799 Erik Björn: 0000-0001-9570-8738 8194


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Environmental Science & Technology Notes

(16) Szczuka, A.; Morel, F. M. M.; Schaefer, J. K. Effect of Thiols, Zinc, and Redox Conditions on Hg Uptake in Shewanella oneidensis. Environ. Sci. Technol. 2015, 49 (12), 7432−7438. (17) Thomas, S. A.; Tong, T. Z.; Gaillard, J. F. Hg(II) bacterial biouptake: the role of anthropogenic and biogenic ligands present in solution and spectroscopic evidence of ligand exchange reactions at the cell surface. Metallomics 2014, 6 (12), 2213−2222. (18) Graham, A. M.; Bullock, A. L.; Maizel, A. C.; Elias, D. A.; Gilmour, C. C. Detailed Assessment of the Kinetics of Hg-Cell Association, Hg Methylation, and Methylmercury Degradation in Several Desulfovibrio Species. Appl. Environ. Microbiol. 2012, 78 (20), 7337−7346. (19) Lin, H.; Lu, X.; Liang, L. Y.; Gu, B. H. Cysteine Inhibits Mercury Methylation by Geobacter sulfurreducens PCA Mutant Delta omcBESTZ. Environ. Sci. Technol. Lett. 2015, 2 (5), 144−148. (20) Lu, X.; Liu, Y. R.; Johs, A.; Zhao, L. D.; Wang, T. S.; Yang, Z. M.; Lin, H.; Elias, D. A.; Pierce, E. M.; Liang, L. Y.; Barkay, T.; Gu, B. H. Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem. Environ. Sci. Technol. 2016, 50 (8), 4366−4373. (21) An, J.; Zhang, L.; Lu, X.; Pelletier, D. A.; Pierce, E. M.; Johs, A.; Parks, J. M.; Gu, B. Mercury Uptake by Desulfovibrio desulfuricans ND132: Passive or Active? Environ. Sci. Technol. 2019, 53, 6264− 6272. (22) Sunda, W. G.; Guillard, R. R. L. THE RELATIONSHIP BETWEEN CUPRIC ION ACTIVITY AND TOXICITY OF COPPER TO PHYTOPLANKTON. Journal of Marine Research 1976, 34 (4), 511−529. (23) Morel, F. M. M. Principles of Aquatic Chemistry; JohnWiley: New York, NY, 1983. (24) Slaveykova, V. I.; Wilkinson, K. J. Predicting the bioavailability of metals and metal complexes: Critical review of the biotic ligand model. Environ. Chem. 2005, 2 (1), 9−24. (25) Di Toro, D. M.; Allen, H. E.; Bergman, H. L.; Meyer, J. S.; Paquin, P. R.; Santore, R. C. Biotic ligand model of the acute toxicity of metals. 1. Technical basis. Environ. Toxicol. Chem. 2001, 20 (10), 2383−2396. (26) Pagenkopf, G. K.; Russo, R. C.; Thurston, R. V. EFFECT OF COMPLEXATION ON TOXICITY OF COPPER TO FISHES. J. Fish. Res. Board Can. 1974, 31 (4), 462−465. (27) Aristilde, L.; Xu, Y.; Morel, F. M. M. Weak Organic Ligands Enhance Zinc Uptake in Marine Phytoplankton. Environ. Sci. Technol. 2012, 46 (10), 5438−5445. (28) Flynn, S. L.; Szymanowski, J. E. S.; Fein, J. B. Modeling bacterial metal toxicity using a surface complexation approach. Chem. Geol. 2014, 374, 110−116. (29) Fein, J. B. Advanced biotic ligand models: Using surface complexation modeling to quantify metal bioavailability to bacteria in geologic systems. Chem. Geol. 2017, 464, 127−136. (30) Xu, Y.; Shi, D. L.; Aristilde, L.; Morel, F. M. M. The effect of pH on the uptake of zinc and cadmium in marine phytoplankton: Possible role of weak complexes. Limnol. Oceanogr. 2012, 57 (1), 293−304. (31) Morel, F. M. M.; Hudson, R. J. M.; Price, N. M. LIMITATION OF PRODUCTIVITY BY TRACE-METALS IN THE SEA. Limnol. Oceanogr. 1991, 36 (8), 1742−1755. (32) Thomas, S. A.; Rodby, K. E.; Roth, E. W.; Wu, J.; Gaillard, J.-F. Spectroscopic and Microscopic Evidence of Biomediated HgS Species Formation from Hg(II)−Cysteine Complexes: Implications for Hg(II) Bioavailability. Environ. Sci. Technol. 2018, 52 (17), 10030− 10039. (33) Hand, C. E.; Honek, J. F. Biological chemistry of naturally occurring thiols of microbial and marine origin. J. Nat. Prod. 2005, 68 (2), 293−308. (34) Caccavo, F.; Lonergan, D. J.; Lovley, D. R.; Davis, M.; Stolz, J. F.; McInerney, M. J. Geobacter Sulfurreducens sp-nov, a hydrogenoxidizing and acetate oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 1994, 60 (10), 3752−3759. (35) Liem-Nguyen, V.; Bouchet, S.; Bjorn, E. Determination of SubNanomolar Levels of Low Molecular Mass Thiols in Natural Waters

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Swedish Research Council via the project Sino-Swedish Mercury Management Research Framework−SMaReF (2013-6978) and the Kempe Foundations (JCK-1501, SMK-2745, and SMK-1243).

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