Article pubs.acs.org/est
Divalent Base Cations Hamper HgII Uptake Valérie Daguené,† Emily McFall,† Emmanuel Yumvihoze,† Shurong Xiang,† Marc Amyot,‡ and Alexandre J. Poulain*,† †
Department of Biology, University of Ottawa, 30 rue Marie Curie, Ottawa, ON, K1N 6N5, Canada GRIL, Université de Montréal, 90 rue Vincent d’Indy, Montréal, QC, H2 V 2S9, Canada
‡
S Supporting Information *
ABSTRACT: Despite the alarming trends of declining base cation concentrations in boreal lakes, no studies have attempted to predict the consequences of this decline on the geochemical cycle of mercury, a top priority contaminant worldwide. In this study, we used a whole-cell gram-negative bioreporter to evaluate the direction and magnitude of changes in net accumulation of HgII by bacteria in response to changing base cation concentrations. We show that regardless of the speciation of HgII in solution, increasing divalent base cation concentrations decrease net HgII accumulation by the bioreporter, suggesting a protective effect of these cations. Our work suggests that the complexity of the cell wall of gram-negative bacteria must be considered when modeling Hg uptake pathways; we propose that base divalent cations contribute to hamper net HgII accumulation by decreasing outer membrane permeability and, therefore, the passive diffusion of HgII species to the periplasmic space. This work points to an unsuspected and likely harmful consequence of a delay in recovering from acidification in boreal lakes, in that uptake of HgII by bacteria is not only enhanced by the reduced pH but can also be enhanced by a decline in base cation levels.
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INTRODUCTION The structure and function of ecosystems worldwide are threatened due to the influence of anthropogenic activities on the biogeochemical cycles of nutrients (e.g., C, N, S, P) and contaminants (e.g., Hg).1 In addition to the alteration of nutrient cycles in aquatic systems, it is also suggested that the concentrations of major ions such as acid anions (e.g., NO3−, SO42−) and divalent base cations (e.g., Ca2+, Mg2+) are affected and may be declining worldwide.2 These declines are thought to be the consequence of the depletion of exchangeable base cations from watershed soils. It has recently been shown that the concentration of Ca2+ is declining in softwater lakes in many boreal regions.3 One ecological consequence of this decline is decreased survival and fecundity of crustacean populations, such as Daphnia, in Canadian Shield lakes.4 Many studies have reported the role of base cation concentrations, such as Ca2+ and Mg2+, on toxic metal accumulation and toxicity.5−8 Typically, cations compete for binding sites with toxic metals at the cell surface and hence decrease the bioavailability of toxic metals. Most of these © 2012 American Chemical Society
studies have been conducted with eukaryotic models and data are lacking on how such cations affect metal uptake by bacteria. Hg is a neurotoxic metal that is bioaccumulated by organisms and bioamplified in food webs.9 This bioaccumulation occurs mostly when Hg is present in an organic form such as methylmercury (MeHg). HgII methylation is typically observed under anaerobic conditions10 but recent reports suggest that it may also occur in marine oxic surface waters.11,12 Bacteria, especially sulfate-reducing bacteria (SRB), are thought to be responsible for the formation of the neurotoxin MeHg by methylation of inorganic mercury in anaerobic sediments.13 The metabolic pathways leading to HgII methylation remain unknown,14,15 but because HgII methylation by bacteria is thought to occur in the cytoplasm, an internalization step is required. Received: Revised: Accepted: Published: 6645
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responds to intracellular inorganic divalent Hg (HgII) levels.23 HMS174 (pRB27) constitutively expresses the luciferase enzymatic complex. This strain was used as a control to assess whether an alteration in light emission pattern was due to an alteration of the overall cell physiology.23 Growth and assay media composition were adapted from Golding et al. with the final concentration of (NH4)2SO4 in the assay medium set at 0.9 mM23 (see Table S1 for detailed assay media composition). Kanamycin was added to a final concentration of 100 μg·mL−1 in all growth media. Briefly, 5 mL of lysogeny broth (LB) was inoculated with a single colony and incubated at 37 °C with shaking until late log phase (typically, 6−7 h). Fifty μL of the LB culture was transferred to a serum bottle containing 5 mL of glucose minimal medium (GMM), as described in ref 23 and incubated overnight. In the morning, 20 mL of fresh GMM was added to the serum bottles and incubated for another 3 h. Cells were harvested by centrifugation and resuspended in phosphate buffer (67 mM Pi composed of NaH2PO4 and K2HPO4 set at pH = 6.8). Cell density was set at a final OD600 of 0.4. A dilution of 1/100 of this cell suspension was used for the assay. Bioassay. Assays were prepared in borosilicate scintillation vials filled with 20 mL of phosphate assay medium (Table S1). Major ions, Ca2+, Mg2+, Sr2+, and K+ were added to the assay medium from working solutions (typically 0.1−1.0 M MgSO4, Na2SO4, MgCl2, SrCl2, CaCl2, and NaCl) prepared fresh daily. Twenty minutes after the addition of the cells and the cations, HgII was added as HgCl2 to a final concentration of 250 pM (corresponding to 50 ng·L−1). The cell suspension was then transferred to a 96-well plate made of Teflon PFA; the well volume was 200 μL. Light production was measured using a multimode plate reader set in luminescence mode (Tecan F200 Pro). Light production was recorded individually for each well for 5 s. Wells were continuously monitored for up to 3 h. As previously described,23 net HgII accumulation is proportional to both the maximum light produced for a given Hg II concentration as well as the maximum slope of induction in light production (see Figures S1 and S2). In this study, we used the maximum amount of light produced by the bioreporter as a proxy for the net accumulation of HgII in the cytoplasm. Lanthanum Experiment. Lanthanum is a known inhibitor of Ca2+ channels that has previously been used to hamper Ca2+ uptake in bacteria.33,34 To test whether such channels were involved in HgII uptake we used a two-pronged approach. First La(III) was added directly to the assay medium to a final concentration of 250 μM as LaCl3. In the second approach, cells grown in glucose minimal medium23 were harvested by centrifugation and resuspended in a solution buffered with 2(N-morpholino)ethanesulfonic acid (MES) (20 mM) at pH = 6.5 amended with LaCl3 to a final concentration of 500 μM; cells were pre-exposed to La(III) in a medium buffered with MES and in the absence of phosphate to remove possible confounding effects due to the precipitation of lanthanum and phosphate (see details in SI). After a 10-min incubation, traces of La(III) were removed by washing the cells twice in phosphate buffer (67 mM, pH = 6.8). Cells were finally suspended in phosphate buffer at a final OD600 of 0.4. A dilution of 1/100 of this cell suspension was used for the assay. Modeling of HgII and Divalent Cation Speciation. Inorganic mercury speciation in solution at equilibrium was computed using the program visual MINTEQ.35 The program was used to determine the relative abundance of HgII species as the chemical composition of the medium varied, especially over gradients of Cl− and NH4+ concentrations. The complete
Microbes (e.g., bacteria, archaea, and algae) are involved in almost all reactions affecting the Hg cycle.16 Not only are microbes involved in methylation−demethylation reactions, they can also control the redox state of Hg and hence its mobility as the reduced form, Hg0, is volatile. For instance, microbes can reduce HgII to Hg0 via specific (i.e., that of the mer-operon) 16 or nonspecific pathways,17 or exhibit the ability to oxidize Hg0.18,19 In most cases, however, an internalization step is also likely required. The mechanism of HgII availability to the microbes remains unclear, but both passive diffusion20−22 and facilitated transport23−25 are thought to be involved. Therefore, being able to predict how key environmental variables affect HgII uptake by microbes is relevant to all microbially mediated reactions involved in Hg transformations and will eventually contribute to strengthen predictive models and mitigating initiatives. Early reports suggested that water hardness,8,26,27 pH,28 dissolved organic carbon,29,30 or chloride concentrations31 affected mercury levels in fish. Multivariate regression models generally indicate that mercury levels in fish are inversely correlated to calcium, chloride, dissolved organic carbon concentrations, and pH.28 These variables may affect Hg fish concentrations in two ways: (i) they may influence the bacterial production of MeHg (at the uptake or methylation steps) or (ii) they may affect the relative accumulation of HgII and MeHg at higher trophic levels. Similar to observations for other toxic metals, the effects of DOC and chloride concentrations on HgII and MeHg levels in fish have been attributed to the formation of HgII and MeHg-DOC species or HgII and MeHg− chlorocomplexes that are poorly available to fish.31 The effects of DOC, chloride, and pH on HgII accumulation by a gram-negative bacterium (sulfate-reducing bacteria, thought to be the most important methylators, exhibit a gram-negative type cell wall) have been investigated using a whole-cell bioreporter.23,24,32 Results have shown that DOC and chloride concentrations decreased HgII uptake, likely by forming poorly available HgII complexes. Hence, chloride and DOC would also affect Hg levels in fish by limiting mercury methylation while decreasing the availability of the inorganic mercury substrate to bacteria. The same studies showed that decreasing pH (i.e., increasing [H+]) increased HgII uptake. It is often assumed that the observed inverse correlation between Hg levels in fish and Ca2+ is attributed to pH because of the nature of the bedrock, such that acidic lakes, where Hg levels in fish tend to be greater, tend to exhibit lower Ca2+ levels . No studies attempted to tease apart the role of pH vs that of base cations. In this study, we used a whole-cell gram-negative bioreporter to evaluate the direction and magnitude of changes in Hg II uptake in response to changing base cation concentrations. Our results show that a decline in divalent base cation concentrations could lead to an enhancement in HgII availability to microbes. This is a potential threat, as gramnegative bacteria are involved in HgII methylation.
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MATERIALS AND METHODS Detailed procedures and control experiments are described in Supporting Information (SI). Bacteria Strains, Media, and Culture. We used E. coli HMS174 (pRB28) and E. coli HMS174 (pRB27) as bioreporters to assess HgII bioavailability. In HMS174 (pRB28) expression of the genes LuxABCDE that encode for the luciferase enzymatic complex are under the control of MerR, a transcription regulator that specifically and strongly 6646
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Table 1. Inorganic Mercury Speciation at Equilibrium, Ionic Strength, and Decrease in Net HgII Accumulation Compared to the Control in the Presence of Various Counter Ions Used in the Manipulation of Mono- and Divalent Cation Content in the Assay Mediuma ionic strength (M)b
insoluble forms %
% decrease in HgII uptake
0.125 0.125 0.125 0.126 0.127 0.129
0 0 0 0 27e 70f
CTL 0.4 18.5 98.1 98.8 99.0
37.9 37.9 37.9 37.9 37.9 37.9
62.1 62.1 62.1 62.1 62.1 62.1
0 0 0 0 0 0
0.125 0.125 0.125 0.125 0.125
0 0 0 0 0
CTL 45.2 68.0 91.8 96.1
37.9 37.2 32.2 25.6 17.8
62.1 61.3 53.1 42.1 29.3
0 1.5 13.4 26.7 37.1
0.125 0.127 0.129 0.133 0.141
0 0 0 0 0
CTL - 9e −5 −5 −15
37.9 25.6 17.8 7.5 2.8
62.1 42.1 29.3 12.3 4.6
0 26.7 37.1 38.8 29.0
Hg(OH)2 Hg(NH3)22+
HgClOH
HgCl2
HgCl3− HgCl42−
b
2+
[Mg ] as MgSO4 (mM) none added 0.01 0.1 1 2 5 [Mg2+] as MgCl2 (mM)c none added 0.01 0.1 0.25 0.5 NaCl and Na2SO4 additiond Na+ (mM) Cl− (mM) 23 0 24.5 0.5 26 1 30.5 2.5 38 5
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 1.12 5.6 15.5
0 0 0 0 0.15
0 0 0 0 0
0 5.6 15.5 40.4 60.4
0 0 0.15 1.0 3.0
0 0 0 0 0.1
a
Ionic strength was calculated by visual MINTEQ. Negative values in % decrease in Hg uptake indicate a greater amount of bioavailable mercury than in the control experiment. In all cases [HgII] = 250 pM. bCorresponds to Figure 1A. cCorresponds to Figure 2. dCorresponds to Figure 1C. e Indicates the presence of insoluble Mg species at equilibrium; in this case MgHPO4, 3H2O (s); dissolved [Mg2+] = 0.73 mM. fIndicates the presence of insoluble Mg species at equilibrium; in this case MgHPO4, 3H2O (s); dissolved [Mg2+] = 0.3 mM.
Table 2. Inorganic Mercury Speciation at Equilibrium, Ionic Strength, and Decrease in HgII Uptake in the Presence of a Fixed Concentration of MgSO4 but Varying Levels of Counter Ionsa compound [NH4+]
= 1.8 mM [NH4+] = 5 mM [NH4+] = 10 mM [Cl−] = 25 mM [Cl−] = 100 mM a
[Mg2+]b
I (M)c
% neutrally charged HgII species
% decrease in HgII uptake
Hg(OH)2
1 mM
0.126
37.9
71.4
37.9
62.1
0
1 1 1 1
0.130 0.138 0.148 0.205
7.4 2.0 79 37.6
45.9 50.2 78.3 77.5
7.4 2.0 0.2 0
92.6 98.0 0.2 0
0 0 7 0.59
mM mM mM mM
Hg(NH3)22+ HgClOH
HgCl2
HgCl3− HgCl42−
0
0
0
0 0 71.8 37.0
0 0 17.9 37.0
0 0 2.9 25.3
Table corresponds to Figure 3. In all cases [HgII] = 250 pM. b[Mg2+] as MgSO4. cIonic strength calculated by software visual MINTEQ.
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speciation of HgII in solution for each experiment is detailed in Tables 1 and 2. The program was also used to determine at which concentrations precipitation of medium components would occur. Typically, calcium and magnesium were used within their range of solubility in the presence of phosphate at pH = 6.8 (see Tables 1 and S2). However, we also performed experiments when both Ca2+ and Mg2+ were predicted to form insoluble species (Tables 1 and S2). The chemical composition of the assay media is presented in Table S1. We tested for a potential role of insoluble species to affect light production and detection by the luminometer; within the range of concentration predicted to trigger the precipitation of medium component, light production and detection was not affected even when cells were diluted 104 fold (see SI and Figure S3). We also tested for the adsorption of HgII onto the wall of our borosilicate vessel during the 2 h duration of our experiment in the presence of 1 mM of Mg2+. While some HgII was adsorbed onto the wall of the assay container, loss of Hg by adsorption cannot account for the effect of base cations described in this study (see SI and Figure S4).
RESULTS AND DISCUSSION Divalent Base Cations Hamper HgII Uptake. Divalent base cation concentrations are declining in boreal lakes, preventing recovery from acidification. To further gain insights into the impact of an alteration in base cation concentrations in boreal lakes, we tested the role of base cations on the uptake of inorganic mercury under controlled laboratory conditions. We first exposed bacterial cells to increasing concentrations of Mg2+ added as MgSO4 (Figure 1A) and followed light production using a luminescent biosensor. Light production started to decline at 0.01 mM < [Mg2+] < 0.1 mM and decreased by 98% at [Mg2+] ≥ 1 mM (Figure 1A, Figure S2). Our control strain did not show any signs of being physiologically affected by [Mg2+] (Figure 1B); a slight stimulation of light production was noted. Throughout the range of Mg2+ tested, the modeled inorganic Hg speciation did not change and was mostly comprised of 62.1% positively charged Hg(NH3)22+ and 37.9% neutrally charged Hg(OH)2 (Figure 1A and Table 1). At [Mg2+] > 2 mM, some insoluble MgHPO4, 3H2O(s) is expected to form 6647
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Figure 1. Increasing concentrations of Mg2+ and not Na+ hamper net HgII accumulation. (A) Effect of increasing concentrations of Mg2+ added as MgSO4 on the net accumulation of HgII by the bioreporter; the open and closed circles represent the speciation of inorganic HgII at equilibrium. (B) Effect of increasing concentrations of Mg2+ added as MgSO4 on the constitutive production of light by the control strain. (C) Effect of increasing concentrations of Na+ added as NaCl and Na2SO4 on the net accumulation of Hg by the bioreporter. Open and closed symbols represent the speciation of inorganic HgII at equilibrium. BioHg stands for net HgII accumulation. Experiments were performed at [HgII] = 250 pM and error bars represent the standard deviation of at least 7 analytical replicates and are representative of experiments performed at least in duplicate. Letters on top of the bar graphs represent statistically different results tested by a one-way ANOVA (p < 0.05) and a Tukey’s HSD posthoc test; in panel A, because of unequal variances, results were separated in two groups of equal variance to run the statistical analyses.
monovalent cations. While no divalent cations were present in the assay medium, [Na+] was already set at [Na+] = 23 mM and [K+] = 81.5 mM as per contribution of the phosphate buffer and [NH4+] = 1.8 mM (Table S1). The total monovalent cation concentration in solution (Na+ + K+ + NH4+) in the unamended assay medium was therefore 106.3 mEq·L−1. While we were able to vary divalent cation concentrations by several orders of magnitude, it was not the case for monovalent cations without dramatically altering the ionic strength and buffering capacity of the assay medium. In an attempt to alter total monovalent cation levels, we increased [Na+] by 14% in the
(Table 1); however this did not affect the production of light by the constitutive control strain (Figure 1B). Net Hg II accumulation had already decreased by 98% when [Mg2+] reached 1 mM, at which all Mg2+ is predicted to be soluble (Figure 1A and Table 1). Hence, loss of bioavailable mercury due to adsorption onto newly formed mineral phase is likely not driving the observed divalent base cation effect. Under these conditions, while net HgII accumulation decreased by 98.1%, ionic strength varied by less than 1% (Table 1). To test whether the decrease in net HgII accumulation was solely due to divalent cations, we assessed the role of 6648
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Figure 2. Increasing concentrations of divalent cations hamper net HgII accumulation by a gram-negative bacterium. (A) Effect of increasing concentrations of Mg2+, Ca2+, and Sr2+ added as chloride salts on the net accumulation of HgII by the bioreporter. Both axes are on logarithmic scale. The gray area corresponds to levels of base cations typically found in natural freshwaters. (B) Effect of increasing concentrations of Mg2+, Ca2+, and Sr2+ added as chloride salts on the constitutive production of light by the control strain. Experiments were performed at [HgII] = 250 pM and error bars represent the standard deviation of at least 7 analytical replicates and are representative of experiments performed at least in duplicate.
Figure 3. Base cations affect net HgII accumulation, regardless of the speciation of Hg at equilibrium. Effect of altering medium component on net HgII accumulation by the inducible strain (containing plasmid pRB28) in the absence and presence of [Mg2+] = 1 mM. Experiments were performed at various NH4+ (A) and Cl− (B) concentrations to alter inorganic HgII speciation in solution. Typical assay medium composition (control highlighted in gray is valid for both A and B panels) is given in Table S1 and corresponds to a [NH4+] = 1.8 mM and no Cl− added. Compounds for which concentrations were altered are provided on the x-axis. Note the scale on the y-axes. Letters on top of the bar graphs represent statistically different results tested by a one-way ANOVA (p < 0.05) and a Tukey’s HSD posthoc test. Asterisks on top of the gray bars show statistically different means between treatment with and without Mg2+ added (t test, p < 0.05). Experiments were performed at [HgII] = 250 pM and error bars represent the standard deviation of at least 3 analytical replicates and are representative of experiments performed at least in duplicate.
divalent base cations did. Indeed, light production that was already strong at [total monovalent cations] = 106.3 mEq·L−1 (corresponding to the control treatments) increased when [total monovalent cations] rose to 121.3 mEq·L−1 (Figure 1C). This is in stark contrast with the addition of Mg2+ to a final concentration of 1 mM ([total divalent cations] = 2 mEq·L−1, ionic strength variation of 1%), which virtually stopped net HgII accumulation (98% decreased compared to the control). This experiment also showed that sulfate ions could not be responsible for changes observed in net HgII accumulation (Figure 1A and C).
presence of both Na2SO4 and NaCl so the ionic strength would not increase too rapidly (Table 1). As [Na+] increased from 23 to 38 mM, [total monovalent cation] increased from 106.3 to 121.3 mEq·L−1, [Cl−] increased from 0 to 5 mM, and the ionic strength increased by 13% (Figure 1C and Table 1). The inorganic HgII speciation greatly changed as chloride levels rose (Figure 1C and Table 1). However, regardless of these changes, no decline in net HgII accumulation was observed and a slight increase was even noted (Figure 1C and Table 1). Together these data suggest that monovalent cations were unlikely to affect net HgII accumulation to the same extent that 6649
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Figure 4. (A) Effect of preincubation time in the presence of base cations on the net accumulation of HgII by the bioreporter; [Ca2+] = 100 μM (as CaCl2) and [Mg2+] = 1 mM (as MgSO4). HgII + cation means that both HgII and Ca2+ or Mg2+ were added simultaneously. (B) Test of an inhibitor of a Ca2+ transporter on net HgII accumulation. Experiments were performed with the inhibitor, LaCl3, directly to the assay medium to a final concentration of 250 μM. The production of light by the inducible strain (containing plasmid pRB28) and the control strain (containing plasmid pRB27) in the absence and presence of [Ca2+] = 100 μM is presented. Letters on top of the bar graphs represent statistically different results tested by a one-way ANOVA (p < 0.05) and a Tukey’s HSD posthoc test. Experiments were performed at [HgII] = 250 pM and error bars represent the standard deviation of at least 3 analytical replicates and are representative of experiments performed at least in duplicate.
Increasing [Cl−] concentrations up to 5 mM in the presence of Na+ did not hamper net HgII accumulation (Figure 1C, Table 1). This supported a role for divalent cations, and not for neutral Hg chlorocomplexes, in hampering Hg accumulation. When Hg is mostly present as an uncharged chlorocomplex such as HgCl2, which is the case when [NaCl] = 5 mM, previous studies have shown that bioavailability is actually not different from what can be observed with Hg(OH)2,36 although their lipophilicity differ; note that HgCl2 is actually considered a key bioavailable species to phytoplankton.37 While our first series of experiments focused on Mg2+ (added as MgSO4), we also performed experiments using Ca2+, Sr2+, and Mg2+ (added as CaCl2, SrCl2 and MgCl2; Figure 2A). In all instances, as base cation concentrations increased, net HgII accumulation decreased by up to 10-fold (Figure 2A). The constitutive production of light by the control strain was not affected by the increasing concentration of base cations (Figure 2B). The range of base cation concentrations for which the hampering effect was most marked corresponded to levels typically found in natural freshwater systems2,4,38 (gray area, Figure 2A). For instance, the presence of only [Ca2+] = 50 μM (ca. 2.2 mg·L−1) and [Mg2+] = 100 μM (ca. 2.4 mg·L−1) corresponded respectively to a 65% and 68% decrease in net HgII accumulation compared to the control without any divalent base cations added (Figure 2). Divalent Base Cations Affect Hg Uptake Regardless of the Dominant Species of Hg Present in Solution. To further test for the role of inorganic HgII speciation in the presence of base cations on HgII availability, we set up a series of experiments with varying concentrations of Cl− (as [NaCl]) and NH4+ (as [(NH4)2SO4]). We first tested for the role of varying Cl− and NH4+ on the net accumulation of HgII in the absence of base cations. It is often assumed that negatively charged chlorocomplexes are poorly available or unavailable to the cell.36 Increasing chloride concentrations led to an increase in the relative abundance of negatively charged chlorocom-
plexes (Table 2) that corresponded to a large decrease in the net accumulation of HgII (Figure 3A and B, note the y-axis scale). However, the presence of these negatively charged complexes could not solely account for the decrease observed in net HgII accumulation. Indeed, at [Cl−] = 25 mM, only 21% of the total pool of HgII was present as negatively charged chlorocomplexes, the remaining was accounted for by 72% of HgCl2 and 7% HgClOH. Yet, it was associated with a decrease of 90% in net HgII accumulation compared to the control without chloride (Figure 3A and B, Table 2). Barkay et al.36 and Farrell et al.39 previously suggested that, while negatively charged chlorocomplexes are poorly available, chloride ions, alone, may have a key but complex role on HgII accumulation and toxicity that could potentially be associated with bacterial membrane permeability. The role of chloride remain unclear and clearly deserves more work but we provide evidence that the effect of chloride is only partially associated with mercury speciation. As [NH4+] increased from 1.8 mM to 5 and 10 mM, a 30% decrease in the net HgII accumulation was observed (Figure 3A). This increase in NH4+ in solution led to a 30−35% decrease of the abundance of neutrally charged hydroxocomplexes corresponding to an increase in positively charged Hg(NH3)22+ (Table 2). Light production by the control strain never decreased when experiments were conducted over the gradient of Cl− and NH4+ tested (Figure S5A); at [Cl−] = 100 mM, constitutive light production appeared to be stimulated. Regardless of the speciation of HgII in solution, the addition of Mg2+ to a final concentration of 1 mM led to a sharp decrease in the net accumulation of HgII (Figure 3A and B); the magnitude of the decline appeared to be dependent on the speciation of HgII in solution. When compared to the control, the decline in HgII accumulation ranged from 45.9% and 50.2% with Hg II speciation largely dominated by positively charged complexes but reached 71.4% and 78.3% with neutrally charged and 6650
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HgII for ligands is also different. HgII is a soft metal preferentially associated with N or S containing moieties whereas Ca2+ preferentially binds to O donor ligands. Calcium channels typically contain a EEEE-locus (4 negatively charged glutamate) and are among the most selective cation channels known.47 It is unlikely that HgII can efficiently compete with Ca2+ for binding to the carboxylic moieties of the glutamate and hence this specific transporter is unlikely to be an uptake route for HgII. Other inhibitors of base cation transporters exist; for instance ω-conotoxin is commonly used.48 However, because of their peptidic nature and the lack of binding constant existing with HgII we postponed their use. Previous work suggested that the uptake of HgII occurs either by passive diffusion or facilitated transport,21,23,25 but previous work did not take into consideration the complexity of the cell wall associated with the two lipid bilayers existing in gramnegative bacteria. Clearly, further experiments are required to provide mechanistic details of how the cell wall is involved in controlling net HgII accumulation. In light of our work, however, we propose that divalent base cations contribute to hamper net HgII accumulation by decreasing outer membrane permeability and, therefore, the passive diffusion of HgII species to the periplasmic space; how HgII crosses the cytoplasmic membrane remains to be determined. E. coli, the bacterium model used in this study, is capable of HgII methylation.49 However, mechanisms of HgII uptake may or may not be the same between E. coli and the well-known producers of MeHg in the environment, sulfate-reducing bacteria; further experiments assessing the role of base cations on HgII methylation by sulfate reducing bacteria and in environmental samples are warranted. These findings also point to an unsuspected and possibly harmful consequence of a delay in recovering from acidification in boreal lakes, in that net accumulation of HgII by bacteria is enhanced not only by reduced pH but also by a decline in base cation concentrations. While great variability is observed for a given lake with respect to the magnitude of the changes in base cations levels,3,50 our work suggests that models may better predict methylmercury availability to food webs by taking into consideration processes that directly or indirectly control base cation concentrations in boreal freshwaters.
negatively charged complexes present (Figure 3A and 3B, Table 2). We showed that base cations hamper net HgII accumulation (Figure 1 and 2) regardless of the speciation of Hg in solution (Figure 1 and Figure 3). These observations prompted us to test how divalent cations may alter net HgII accumulation. Investigations on How Base Cations Affect Net HgII Accumulation. We first compared the response of the bioreporter when cells were preconditioned for 20 min in the presence of base cations prior to the addition of HgII, with cells exposed to base cations and HgII simultaneously (Figure 4A). In both cases, net HgII accumulation declined (Figure 4A). When cells were preincubated with base cations, the decline in net HgII accumulation was greater than when cells were exposed to HgII and base cations simultaneously. This was particularly true when Ca2+ was added as CaCl2 compared to Mg2+ added as MgSO4. The lipopolysaccharide (LPS) layer of the outer membrane of gram-negative bacteria has been shown to contain high affinity binding sites for Ca2+ and Mg2+ 40 and divalent ions (Mg2+ or Ca2+) are critical in stabilizing the outer membrane of bacteria.40,41 In the presence of Ca2+ and Mg2+ ions, LPS exists in an ordered quasi-crystalline arrangement constituting a monolayer that hydrophobic molecules have difficulty penetrating;42 as such, divalent cations typically decrease outer membrane permeability.43,44 Although further work is required to test it, such a decrease in membrane permeability in the presence of base cations could be responsible for the observed decrease in net HgII accumulation, as it would hamper Hg species crossing the first lipid bilayer. Because the presence of divalent cations hampered net HgII accumulation (Figures 1A and 2A), through a process that appeared to be fast (Figure 4A) and because divalent base cations can compete with toxic metals for uptake,45 we also performed a series of experiments to address the possibility that divalent base cations and mercury compete for a common transporter. We performed experiments using lanthanum, La(III), a known inhibitor of an active calcium channel transport in bacteria.33,34 We used La(III) at concentrations of 250 and 500 μM; these levels are 10−100 times greater than what has been previously used.33,34 We did not observe any decline in net HgII accumulation in the presence of lanthanum in the assay medium (Figure 4B); in fact, net HgII accumulation appeared to be stimulated in the presence of La(III) (Figure 4B). Whether or not La(III) was present, net HgII accumulation decreased when Ca2+ was added to the medium, suggesting that direct competition between HgII and Ca2+ at a Ca2+ uptake site is not responsible for the observed base cation effect. In a subsequent experiment, to avoid adding La(III) directly to the medium, cells were pretreated with La(III) prior to the assay. Both constitutive and inducible cells showed a decline in light production after the washing step. After correction for this decline, only a slight decrease (ca. 10%) in HgII uptake was observed when cells were pretreated with La(III) (see SI Figure S6). Glynn et al.46 suggested that uptake of toxic metals such as HgII or Cd2+ could use calcium channels and Chen et al. showed that Cd2+ uptake by a Bacillus species was hampered with 5 μM of La(III) in the assay medium.33 Our results suggest that HgII is not taken up by a Ca2+ transport system that can be inhibited by lanthanum at concentrations 10−100 fold higher than those used in previous studies.33,34 This observation is further supported by the fact that the ionic radius of HgII is different than that of Ca2+ and the affinity of Ca2+ and
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ASSOCIATED CONTENT
S Supporting Information *
Details of the experimental design, composition of the assay medium (Table S1), calibration curve (Figure S1), experimental induction curves (Figure S2), potential role of medium component precipitation on light detection (Figure S3), test for Hg adsorption on the wall of borosilicate container walls (Figure S4), effect of altering the medium chemistry on constitutive light production by the control strain (Figure S5), and test for the role of a Ca2+ inhibitor added to the washing step on net HgII accumulation (Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: 613-562-5800, x2373. Notes
The authors declare no competing financial interest. 6651
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ACKNOWLEDGMENTS We thank R. Sparling and C. Kelly from the University of Manitoba as well as T. Barkay from Rutgers University for training on the application of the Hg bioreporter under trace metal conditions, for providing the original constructs and strains, and for stimulating discussions. We thank the Poulain lab members and B.E. Keatley for comments on earlier versions of this manuscript. We thank the anonymous reviewers who evaluated the manuscript and contributed to its improvement. A Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to A.J.P. funded this study.
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