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Environmental Processes
Mercury Uptake by Desulfovibrio desulfuricans ND132: Passive or Active? Jing An, Lijie Zhang, Xia Lu, Dale A Pelletier, Eric M. Pierce, Alexander Johs, Jerry M. Parks, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00047 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019
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Mercury Uptake by Desulfovibrio desulfuricans ND132: Passive or Active?
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Jing An,1,2 Lijie Zhang,1 Xia Lu,1 Dale A. Pelletier,3 Eric M. Pierce,1 Alexander Johs,1
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Jerry M. Parks,3 Baohua Gu1,4*
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Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States
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Key Laboratory of Pollution Ecology and Environment Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
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Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States
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Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN 37996, United States
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ABSTRACT Recent studies have identified HgcAB proteins as being responsible for mercury
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[Hg(II)] methylation by certain anaerobic microorganisms. However, it remains controversial
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whether microbes take up Hg(II) passively or actively. Here we examine the dynamics of
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concurrent Hg(II) adsorption, uptake, and methylation by both viable and inactivated cells
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(heat-killed or starved) or spheroplasts of the sulfate-reducing bacterium Desulfovibrio
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desulfuricans ND132 in laboratory incubations. We show that, without addition of
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thiols, >60% of the added Hg(II) (25 nM) was taken up passively in 48 h by live and
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inactivated cells and also by cells treated with the proton gradient uncoupler,
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carbonylcyanide-3-chlorophenylhydrazone (CCCP). Inactivation abolished Hg(II)
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methylation, but the cells continued taking up Hg(II), likely through competitive binding or
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ligand exchange of Hg(II) by intracellular proteins or thiol-containing cellular components.
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Similarly, treatment with CCCP impaired the ability of spheroplasts to methylate Hg(II) but
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did not stop Hg(II) uptake. Spheroplasts showed a greater capacity to adsorb Hg(II) than
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whole cells, and the level of cytoplasmic membrane-bound Hg(II) correlated well with MeHg
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production, as Hg(II) methylation is associated with cytoplasmic HgcAB. Our results indicate
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that active metabolism is not required for cellular Hg(II) uptake, thereby providing improved
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understanding of Hg(II) bioavailability for methylation.
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INTRODUCTION Methylmercury (MeHg) is produced predominantly by a small group of anaerobic
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microorganisms possessing the gene pair, hgcAB, that confers the ability to convert inorganic
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mercury (Hg) to MeHg.1-4 In natural aquatic environments, MeHg bioaccumulates and
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biomagnifies at high levels in food webs, particularly in fish and rice,5-10 and is thus a
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significant threat to human health and the environment. Although we now know that Hg
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methylation is carried out by HgcAB proteins located in the cytoplasm,1-3, 11 our
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understanding of the pathways and factors that control mercuric mercury [Hg(II)] uptake by
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these organisms remains limited. Currently, there are two proposed Hg(II) uptake
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mechanisms: (1) passive permeation of Hg(II) species such as Hg(SR)2, HgCl2, HgSaq, and
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HgS nanoparticles,12-18 and (2) energy-dependent active uptake of Hg(II).19-22 The former is
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supported by equilibrium analyses showing that the octanol-water partition coefficient (Kow)
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of a Hg(II) complex is proportional to its ability to permeate the cell membrane and may thus
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be used as a measure of Hg(II) bioavailability.15 In addition, concentrations of neutral Hg(II)-
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sulfide species show a positive correlation with MeHg production by several sulfate-reducing
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bacteria.12-14 Thermodynamic calculations and molecular dynamics simulations also indicate
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that the energy barrier required for passive permeation of small neutral Hg(II) species
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through bacterial cytoplasmic membranes is low (~ 2–3 kcal/mol).16, 18 The active uptake
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mechanism is supported mainly by experiments with iron-reducing bacteria (e.g., Geobacter
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sulfurreducens PCA) showing that the presence of certain thiol compounds such as cysteine
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enhances Hg(II) uptake and methylation, whereas others (e.g., glutathione and penicillamine)
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inhibit Hg(II) methylation.20-23 However, this effect is less clear in experiments with sulfate3 ACS Paragon Plus Environment
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reducing bacteria, such as D. desulfuricans ND132.20-23 The energy requirement was
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demonstrated by inhibited Hg(II) methylation in the presence of carbonylcyanide-3-
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chlorophenylhydrazone (CCCP), a proton gradient uncoupler, but its direct effect on Hg(II)
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uptake has not been established.21 Furthermore, Hg(II) uptake is inhibited by certain divalent
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metal cations such as Zn2+ and Cd2+, leading to the hypothesis that certain metal cation
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transporters may be involved in the active uptake of Hg(II).22 However, direct evidence is
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still lacking, and the proposed mechanism is inconsistent with the generalized theory of
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biouptake of metals,24, 25 unless Hg(II) is taken up as intact Hg(II)-cysteine complexes. The
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inhibition of Zn2+ and Cd2+ could arguably be attributed to the indirect competitive effects of
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these metal ions on Hg(II) adsorption and uptake.23, 26 Importantly, we also note that all experiments supporting an active uptake mechanism
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were performed in the presence of thiols, which form strong complexes with Hg(II), but it is
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unknown whether active uptake would occur in the absence of thiols.27 Recent studies have
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shown that thiols can effectively compete with cells for Hg(II) binding and uptake (or
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methylation), depending on the specific microbial strains and the type and concentration of
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thiols in the extracellular environment.23, 26, 28-31 This dependence was attributed to a
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decreased concentration of cell-associated Hg(II) as a result of competitive ligand exchange
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between thiols in solution and bacterial cells. For example, D. desulfuricans ND132
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possesses a higher content of thiol functional groups on its cell envelope29, 32 and thus shows
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a higher binding affinity and ability to compete with thiols such as glutathione, cysteine, and
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penicillamine for Hg(II) uptake and methylation than G. sulfurreducens PCA.23, 29, 31
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However, the presence of more strongly binding thiols, such as 2,3-dimercapto-14 ACS Paragon Plus Environment
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propanesulfonate (DMPS), completely inhibits Hg(II) uptake and methylation by both D.
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desulfuricans ND132 and G. sulfurreducens PCA.23, 31 D. desulfuricans ND132 is an anaerobic Gram-negative bacterium in the class of
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Deltaproteobacteria and a known strong Hg(II) methylator.33, 34 The cell envelope of Gram-
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negative bacteria consists of both an outer membrane (OM) and a cytoplasmic membrane
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separated by the periplasmic space. In general, the OM of Gram-negative bacteria is
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composed of phospholipids, lipopolysaccharides, and proteins (including porins, receptors,
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etc.)35 and is generally very permeable for small (2 h and subsequently kept in an anoxic chamber (Coy Lab Products, Grass
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Lake, MI) for at least 24 h before use. All washing steps and subsequent methylation assays
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were conducted in the anoxic chamber containing a mixture of 98% N2 and 2% H2.23, 29, 31, 44 Whole-cell Hg(II) uptake and methylation assays. Hg(II) uptake and methylation
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assays were conducted in PBS in 4 mL amber glass vials.23, 31, 44 Briefly, each vial contained
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a final concentration of washed cells of 5×1011 cell/L and was supplemented once (at time
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zero) with 1 mM each of pyruvate and fumarate as the electron donor and acceptor. The
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Hg(II) working solution was freshly prepared from a stock solution of 50 μM HgCl2 in 1%
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HCl and added to the cell suspension to give a final concentration of 25 nM Hg(II) and a total 6 ACS Paragon Plus Environment
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volume of 1 mL in PBS. All vials were sealed immediately with polytetrafluoroethylene
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(PTFE)/silicone screw caps and kept in the dark on an orbital shaker. At selected time points,
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six sample vials were taken out of the anoxic chamber and analyzed for both inorganic Hg(II)
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and MeHg species distributions as follows. Two of the samples were preserved immediately
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in HCl (0.5% v/v) for total inorganic Hg(II) (THgi) and MeHg analyses (described below),
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and two samples were filtered through 0.2-μm syringe filters to remove cells and then
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preserved for soluble Hg(II) (Hgsol) and MeHgsol analyses. A small aliquot (10 µL) of DMPS
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was added to each of the two remaining samples to obtain a final concentration of 0.1 mM.
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These samples were equilibrated for an additional 15 min to wash off cell-surface-adsorbed
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Hg(II) (Hgads) and MeHg (MeHgads), as they form strong complexes with DMPS.23, 45
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Samples were again filtered and analyzed, so that the intracellular Hg(II) (Hgcell) (or Hg(II)
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uptake) could be calculated by subtracting Hgsol and Hgads from THgi (i.e., Hgcell = THgi –
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Hgsol – Hgads),23, 44 where Hgads was calculated as the difference between amounts of Hg(II) in
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filtrate solutions with and without DMPS wash. The adsorbed and intracellular MeHg were
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not reported, as we focus on inorganic Hg(II) uptake. In addition, MeHg is known to be
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mostly excreted to the solution phase even without the addition of thiols.46, 47 To determine the effectiveness of DMPS for washing off adsorbed Hg(II) on cell
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membranes and to prevent Hg(II) from uptake and methylation, lysed cells (ruptured by ultra-
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sonification) were also evaluated for Hg(II) adsorption, uptake, and desorption. The same
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washing procedure was used as above, except that lysed cell debris was separated by ultra-
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centrifugation (at 123,700×g for 1 h at 4°C), rather than filtration, following DMPS washing
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at various time points. Experiments were also performed by reacting Hg(II) with DMPS first 7 ACS Paragon Plus Environment
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before adding the cell lysate to determine if cell membranes or debris are still capable of
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adsorbing Hg(II). After 2-h and 24-h equilibrations, the supernatant was again collected and
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analyzed to determine the effectiveness of DMPS in preventing Hg(II) from being adsorbed
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by lysed cellular components or debris.
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Hg(II) uptake and methylation assays were also performed in the same manner with
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heat-killed cells prepared by heating the cell suspension in PBS to 60°C for 3 h,21, 22 or with
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starved cells by letting washed cells rest in PBS for 15 h and avoiding the use of the electron
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donor or acceptor during the methylation assays. In addition, experiments were performed in
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the presence or absence of 20 μM CCCP to determine its effects on Hg(II) uptake and
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methylation.21, 22 In the CCCP assays, washed cells were preincubated with CCCP for 1 h
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before Hg(II) was added (either with or without 50 μM glutathione). Hg(II) uptake and methylation assays with spheroplasts. Spheroplasts were
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prepared by removing the outer membrane and the peptidoglycan layer of D. desulfuricans
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ND132 following a previously established method.22, 32, 48 The cells were first suspended in 4
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mL of 250 mM Tris-HCl buffer (pH 7.5), to which 0.4 mL of 500 mM EDTA was added and
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reacted for 1 min (to chelate structural ions in the peptidoglycan layer) before the addition of
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4 mL of 700 mM sucrose and 75 mg lysozyme. The cells were then incubated at room
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temperature for 6 h in the anoxic chamber, followed by the addition of 8 mL of deoxygenated
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ultrapure water to induce osmotic shock. The resulting spheroplasts were immediately
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harvested by centrifugation at 20,000×g for 10 min and washed twice with PBS to remove
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residual lysozyme and chemicals from the incubation. Spheroplasts were then resuspended in
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PBS and centrifuged at 1,200×g for 10 min to remove outer-membrane fragments and 8 ACS Paragon Plus Environment
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impurities before spheroplasts were used for Hg(II) uptake and methylation assays, as
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described for the whole cell experiments.
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Hg(II) and MeHg Analyses. All samples were preserved in HCl (0.5% v/v) at 4°C
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until analysis. An aliquot (0.1–0.2 mL) was analyzed for MeHg, and the remaining aliquot
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was oxidized with BrCl (5% v/v) overnight at 4°C and analyzed for THg by reduction with
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SnCl2 and detection of purgeable elemental Hg(0) using a Lumex RA-915+ analyzer (Ohio
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Lumex Co., Cleveland, OH). MeHg was analyzed following a modified version of EPA
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Method 1630, as described previously.23, 29, 31, 44 Briefly, MeHg was extracted from samples
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via distillation and ethylation, in which corrections for potential matrix interference were
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performed by adding isotopically labeled Me200Hg to each sample as an internal standard.
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The extracted MeHg was quantified using an automated MERX purge and trap system
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(Brooks Rand, Seattle, WA) followed by detection on an inductively coupled plasma mass
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spectrometer (Elan DRCe, PerkinElmer, Shelton, CT). The recovery of the spiked MeHg
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standard was 100±10%, and the detection limit was ~3×10−5 nM MeHg. Negligible loss of
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Hg(II) or MeHg was observed during incubation, as previously reported,29, 49 since all
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experiments were performed in PBS containing a high chloride concentration. Most batch
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experiments were repeated once or twice, each with duplicate samples. All data were then
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reported as an average of all replicate samples, and error bars represent one standard
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deviation.50
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RESULTS
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Determining Hg(II) adsorption and uptake by D. desulfuricans ND132
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The robustness of the DMPS washing technique was evaluated to distinguish Hg(II)
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adsorption onto the cell surface from cellular uptake of Hg(II).23 Cells do not adsorb or take
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up DMPS because of its negatively charged sulfonate head group.23 Therefore, when DMPS
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(0.1 mM) was first equilibrated with Hg(II) (before being mixed with cells), nearly 100% of
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the Hg(II) remained in solution or in the cell filtrate, and a negligible amount of MeHg was
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produced in 72 h (Figure 1a), indicating that DMPS effectively prevented Hg(II) from being
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adsorbed, taken up, or methylated. However, when cells were incubated with Hg(II) for 10
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min in the absence of DMPS, only ~43% of the Hg(II) was found in solution (Figure 1b) due
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to cell adsorption and uptake (with negligible methylation within 10 min). Following the
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DMPS wash, the soluble Hg(II) increased to ~60%, indicating that ~17% of the Hg(II) was
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washed off or desorbed from the cell surface (by difference to samples not treated with
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DMPS), and ~40% of the Hg(II) was taken up by the cells. When cells were incubated with
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Hg(II) for 24 h, a negligible amount of the Hg(II) was found in solution or in the DMPS-
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washing solution (
