Effects of Cellular Sorption on Mercury Bioavailability and

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Effects of Cellular Sorption on Mercury Bioavailability and Methylmercury Production by Desulfovibrio desulfuricans ND132 Yurong Liu, Xia Lu, Linduo Zhao, Jing An, Ji-Zheng He, Eric M. Pierce, Alexander Johs, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04041 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Environmental Science & Technology

30 25 20 r = 0.98

Methylmercury (% of HgT)

Δ (Methylmercury produced) (% of HgT)

TOC Graphic

60 40 20 0 No cysteine added

15

0

24

Cysteine addition time (h)

10 5

Cysteine added = 50 μM

0 -30

-25

-20 -15 -10 -5 0 Δ (Inorganic Hg desorbed) (% of HgT)

5

ACS Paragon Plus Environment

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Effects of Cellular Sorption on Mercury Bioavailability and Methylmercury

2

Production by Desulfovibrio desulfuricans ND132

3 4 5

Yu-Rong Liu,‡,† Xia Lu,† Linduo Zhao,† Jing An,† Ji-Zheng He‡,¶, Eric M. Pierce,† Alexander Johs,† and Baohua Gu†,*

6 7 8 9 10 11 12

‡

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, China

†

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States

¶

Department of Veterinary and Agricultural Sciences, the University of Melbourne, Victoria, Australia

13 14 15 16 17 18 19 20 21

*

Corresponding Author: Email: [email protected]; Phone: (865)-574-7286; Fax: (865)-576-8543

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Abstract

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Microbial conversion of inorganic mercury (IHg) to methylmercury (MeHg) is a

24

significant environmental concern because of the bioaccumulation and biomagnification of

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toxic MeHg in the food web. Laboratory incubation studies have shown that, despite the

26

presence of large quantities of IHg in cell cultures, MeHg biosynthesis often reaches a plateau

27

or a maximum within hours or a day by an as yet unexplained mechanism. Here we report

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that mercuric Hg(II) can be taken up rapidly by cells of Desulfovibrio desulfuricans ND132,

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but a large fraction of the Hg(II) is unavailable for methylation because of strong cellular

30

sorption. Thiols, such as cysteine, glutathione, and penicillamine, added either

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simultaneously with Hg(II) or after cells have been exposed to Hg(II), effectively desorb or

32

mobilize the bound Hg(II), leading to a substantial increase in MeHg production. The amount

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of thiol-desorbed Hg(II) is strongly correlated to the amount of MeHg produced (r = 0.98).

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However, cells do not preferentially take up Hg(II)-thiol complexes, but Hg(II)-ligand

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exchange between these complexes and the cell-associated proteins likely constrains Hg(II)

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uptake and methylation. We suggest that, aside from aqueous chemical speciation of Hg(II),

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binding and exchange of Hg(II) between cells and complexing ligands such as thiols and

38

naturally dissolved organics in solution is an important controlling mechanism of Hg(II)

39

bioavailability, which should be considered when predicting MeHg production in the

40

environment.

2



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Introduction

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Increased mercury (Hg) input and its subsequent conversion to neurotoxic

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methylmercury (MeHg) in the environment is a growing concern because of the

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bioaccumulation and biomagnification of MeHg in the food web.1-4 Methylmercury is formed

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by a group of anaerobic microorganisms possessing the key gene cluster hgcAB.4-6 It was

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shown that Hg methylation is an intracellular process, although significant knowledge gaps

47

exist with respect to Hg(II) uptake and the controls of Hg(II) uptake and methylation by these

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organisms.7-10 The HgcA protein, which is required for Hg(II) methylation, consists of a

49

putative transmembrane domain and a corrinoid binding domain facing the cytosol,4,6 which

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has been implicated with the transfer of methyl groups derived from one-carbon metabolic

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processes to Hg(II).4,11 The uptake and transport of Hg(II) into the cytoplasm are thus

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essential and thought to be controlled by certain metal transporters such as those related to

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divalent metal uptake.10 Previous studies have proposed that neutral HgCl2 and Hg(HS)2

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species passively diffuse into the cell, where they are methylated.12-15 However, recent

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studies questioned this hypothesis and suggested that Hg(II) uptake is an active process

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mediated by facilitated transport and exchange mechanisms.10,16-18 Certain thiols such as

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cysteine were found to greatly enhance Hg(II) uptake and methylation in Geobacter

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sulfurreducens, and it was proposed that G. sulfurreducens strains have a specific uptake

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mechanism for the Hg(II)-cysteine complex.17 Additional studies, however, question this

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hypothesis because cysteine enhanced methylation is found to be specific to microbial species

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and depends on both the cysteine concentration and reaction time.7-9 For example, cysteine

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was shown to inhibit Hg(II) methylation by a ∆omcBESTZ mutant of G. sulfurreducens PCA, 3


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which is deficient in five outer-membrane c-type cytochromes required for dissimilatory

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metal reduction.9 Thiols such as glutathione and penicillamine were found to enhance Hg(II)

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methylation by D. desulfuricans ND132 but completely inhibited Hg(II) methylation by G.

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sulfurreducens PCA.7,8,18 Recent studies with E. coli strains have also suggested that cells do

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not take up the entire Hg-thiol complex, but rather the Hg(II) in the Hg-thiol complex is

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transferred to a transport protein on the cell membrane and subsequently internalized.19

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In pure culture studies, MeHg biosynthesis usually reaches a plateau within a few hours

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to one day, and methylation often stalls even when a large quantity of inorganic Hg(II) exists

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in the system.7,8,20,21 Previous studies speculated that this incomplete conversion of inorganic

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Hg(II) to MeHg may result from the saturation of methylating enzymes22 or from limitations

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in the methyl donor.8 However, subsequent studies ruled out this possibility because assays

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conducted either with or without a carbon source, an electron donor/acceptor, or essential

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nutrients showed little difference in Hg(II) methylation.8 Furthermore, Hg(II) methylation

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rates by D. desulfuricans ND132 are proportional to added Hg(II) concentrations over a wide

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range (0.25 to 40 nM),7,8 and starved cells methylate Hg(II) as quickly as cells provided with

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energy-generating substrates, suggesting that energy requirements for the methylation of

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nanomolar Hg(II) levels are small. Given these considerations, it was speculated that stalled

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or incomplete Hg(II) methylation may be a result of Hg(II) conversion into a form that is

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unavailable for intracellular uptake.8 Strong sorption of Hg(II) to cell surface binding sites

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may serve as a sink for Hg(II), lowering its bioavailability. Moreover, competition between

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cell surface sorption and intracellular uptake may limit Hg(II) methylation.

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We hypothesize that Hg(II) cellular sorption is the main cause of limited Hg(II) 4



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bioavailability, and that the presence of thiols can result in desorption or liberation of the

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sorbed Hg(II) enhancing methylation. Using the strain Desulfovibrio desulfuricans ND132 as

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a model organism,7 we examined the cellular sorption, desorption, and exchange of Hg(II) in

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the presence or absence of thiol ligands including cysteine, glutathione (GSH), and

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penicillamine (PEN). We propose that cellular binding and exchange of Hg(II) with these

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complexing ligands in solution is an important controlling mechanism of Hg(II) uptake and

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bioavailability in the environment.

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MATERIALS AND METHODS

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Bacterial culture and assay conditions

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Desulfovibrio desulfuricans ND132 was cultured in a modified MOY medium containing

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40 mM fumarate and 40 mM pyruvate at 30°C.20,23 Cells were harvested during the

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mid-exponential phase with an optical density (OD) of 0.5 to 0.6 and washed three times by

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repeated centrifugation (at 1200g, 10 min., 25°C) and resuspension in a deoxygenated

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phosphate-buffered saline (PBS) at pH 7.4. PBS consisted of 0.14 M NaCl, 3 mM KCl, 10

100

mM Na2HPO4, and 2 mM KH2PO4. The buffer was first autoclaved and deoxygenated by

101

boiling and subsequently kept in an anaerobic glove chamber (Coy) with ~98% N2 and 2%

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H2 for at least 24 h before use. All the washing steps and Hg(II) methylation assays were

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conducted in the glove chamber as previously described.20,21

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Hg(II) uptake and methylation assays

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Hg(II) uptake and methylation assays were conducted in PBS in 4 mL amber glass vials

106

(National Scientific). Each vial contained washed ND132 cells (5×108 cell/mL), 5


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supplemented once (at time zero) with 1 mM each of pyruvate and fumarate as the respective

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electron donor and acceptor. The Hg(II) working solution was freshly prepared daily from a

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stock solution of 50 µM HgCl2 in 1% HCl and added last to the cell suspension to provide a

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final concentration of 25 nM Hg(II). All the vials were immediately sealed with a

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PTFE/silicone screw cap and kept in the dark on an orbital shaker. At selected time points,

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replicate sample vials (6-8) were taken out of the anoxic chamber and analyzed for Hg and

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MeHg species distributions as follows. Half of the samples (3–4) were filtered through

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0.2-µm syringe filters to remove cells and then preserved in HCl (0.5% v/v) at 4°C until

115

analysis. An aliquot (0.2–0.4 mL) was used to analyze the soluble MeHg (MeHgsol)

116

(described below). The remaining aliquot was oxidized with BrCl (5% v/v) overnight at 4°C

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and analyzed for the total soluble Hg. Thus, the soluble inorganic Hg(II) (IHgsol) could be

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calculated by the difference between total soluble Hg and MeHgsol.9,24 An aliquot of the

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unfiltered sample (0.2–0.4 mL) was also analyzed for total Hg (HgT) following its oxidation

120

in

121

2,3-dimercapto-1-propanesulfonic acid (DMPS), a Hg-chelating agent, was added to each of

122

the remaining 3–4 samples (without filtration) to yield a final concentration of 100 µM

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DMPS to wash off cell surface adsorbed Hg(II) (IHgad) and MeHg (MeHgad). Samples were

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equilibrated for ~15 min and filtered, and the filter-passing solutions were analyzed for the

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sum of wash-off soluble Hg: IHgwash (= IHgad + IHgsol) and MeHgwash (= MeHgad + MeHgsol),

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as described above. The internalized or intracellular IHg (IHgcell) and MeHg (MeHgcell) were

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calculated by the difference between IHgtotal or MeHgtotal and the wash-off IHgwash or

128

MeHgwash. All analytical errors were calculated as one standard deviation from 3 or more

BrCl

and

total

MeHg

(without

oxidation).

6


Separately,

0.1

mL

of


129

replicate samples of one or more batch experiments. For differences between 2 or more

130

measurements, the propagated errors were calculated as √ + , where a and b are the

131

analytical errors determined from measurements a and b, respectively.

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Sequential addition of Hg isotopes and thiols and impact on methylation

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To investigate whether the cells remained active and were able to methylate Hg after

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reaching the methylation plateau (~24 h), different Hg isotopes were added sequentially to

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the cell suspension at 0, 24, and 48 h. The washed cells were first incubated with 25 nM

136

201

Hg (as HgCl2) under the same conditions as described above. Another aliquot of 25 nM

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202

Hg was added at time zero (i.e., with

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Me202Hg production was determined at 72 h. To determine whether D. desulfuricans would

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preferentially take up and methylate Hg(II)-thiol complexes, similar methylation assays were

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performed by the sequential addition of pre-mixed 202Hg-cysteine complexes at various Hg(II)

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to cysteine molar ratios (i.e., 1:0, 1:2, 1:10 and 1:100) 24 h after cells were first incubated

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with 201Hg (without cysteine). Parallel experiments were performed by sequential additions of

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cysteine taking place after cells were incubated with Hg(II) at various times. Additional

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studies were carried out in a similar manner to determine the effects of thiols (cysteine, GSH,

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or PEN, 50 µM each) on Hg methylation and species distribution.

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Hg and MeHg analyses

201

Hg) or at 24 h and 48 h. Then, Me201Hg or

147

The total Hg was determined after samples were oxidized with BrCl overnight followed

148

by a reduction with SnCl2 using a Brooks Rand MERX automated system (Seattle, WA)

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interfaced to an inductively couple plasma mass spectrometer (ICP-MS) (Elan-DRCe,

150

Perkin-Elmer, Inc. Shelton, CT).4,20,21 For 201Hg and 202Hg isotope analyses, ambient IHg was 7


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added as an internal standard. MeHg was analyzed using a modified EPA Method 1630, with

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Me200Hg added as an internal standard, as described previously.4,20,21 MeHg was first

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extracted from the sample matrix via distillation, ethylation, and trapping on a Tenax column

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via N2-puring. Thermal desorption and separation by gas chromatography were applied,

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followed by the detection of Hg by ICP-MS. The recovery of spiked MeHg standards was

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100±10%, and the detection limit was about 3×10-5 nM MeHg.

157 158

Results and Discussion

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Hg(II) methylation and species distribution

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Reactions between Hg(II) (as HgCl2, 25 nM) and washed cells of D. desulfuricans

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ND132 (5×108 cells/mL) in PBS resulted in rapid MeHg production within hours, and about

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28% of the Hg(II) was converted to MeHg (as MeHgtotal) within 24 h (Figure 1a). However,

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methylation stalled or approached a plateau after 24 h, although a large percentage of the

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Hg(II) (~72% IHgtotal) was in the system (Figure 1b). An analysis of the total Hg (MeHgtotal +

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IHgtotal) in cell suspension indicated a good mass balance (Figure 1b), suggesting that loss of

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Hg(II) to container walls was small (within 2–7%), as previously reported.20,25 Additionally,

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we did not observe Hg(II) reduction or loss of Hg(0). The observed methylation activity is

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however consistent with previous studies with an as yet unexplained mechanism.7,8,21,24

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To elucidate this mechanism, we determined Hg(II) and MeHg sorption or uptake and

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species distribution on cells using DMPS as an effective washing agent for distinguishing

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cell-surface adsorbed Hg(II) or MeHg from those entered inside the cells.18,25 DMPS is a

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strong chelator for Hg(II),26 owing to its two ortho-positioned –SH functional groups. The 8



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presence of as little as 50 µM DMPS effectively prevented Hg(II) sorption, uptake or

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methylation by ND132 cells, resulting in ~100% of the added Hg(II) in solution (SI Figure

175

S1a). When Hg(II) was reacted with the cells first, the sorbed Hg(II) could also be mostly

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desorbed after washing with 100 µM DMPS (SI Figure S1b). Additionally, the cells showed

177

no interaction or uptake of DMPS, probably because of its negatively charged sulfonate head

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group (SI Figure S1b). Using this technique, we demonstrated that MeHg was rapidly

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exported to the solution phase (~80% of the synthesized MeHg), leaving a small percentage

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either adsorbed on or remaining inside the cells (Figure 1a). For inorganic Hg(II), a large

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portion (IHgcell, ~90% of the total IHg) was rapidly internalized into the cell and could not be

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washed off with DMPS (Figure 1b). Only a small percentage of the IHg (~5%) was sorbed on

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the cell surface. As a result, the soluble Hg(II) (IHgsol) decreased concomitantly to < 10%

184

after 6 h. These results suggest that the internalized IHgcell must be immobilized to

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intracellular materials and thus unavailable for methylation after 24 h.

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We further examined if the stalled methylation may result from decreased microbial

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activity after 24 h. In this experiment, MeHg production and Hg species distribution were

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followed using stable isotopes of

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either together or sequentially, with one of the isotopes (202Hg) added 24 h after cells were

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incubated with the

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amounts of Me201Hg and Me202Hg (~6 nM or 25%) were produced after 3 days (SI Figure

192

S2a, left columns), as expected. The distribution of IHg species after 72 h was also similar

193

(SI Figure S2b, left columns). When

194

201

201

201

Hg and 202Hg (as HgCl2, 25 nM each). They were added

Hg isotope. When both isotopes were added at time zero, similar

202

Hg was added 24 h after cells were incubated with

Hg, slightly lower amounts of Me202Hg (~4.5 nM or 19%) were produced (SI Figure S2a, 9


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middle columns), indicating that cells remained active and were still capable of methylating

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similar amounts of Hg(II). Slightly higher amounts of Me201Hg were formed because

197

was added at time zero; the addition of

198

previously complexed

199

48 h (after the first addition of

200

202

201

much as those formed with one-time addition of 202Hg at time zero or at 24 h (SI Figure S2a,

202

right columns). These results clearly indicate that the stalled methylation activity after 24 h

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(Figure 1) cannot be attributed to loss of microbial activity, but rather to a limited availability

204

of substrate or Hg(II); cells remained active methylating Hg(II) for at least 3 days.

205

Effects of thiols on Hg(II) species distribution and enhanced methylation

201

202

Hg

Hg may have displaced or mobilized some

Hg inside the cell. When another aliquot of 202

201

202

Hg(II) was added at

Hg at 24 h), cells again methylated a similar amount of

Hg and produced a total of 11 nM

202

MeHg the next day; this amount was about twice as

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Complexes between Hg(II) and thiols such as cysteine and glutathione can enhance

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methylation by D. desulfuricans ND132,8,18,23 but it is unclear whether enhanced methylation

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results from preferential cellular uptake of Hg(II)-thiol complexes, or from thiol-induced

209

desorption and thus increased Hg(II) bioavailability, or both. Our results indicate that, when

210

Hg(II) methylation reached its plateau at 24 h, addition of cysteine (50 µM) nearly doubled

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MeHg production, and more than 50% of the Hg(II) became methylated (Figure 2a). Addition

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of a higher concentration of cysteine (500 µM) resulted in more MeHg production (~70%)

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(Figure 2a), but sequential addition of the same concentration of cysteine (50 µM or 500 µM)

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at 48 h yielded only slightly increased MeHg production. Furthermore, similar amounts of

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MeHg were produced regardless of whether cysteine was added with Hg(II) at time zero or at

216

a later time (i.e., 2 or 24 h after cells were incubated with Hg[II]) (Figure 2b). These 10



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observations confirm that the stalled Hg(II) methylation is largely a result of Hg(II) binding

218

to cellular materials because the stalled methylation could be restored by the addition of

219

thiols (e.g., cysteine), regardless of whether the thiol is added along with Hg(II) at time zero

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or after Hg(II) is immobilized on the cells. Relatively high thiol concentrations were more

221

effective in desorbing or mobilizing the sorbed Hg(II) and thus produced more MeHg. We also determined Hg(II) methylation by incubating cells with pre-mixed

222 223

202

224

after cells were first reacted with

225

These experiments were designed to further clarify whether the thiol-enhanced Hg(II)

226

methylation is a result of preferential uptake of the Hg(II)-thiol complexes or of thiol induced

227

mobilization of the cell-bound Hg(II). Speciation calculations indicate that 1:1 Hg-cysteine

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complexes dominate (72–97%) at Hg:Cysteine ratios of 1:10 or higher, and 1:2 complexes

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dominate (74%) at the Hg:Cysteine ratio of 1:100 in PBS.9,17 Compared to samples without

230

cysteine (or Hg:Cysteine at 1:0, Figure 2c), the addition of

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1:100 Hg:Cysteine) slightly increased the production of Me201Hg 24 h after cells reacted with

232

201

233

increase was small and remained unchanged at Hg:Cysteine ratios between 1:2 and 1:100.

234

There was also a negligible increase in Me202Hg production at Hg:Cysteine ratios between

235

1:0 and 1:10, and only a slight increase in methylation at the Hg:Cysteine ratio of 1:100

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(Figure 2c). These results indicate that, even with an excess amount of cysteine, a low

237

cysteine concentration (< 2.5 µM, or 1:100 Hg:Cysteine) is ineffective in enhancing Hg(II)

238

methylation, as observed in a previous study.9 High thiol concentrations are necessary to

Hg-cysteine complexes at various Hg(II) to cysteine molar ratios (i.e. 1:2, 1:10 and 1:100) 201

Hg for 24 h (or approached its methylation plateau).

202

Hg-cysteine complexes (1:2 to

Hg (Figure 2c), since cysteine may have mobilized some cell-bound

11


201

Hg. However, this

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result in mass action or ligand exchange and desorption of the sorbed Hg(II) (Figure 2a). The

240

results thus support the notion that Hg(II)-cysteine complexes are not the preferred species

241

for Hg(II) uptake and methylation. Instead, the observed enhancement of Hg(II) methylation

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by thiols is caused mainly by remobilizing or exchanging the cell-bound Hgcell. This

243

observation corroborates with the proposed uptake mechanism of Hg(II) in the Hg-thiol

244

complex by the E. coli strain.19

245

The effect of cysteine or thiols in mobilizing cellular bound Hg(II) is further illustrated

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by detailed studies of Hg(II) methylation and species distribution during Hg-cell interactions

247

(Figure 3; SI Figure S3). Compared to experiments without cysteine addition (Figure 1),

248

Hg(II) methylation increased substantially when a large excess amount of cysteine (50 µM)

249

was added 2 h after commencing the Hg(II) methylation assay (Figure 3a). After 24 h the

250

total MeHg production increased to ~53% of the total Hg added, or ~2 times higher than that

251

in the absence of cysteine. Similarly, we found that most of the synthesized MeHg was

252

exported to the extracellular environment, leaving only a small percentage either sorbed

253

(MeHgad) or remaining inside the cells (MeHgcell) (Figure 3a). Most of the added inorganic

254

Hg(II) was either taken up (IHgcell) or methylated, and little IHg (

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