Effects of Cellular Sorption on Mercury Bioavailability and

Nov 14, 2016 - However, cells do not preferentially take up Hg(II)–thiol complexes, but Hg(II)–ligand exchange between these complexes and the cel...
1 downloads 11 Views 467KB Size
Subscriber access provided by UNIV OF REGINA

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

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

10

Environmental Science & Technology

1

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

1

ACS Paragon Plus Environment

Page 2 of 23

Page 3 of 23

Environmental Science & Technology

22

Abstract

23

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

24

significant environmental concern because of the bioaccumulation and biomagnification of

25

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

28

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

29

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

31

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

33

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

34

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

35

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

36

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

37

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

ACS Paragon Plus Environment

Environmental Science & Technology

41

Introduction

42

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

43

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

44

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

45

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

46

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

48

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

ACS Paragon Plus Environment

Page 4 of 23

Page 5 of 23

Environmental Science & Technology

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

ACS Paragon Plus Environment

Environmental Science & Technology

85

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

86

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

87

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

88

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

89

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

90

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

91

bioavailability in the environment.

92 93

MATERIALS AND METHODS

94

Bacterial culture and assay conditions

95

Desulfovibrio desulfuricans ND132 was cultured in a modified MOY medium containing

96

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

97

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

98

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

99

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%

102

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

103

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

104

Hg(II) uptake and methylation assays

105

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

ACS Paragon Plus Environment

Page 6 of 23

Page 7 of 23

Environmental Science & Technology

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

117

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

118

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

119

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

123

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

124

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

125

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

126

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

127

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

ACS Paragon Plus Environment

Separately,

0.1

mL

of

Environmental Science & Technology

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.

132

Sequential addition of Hg isotopes and thiols and impact on methylation

133

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

134

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

135

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

137

202

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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)

149

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

ACS Paragon Plus Environment

Page 8 of 23

Page 9 of 23

Environmental Science & Technology

151

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

152

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

153

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

154

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

155

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

156

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

157 158

Results and Discussion

159

Hg(II) methylation and species distribution

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

ACS Paragon Plus Environment

Environmental Science & Technology

173

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

174

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

176

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

178

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

179

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

180

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

181

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

182

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

183

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

185

intracellular materials and thus unavailable for methylation after 24 h.

186

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

187

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

188

followed using stable isotopes of

189

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

190

incubated with the

191

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

ACS Paragon Plus Environment

Page 10 of 23

Page 11 of 23

Environmental Science & Technology

195

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

196

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

203

(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

206

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

207

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

208

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

211

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

212

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

213

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

214

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

215

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

ACS Paragon Plus Environment

Environmental Science & Technology

Page 12 of 23

217

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

220

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

228

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

229

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

231

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

236

(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

ACS Paragon Plus Environment

201

Hg. However, this

Page 13 of 23

Environmental Science & Technology

239

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

242

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

246

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 (