Arsenic Methyltransferase is Involved in Arsenosugar Biosynthesis by

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Arsenic Methyltransferase is Involved in Arsenosugar Biosynthesis by Providing DMA Xi-Mei Xue, Jun Ye, Georg Raber, Kevin A. Francesconi, Gang Li, Hong Gao, Yu Yan, Christopher Rensing, and Yong-Guan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04952 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Environmental Science & Technology

1

Arsenic Methyltransferase is Involved in Arsenosugar

2

Biosynthesis by Providing DMA

3

Xi-Mei Xue1, Jun Ye1, Georg Raber2, Kevin A. Francesconi2, Gang Li1, Hong

4

Gao3, Yu Yan1, Christopher Rensing4 and Yong-Guan Zhu1,5*

5 6

1

7

Environment, Chinese Academy of Sciences, Xiamen 361021, China.

8

2

Institute of Chemistry, University of Graz, Graz, Austria

3

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of

9

Key Laboratory of Urban Environment and Health, Institute of Urban

10

Hydrobiology, Chinese Academy of Sciences, Wuhan, China

11

4

12

China

13

5

14

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou,

State Key Laboratory of Urban and Regional Ecology, Research Center for

15 16

*

Address Correspondence to Yong-Guan Zhu,

17

Address: Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei

18

Road, Xiamen 361021, China

19

Phone number: +86(0)592 6190997

20

Fax number: +86(0)592 6190977

21

Email address: [email protected] 1

ACS Paragon Plus Environment


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Abstract

23

Arsenic is an ubiquitous toxic element in the environment, and organisms have

24

evolved

25

biotransformation mechanisms have mainly focused on arsenate (As(V)) reduction,

26

arsenite (As(III)) oxidation, and arsenic methylation; little is known, however, about

27

the pathway for the biosynthesis of arsenosugars, which are significant arsenic

28

transformation products. Here, the involvement of As(III) S-Adenosylmethionine

29

methyltransferase (ArsM) in arsenosugar synthesis is demonstrated for the first time.

30

Synechocystis sp. PCC 6803 incubated with As(III) or monomethylarsonic acid

31

(MMA(V)) produced dimethylarsinic acid (DMA(V)) and arsenosugars, as

32

determined by high performance liquid chromatography–inductively coupled plasma

33

mass spectrometry (HPLC/ICPMS). Arsenosugars were also detected in the cells

34

when they were exposed to DMA(V). A mutant strain Synechocystis ∆arsM was

35

constructed by disrupting arsM in Synechocystis sp. PCC 6803. Methylation of

36

arsenic species was not observed in the mutant strain after exposure to arsenite or

37

MMA(V); when Synechocystis ∆arsM was incubated with DMA(V), arsenosugars

38

were detected in the cells. These results suggest that ArsM is a required enzyme for

39

the methylation of inorganic arsenicals, but not required for the synthesis of

40

arsenosugars from DMA, and that DMA is the precursor of arsenosugar biosynthesis.

41

The findings will stimulate more studies on the biosynthesis of complex

different

arsenic

detoxification

strategies.

Studies

on

arsenic

2


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organoarsenicals, and lead to a better understanding of the bioavailability and function

43

of the organoarsenicals in biological systems.

44

45

TOC artwork

46

47

Keywords: arsenosugar, ArsM, Synechocystis sp. PCC 6803

48

Introduction

49

Arsenic is distributed in the environment in various species including inorganic

50

arsenic species As(III) and As(V), and many organic arsenic species such as DMA,

51

arsenobetaine, arsenocholine, arsenosugars, and arsenolipids 1. Some organisms, in

52

particular marine organisms, can methylate inorganic arsenic and synthesize a range

53

of arsenic compounds containing riboses, collectively termed arsenosugars. The

54

arsenosugars are present at high concentrations in marine algae, and have also been

55

found in freshwater organisms

56

and fungi 5. Although more than 20 arsenosugars have been reported, the four most

57

common forms are: glycerol arsenosugar (sugar 1), phosphate arsenosugar (sugar 2),

58

sulfonate arsenosugar (sugar 3), and sulfate arsenosugar (sugar 4) (Fig. S1).

2, 3

, and in terrestrial organisms such as earthworms

4

3



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The arsenosugars appear to play a central role in arsenic transformation

60

processes. They are considered to be the likely precursors to arsenobetaine 6, the main

61

arsenic compound in marine animals 7, which has low toxicity and is not metabolized

62

in animals

63

10

64

biotransformation in primary producers. In view of their chemical structures,

65

arsenosugars are the likely immediate precursors of arsenosugar phospholipids

66

Because of the arsenosugars’ pivotal role in the cycling of arsenic, research on the

67

molecular mechanism of their biosynthesis could provide a theoretical basis for

68

studies on the many other organoarsenicals in the environment.

69

8, 9

. Moreover, arsenosugar phospholipids have been found in marine alga

and fresh cyanobacteria

11

, and might be the final products of arsenic

In 1987 Edmonds and Francesconi

14

12, 13

.

proposed a scheme for the biosynthesis of

70

arsenosugars whereby S-adenosylmethionine contributed the two methyl groups and

71

the ribose group. Subsequent studies on arsenosugar biosynthesis have focused on the

72

analysis of arsenic metabolites through either short-term

73

arsenic

74

S-Adenosylmethionine methyltransferase (ArsM) was required for arsenosugar

75

biosynthesis.

2

or long-term exposure to

15

. However, no direct evidence had been reported to indicate that

76

The oxygenic photo-autotrophic unicellular cyanobacterium Synechocystis sp.

77

PCC 6803 is a well-established and widely used experimental model to study

78

molecular mechanisms of metabolic pathways because the complete genomic

79

sequence has been determined

16, 17

and it is able to integrate foreign DNA into its

4


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genome through homologous recombination 18. To date, ArsM homologues have been

81

identified in bacteria 19, archaea 20 and mammals 21, where they catalyze the formation

82

of methylated arsenic species from As(III). In addition, the arsM genes in

83

Cyanidioschyzon sp. isolate 5508

84

Rhodopseudomonas palustris

85

repressor-type repressors (ArsR). Recently, ArsM from Synechocystis sp. PCC 6803

86

was found to catalyze the formation of a number of methylated intermediates from

87

As(III)

88

Synechocystis sp. PCC 6803 3.

19

22

, Methanosarcina mazei Go1

23

, and

appear to be regulated by arsenical resistance operon

24

, and shortly after it was observed that arsenosugars were produced in

89

Based on these earlier studies, we hypothesized that ArsM is involved in

90

arsenosugar synthesis. We therefore disrupted arsM in Synechocystis sp. PCC 6803

91

by deleting the part of arsM and inserting a kanamycin resistance cassette in the

92

coding region of the gene, and supplemented Synechocystis wild type (WT) and

93

Synechocystis ∆arsM with As(III) or monomethylarsonic acid (MMA(V)) to

94

determine if ArsM can catalyze an early step of arsenosugar biosynthesis. Moreover,

95

Synechocystis ∆arsM and Synechocystis WT were treated with DMA(V) to determine

96

if DMA is the starting compound of arsenosugar biosynthesis.

97 98

MATERIALS AND METHODS

99

Reagents and solutions. Ammonium dihydrogen phosphate (NH4H2PO4),

100

ammonium bicarbonate (NH4HCO3), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

5



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acid (HEPES), glycerol, sodium chloride (NaCl), imidazole, sodium arsenite

102

(Na2AsO2), and sodium arsenate (Na3AsO4·12H2O), reagent grade, were purchased

103

from

104

β-D-1-Thiogalactopyranoside (IPTG), and S-(5′-Adenosyl)-L-methionine chloride

105

(SAM) were bought from Sigma-Aldrich Co (Poole, Dorset, UK). Malonic acid and

106

ammonium hydroxide (NH3·H2O) were from Fluka (Buchs, Switzerland). Sodium

107

monomethylarsonate

108

AccuStandard. Inc (New Haven, CT, USA). Arsenosugar standards were extracted

109

and purified from Fucus serratus 25 which has been validated to contain the four most

110

common oxo-arsenosugars (Fig. S1). 5´-Deoxy-5´-dimethylarsinyladenosine was

111

synthesized previously 26.

BZL

(Beijing,

China).

and

sodium

L-Glutathione

reduced

dimethylarsinate

were

(GSH),

Isopropyl

purchased

from

112

Disruption of arsM in Synechocystis sp. PCC 6803. The mutagenesis of

113

Synechocystis arsM (GenBank accession number HM776638) was performed as

114

follows: 977 bp of arsM was amplified with the primers SsArsMF and SsArsMR

115

(Table S1) and cloned into pMD18T simple vector (TaKaRa, Dalian, China) to yield

116

plasmid p18T-arsM. A kanamycin resistance (KamR) cassette amplified from plasmid

117

pHSG299 (TaKaRa, Dalian, China) with the primers kanF and kanR (Table S1) was

118

inserted into the arsM coding region of p18T-arsM at the NcoI and BspMII sites

119

producing plasmid pTarsMKan. The plasmid was transformed Synechocystis WT as

120

previously described 27. Exponential-phase cells (15 mL) were centrifuged at 6,000 g

121

for 10 min, and washed one time with 30 mL fresh BG11 medium. The pellet was

6


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122

suspended in 1.5 mL fresh BG11 medium containing 30 µL pTarsMKan (about 100

123

µg mL-1). The mixture of cells and plasmids was incubated for 5 hours under

124

continuous light, and then spread on the nitrocellulose membranes lying on BG11

125

plates without antibiotic. After 20 hours, the filters were transferred to another BG11

126

plates amended with 30 µg mL-1 kanamycin. The transformant Synechocystis ∆arsM

127

was obtained after four serial streak-purifications of a single colony on BG11 plates,

128

then determined whether arsM in Synechocystis ∆arsM was completely replaced

129

using PCR with diagnostic primers (Fig. S2). Confirmed Synechocystis ∆arsM was

130

cultivated in 100 mL of BG11 medium with 50 µg mL-1 kanamycin at 30oC.

131

Arsenic speciation analysis in Synechocystis. To prevent self-shading of the

132

culture, an optical density at 730 nm (OD730) of 0.1 was used at the beginning of

133

culture. After two weeks of continuous culture, 100 mL of stationary phase

134

Synechocystis cells were harvested by centrifuging and washing with ice-cold

135

phosphate buffer as previously described

136

analysis. The FP120 FastPrep cell disruptor (Bio 101/Savant Instruments, Holbrook,

137

NY, USA) and 500-µm-diameter glass beads were used during lysis. Approximately

138

0.5 g beads and 0.01 g lyophilized samples suspended in 1 mL Milli-Q water were

139

transferred to 2 mL Eppendorf tubes. Bead-beating was performed three times at 6.5

140

m s-1 for 60 s with cooling intervals of 5 min at 4oC between each bead-beating. The

141

homogenates were subsequently centrifuged at 13680 g on a Hermle Z326K (Hermle

142

Labortechnik GmbH, Wehingen, Germany) for 15 min at 4oC. The supernatant was

24

, and lyophilized for arsenic species

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pipetted into 15 mL polypropylene tubes. This procedure was repeated three times and

144

the supernatants combined with that from the first extraction. For each vial,

145

approximately 600 µL of supernatant was filtered through a 0.22 µm membrane filter

146

(MF-Mixed Cellulose Ester Membrane Filter, Millipore, Billerica, MA) into 1 mL

147

crimp/snap polypropylene vials (Agilent Technologies, Palo Alto, CA, USA) for

148

analysis. The culture medium was filtered directly through a 0.22 µm membrane filter

149

for arsenic speciation analysis.

150

HPLC/ICPMS measurements were performed using an Agilent 1200 HPLC for

151

separations coupled with an Agilent 7500cx ICPMS for element detection. A

152

Hamilton PRP-X100 anion-exchange column from Hamilton Company was used in

153

HPLC. The ICPMS was tuned for monitoring of m/z 75 (arsenic). At the same time,

154

m/z 77 and 82 (selenium) were monitored to verify that ArCl+ interferences were not

155

present. Identification of arsenic species was performed by comparing the retention

156

times with those of arsenic standards.

157

Anion-exchange HPLC/ICPMS can separate three of the four main arsenosugars

158

from As(III), but the sugar 1 comes near the void volume together with As(III) and

159

TMAO. To further confirm that Synechocystis treated with DMA(V) can produce

160

arsenosugars, the extract of Synechocystis cells was analyzed using HPLC with

161

simultaneous ICPMS and electrospray ionization mass spectrometry (ESIMS, Agilent

162

6460 Triple Quadrupole LC/MS) detection. The column was PRP-X100

8


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anion-exchange column (4.6×150 mm, 5 µm) with a Hamilton pre-column, and the

164

mobile phase was changed to 5 mM malonic acid (pH 5.6, adjusted with NH3·H2O).

165

Purification of ArsM and in vitro/vivo assays. To generate an arsM expression

166

vector, arsM was inserted into vector pET22b (Novagen, Madison, USA) to produce

167

pET22b-arsM. ArsM was expressed in E. coli strain Rosetta bearing pET22b-arsM,

168

purified by Ni(II)-NTA chromatography. ArsM was eluted with a buffer consisting of

169

20 mM HEPES (pH 7.2) containing 10% (w/v) glycerol, 0.3 M NaCl, and 0.5 M

170

imidazole,

171

electrophoresis (SDS-PAGE). Fractions containing purified ArsM were pooled and

172

concentrated by using a 10-kDa cutoff Amicon Ultrafilter (Millipore), then cooled

173

with liquid nitrogen and stored at -80oC. Protein concentrations were determined by

174

absorbance at 280 nm. As(III) methylation experiment with purified ArsM was

175

performed in a buffer consisting of 40 mM NH4HCO3 buffer (pH 7.5), containing 5

176

mM GSH, 1 mM SAM, 5 µM ArsM, and 10 µM As(III). The experiment were

177

conducted with a temperature gradient overnight to measure the effect of temperature

178

on the enzyme activity or at 37oC to look for the optimum pH.

and identified by sodium

dodecyl sulfate polyacrylamide

gel

179

For in vivo reactions, Rosetta bearing plasmid pET22b or pET22b-arsM was

180

incubated at 37oC overnight. Late exponential phase cells were diluted 100-fold into 5

181

mL Luria–Bertani (LB) medium containing 100 µg mL-1 ampicillin. Cells were grown

182

to an OD600 of 0.5, at which point 0.5 mM IPTG and 1 µM As(III), or MMA(V), or

183

DMA(V) were added to induce expression of ArsM and subsequent transformation of

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arsenic. The neutral species As(III) and the cationic species TMAO both run close to

185

the void volume (and thus are not separated) on the anion column (PRP X-100),

186

arsenic produced by cells and in vitro reaction was treated with H2O2 to oxidize As(III)

187

to As(V), which is well retained on the anion column and can be clearly separated

188

from the other arsenic species, before analysis in order to show the presence of

189

TMAO.

190

Arsenic toxicity assay. To investigate the growth of cyanobacteria exposed to

191

As(V) or As(III), we measured OD730 with a UV-visible spectrophotometer (UV-6300

192

Double Beam Spectrophotometer, Mapada, China) of cyanobacteria during their 14

193

days of incubation at 1 mM As(V), or 1 mM As(III), or arsenic-free. Axenic cultures

194

of Synechocystis WT and Synechocystis ∆arsM were grown in 150 mL Erlenmeyer

195

flasks containing 50 mL sterilized BG-11 medium (50 µg mL-1 kanamycin added for

196

Synechocystis ∆arsM culture) at 30oC with shaking at 96 rpm under continuously

197

white light illumination (40 µmol photons m-2 s-1).

198 199

RESULTS

200

E. coli strain Rosetta bearing pET22b-arsM methylated arsenic in vivo. E.

201

coli strain Rosetta bearing pET22b-arsM was found to methylate As(III) or MMA(V)

202

to DMA(V) after 3 hours of IPTG induction in LB medium. Most of As(III) and part

203

of MMA(V) were converted into DMA(V) (Fig. 1a and b). These results

204

demonstrated that heterologous expression of arsM from Synechocystis in E. coli

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conferred the ability to methylate arsenic. When E. coli Rosetta cells were incubated

206

with DMA(V), trimethylarsine oxide (TMAO) was not generated (Fig. 1c).

207

In vitro arsenic methylation by ArsM. ArsM was purified from Rosetta cells for

208

in vitro assays. ArsM activity at 37oC was tested over a pH range 6.5 to 8.5 and pH

209

7.5 was found to be optimal (data not shown). When assayed at six temperatures

210

ranging from 25oC to 37oC, purified ArsM converted As(III) to MMA(V), DMA(V),

211

and TMAO (Table 1). However, the relative amounts of MMA(V) and TMAO were

212

strongly dependent on temperature, with MMA predominant at lower temperatures.

213

For example, there was only MMA(V) and DMA(V) present at 25oC with

214

MMA/TMAO ratios being 18.5 and 0.16 at 30oC and 37oC, respectively. However at

215

a lower temperature the conversion of As(III) to MMA was faster than the following

216

conversion of DMA to TMAO due to an overall decrease in enzymatic activity; while

217

at higher temperature the latter conversion was much faster. These data suggested that

218

temperature affected the activity of ArsM. ArsM at low temperature could only

219

transform As(III) to MMA, while at higher temperatures further methylation of MMA

220

to DMA or to TMA occurred before releasing methylated arsenic from ArsM.

221

ArsM is involved in arsenosugar biosynthesis. Generation of Synechocystis

222

∆arsM was confirmed by PCR amplification of the DNA region containing arsM. As

223

shown in Fig. S2, PCR amplification with primers whose sequences flank arsM

224

yielded an expected fragment (lane 1) in Synechocystis WT cells; the same

225

amplification also yielded a fragment of 1948-bp (lane 2) from Synechocystis ∆arsM,

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226

which is exactly as expected when a partial arsM gene-containing fragment of 91-bp

227

was replaced by a 1062-bp KamR. The absence of intact arsM in lane 2 confirmed the

228

total replacement of all wild type copies of arsM in Synechocystis ∆arsM. PCR

229

amplification also showed the successful construction of Synechocystis ∆arsM::arsM.

230

In this mutant, the intact arsM and CamR (lane 4) could be detected.

231

After exposure to As(III), Synechocystis WT accumulated As(V) as the most

232

abundant arsenic species, followed by As(III), and small amounts of MMA(V),

233

DMA(V), and sugar 1 & 2 (Fig. 2a). Insertional inactivation of arsM in Synechocystis

234

sp. PCC 6803 (∆arsM), however, resulted in the complete loss of arsenic methylation

235

ability (Fig. 2a, Fig. S3 and S5). When Synechocystis ∆arsM::arsM was exposed to

236

As(III), MMA(V), or DMA(V), further methylated arsenic or arsenosugars were

237

detected in the cells (Fig. S4).

238

To explore the starting point of biosynthesis of arsenosugars, Synechocystis WT

239

and Synechocystis ∆arsM were also exposed to 10 µM MMA(V) or 100 µM DMA(V).

240

In the MMA(V) exposures, Synechocystis WT transformed MMA(V) to DMA(V) and

241

arsenosugars, whereas Synechocystis ∆arsM produced neither DMA(V) nor

242

arsenosugars (Fig. 2b and S5). At the same time As(V) was clearly detected in the

243

samples, indicating that demethylation had taken place (Fig. 2b) though MAs(III)

244

demethylase gene, arsI identified in Bacillus sp. MD1 29 and Nostoc sp. PCC 7120 30,

245

has not been found in Synechocystis. However, when Synechocystis ∆arsM was

246

incubated with 100 µM DMA(V), sugar 1 & 2 were again produced and in quantities

12


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comparable to those produced by Synechocystis WT (Fig. 2c, S6, and S7). The data

248

suggested that the addition of the ribosyl group in arsenosugar biosynthesis proceeds

249

after methylation, and uses DMA as the substrate.

250

Disruption of arsM slightly increased sensitivity to As(III) but not to As(V)

251

in Synechocystis. Cell growth of Synechocystis WT and ∆arsM was monitored with 1

252

mM As(III) or 1 mM As(V). Synechocystis WT and ∆arsM cells exposed to 1 mM

253

As(V) showed similar patterns of growth, with rapid growth of both strains in medium

254

without As(V). When being exposed to 1 mM As(III), growths of Synechocystis WT

255

and ∆arsM were inhibited compared to exposure to 1 mM As(V) (Fig. 3). Moreover,

256

growth of Synechocystis ∆arsM in 1 mM As(III) was slightly decreased compared to

257

Synechocystis WT. At lower exposures (0.1-100 µM), no difference of cell growth

258

were found. The results showing minimal effects even at very high exposure (1 mM)

259

indicated that arsenic methylation was not the main form of detoxification for

260

Synechocystis sp. PCC 6803.

261 262

DISCUSSION

263

The mechanism of arsenic glycosidation is integral for a better understanding of

264

arsenosugars and their roles in the biogeochemical cycle of arsenic. Arsenosugars are

265

considered to be relatively nontoxic compared to inorganic species

266

arsenosugars in animals and the human body are metabolized mainly to DMA(V), the

267

same major human metabolite from toxic inorganic arsenic

31

. However,

31

. Our results

13



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268

demonstrated As(III) methylation and glycosylation by a freshwater organism

269

Synechocystis sp. PCC 6803. As(III) methylation in prokaryotes is considered to be a

270

detoxification mechanism 19. ArsM from cyanobacteria conferred resistance to As(III)

271

in E. coli strain Rosetta due to over-expression of arsM induced by IPTG. Insertional

272

inactivation of arsM in Synechocystis sp. PCC 6803 resulted in a slightly reduced

273

As(III) resistance when compared to Synechocystis WT (Fig. 3). However, As(III)

274

methylation and glycosylation in Synechocystis sp. PCC 6803 does not appear to be a

275

major arsenic detoxification mechanism (Fig. 3), possibly due to the relatively low

276

methylation activity when compared to As(III) oxidation and intracellular As(V)

277

reduction and subsequent efflux

278

beneficial in a high-phosphate freshwater environment but may be significant in the

279

marine environment which is always phosphate limited. Arsenosugars not only exist

280

as the predominant arsenic species in marine algae, but also make up a significant

281

proportion of the total arsenic in herbivorous mollusks

33

282

been found in freshwater organisms such as crayfish

34

283

biosynthesis pathway and function of these compounds in freshwater biota is still

284

unclear.

32

. In addition, arsenosugars appear not to be

. Arsenosugars have also and mussels

35

, but the

285

In this study, arsenosugars and methylated arsenic species were identified in

286

Synechocystis WT exposed to As(III). These findings are consistent with previous

287

reports showing that cyanobacteria can produce arsenosugars when treated with

288

inorganic arsenic 3. In a previous study

24

, which showed accumulation and

14


Page 15 of 27


289

transformation of arsenic in Synechocystis sp. PCC 6803, arsenosugars had not been

290

detected. This discrepancy can be ascribed to the different extraction method used for

291

those samples since in the earlier study treatment with 1% HNO3 and microwave

292

heating most probably chemically degraded any arsenosugars present 36. We disrupted

293

arsM in Synechocystis in order to determine whether ArsM is involved in the

294

biosynthesis of arsenosugars by providing DMA. Neither arsenosugars nor further

295

methylated arsenic species could be detected inside the cells or in the supernatant

296

when Synechocystis ∆arsM was incubated with inorganic arsenic or MMA indicating

297

an important role of ArsM in the biosynthesis of arsenosugars by methylating

298

inorganic arsenic and MMA to DMA. Moreover, the result that complementary

299

mutant Synechocystis ∆arsM::arsM treated with As(III), MMA(V), or DMA(V) can

300

further methylate arsenic showed that the disruption of arsM did not affect the

301

expression of the other downstream genes in the same operon, or the downstream

302

genes were not involved in arsenic methylation.

303

It has been suggested that the formation of arsenosugars in marine algae follows

304

methylation of As(III) 14. In this scenario, instead of final reduction and methylation

305

to trimethylarsine, DMA(V) would first be reduced to DMA(III) and then oxidized by

306

adding an adenosyl group from SAM. The enzymatic, hydrolytic removal of adenine

307

would then follow to form arsenosugars 14. However, no experiments have previously

308

been conducted to test the hypothesis that arsenosugar synthesis is actually initiated

309

from DMA. In the present study, Synechocystis WT could transform MMA(V) into

15



Page 16 of 27

310

DMA(V) and subsequently into arsenosugars. In contrast, neither DMA nor

311

arsenosugars were observed in Synechocystis ∆arsM cells (mutant) (Fig. 2 and S5). In

312

addition, both Synechocystis WT and Synechocystis ∆arsM cells were able to produce

313

arsenosugars, albeit in relatively small amounts, when supplied with 100 µM DMA(V)

314

(Fig. 2 and S6). These results, in combination with the observed in vitro conversion of

315

As(III) to MMA and DMA catalyzed by ArsM, suggest that DMA is the starting

316

compound for the biosynthesis of arsenosugars.

317

Rosetta cells functionally expressing arsM could further methylate As(III) or

318

MMA(V). The arsenic-containing nucleoside, 5´-deoxy-5´-dimethylarsinyladenosine,

319

previously isolated from the kidney of Tridacna maxima

320

intermediate in the proposed biosynthetic pathway for arsenosugars. However, we

321

found no evidence for the presence of arsenosugar intermediates in Rosetta cells

322

bearing pET22b-arsM or in vitro reactions (Fig. 1 and Table 1). In addition, some

323

organisms that have been reported to methylate arsenic did not produce arsenosugars.

324

The results imply that ArsM cannot transfer the adensoyl group from SAM to

325

DMA(III). It was previously shown, in Synechocystis, that As(V) is reduced to As(III)

326

by a cytosolic arsenate reductase (ArsC)

327

converted to DMA(III) by ArsM. We further propose that, DMA(III) is transformed to

328

dimethylarsinyladenosine by an unknown protein that can transfer the adenosyl group

329

to DMA. Finally, dimethylarsinyladenosine undergoes glycosidation to form

330

arsenosugars, as previously proposed 14.

26

, was regarded as a key

37

, and As(III) is then methylated and

16


Page 17 of 27


331

Different plasma membrane systems are responsible for the uptake of different

332

arsenic species. The uptake rates for both DMA(V) or MMA(V) by Synechocystis

333

were clearly slower than for As(V) or As(III) (Fig. S7). Raab et al.

334

arsenic uptake capacity by 46 different plant species exposed to 1 mg L-1 As(V),

335

MMA(V) or DMA(V) for 24 hours, and found that plants on average absorbed As(V)

336

at a significantly higher rate than MMA(V) and DMA(V). A similar pattern was also

337

observed in rice

338

(the aquaporin NIP2;1) is thought to mediate the uptake of undissociated methylated

339

arsenic species in rice roots 41. However, the mechanism for transporting DMA(V) or

340

MMA(V) in cyanobacteria remains to be investigated.

39

and in several other angiosperms

40

38

compared

. The silicon transporter, Lsi1

341

In summary, our study has for the first time shown that ArsM is a required

342

enzyme in the synthesis of arsenosugars by providing DMA as the precursor to

343

arsenosugars. Future studies, aimed at elucidating the metabolic processes of

344

arsenosugar biosynthesis, will try to identify the genes encoding metabolic enzymes

345

proposed to be involved in adensoyl transfer in Synechocystis.

346 347

ACKNOWLEDGMENTS

348

Our research is supported by the National Natural Science foundation of China

349

(21507125 and 31270161), Natural Science Foundation of Fujian Province

350

(2014J01141), and the Austrian Science Fund (FWF) project number 23761-N17.

351 352

REFERENCES 17



353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

Page 18 of 27

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31. Feldmann, J.; Krupp, E. Critical review or scientific opinion paper: Arsenosugars—a class of benign arsenic species or justification for developing partly speciated arsenic fractionation in foodstuffs? Anal. Bioanal. Chem. 2011, 399 (5), 1735-1741; DOI 10.1007/s00216-010-4303-6 32. Yin, X. X.; Wang, L. H.; Bai, R.; Huang, H.; Sun, G. X. Accumulation and Transformation of Arsenic in the Blue-Green Alga Synechocysis sp. PCC 6803. Water Air Soil Pollut. 2012, 223 (3), 1183-1190; DOI 10.1007/s11270-011-0936-0 33. Benson, A.; Summons, R. Arsenic accumulation in great barrier reef invertebrates. Science 1981, 211 (4481), 482-483; DOI 10.1126/science.7455685 34. Devesa, V.; Súñer, M. A.; Lai, V. W. M.; Granchinho, S. C. R.; Martínez, J. M.; Vélez, D.; Cullen, W. R.; Montoro, R. Determination of arsenic species in a freshwater crustacean Procambarus clarkii. Appl. Organomet. Chem. 2002, 16 (3), 123-132; DOI 10.1002/aoc.269 35. Soeroes, C.; Goessler, W.; Francesconi, K. A.; Schmeisser, E.; Raml, R.; Kienzl, N.; Kahn, M.; Fodor, P.; Kuehnelt, D. Thio arsenosugars in freshwater mussels from the Danube in Hungary. J. Environ. Monitor. 2005, 7 (7), 688-692; DOI 10.1039/B503897A 36. Nischwitz V. and Pergantis S. A. Mapping of arsenic species and identification of a novel arsenosugar in giant clams Tridacna maxima and Tridacna derasa using advanced mass spectrometric techniques. Environ. Chem. 2007, 4, 187–196; DOI 10.1071/EN07009 37. López-Maury, L.; Florencio, F. J.; Reyes, J. C. Arsenic sensing and resistance system in the cyanobacterium Synechocystis sp. strain PCC 6803. J. bacteriol. 2003, 185 (18), 5363-5371; DOI 10.1128/JB.185.18.5363-5371.2003 38. Raab, A.; Williams, P. N.; Meharg, A.; Feldmann, J. Uptake and translocation of inorganic and methylated arsenic species by plants. Environ. Chem. 2007, 4 (3), 197-203; DOI 10.1071/EN06079; 39. Abedin, M. J.; Feldmann, J.; Meharg, A. A. Uptake kinetics of arsenic species in rice plants. Plant Physiol. 2002, 128 (3), 1120-1128; DOI 10.1104/pp.010733 40. Schmidt, A. C.; Mattusch, J.; Reisser, W.; Wennrich, R. Uptake and accumulation behaviour of angiosperms irrigated with solutions of different arsenic species. Chemosphere 2004, 56 (3), 305-313; DOI 10.1016/j.chemosphere.2004.02.031 41. Li, R. Y.; Ago, Y.; Liu, W. J.; Mitani, N.; Feldmann, J.; McGrath, S. P.; Ma, J. F.; Zhao, F. J. The rice aquaporin Lsi1 mediates uptake of methylated arsenic species. Plant Physiol. 2009, 150 (4), 2071-2080; DOI 10.1146/annurev-arplant-042809-112152

467 468 469 470 471 472 473

20


Page 21 of 27


474 475 476 477

Legends

21



Page 22 of 27

478 479

Table 1 In vitro methylation of As(III) by ArsM in NH4HCO3 buffer at different temperatures (25~37oC). The reaction products were analyzed

480

by HPLC/ICPMS using an anion exchange column after the samples were treated with H2O2 to convert As(III) to As(V).

481

25 oC

28 oC

30 oC

33 oC

35 oC

37 oC

TMAO (µg L-1)

0

1.3±0.2

5.5±1.7

17.3±2.2

20.5±2.9

38.7±3.4

DMA (µg L-1)

27.5±4.6

92.0±9.3

153±13

264±22

229±18

296±22

MMA (µg L-1)

111±11

115±8

102±9

17±3

7.2±1.3

6.1±0.8

As(V) (µg L-1)

632±36

528±40

472±23

465±33

494±41

449±32

22


Page 23 of 27


482 483

Fig. 1 Arsenic biotransformation in E. coli strain Rosetta cells bearing pET22b-arsM

484

or pET22b. a: Rosetta bearing pET22b-arsM treated with As(III); b: Rosetta bearing

485

pET22b-arsM treated with MMA(V); c: Rosetta bearing pET22b-arsM treated with

486

DMA(V); d: Rosetta bearing pET22b treated with As(III). Arsenic species treated

487

with H2O2 were determined by HPLC/ICPMS. The mobile phase containing 6.6 mM

488

(NH4)2HPO4 and 6.6 mM NH4NO3 (pH 6.2, adjusted with HNO3) was pumped

489

through a Hamilton PRP-X100 anion-exchange column (4.1×250 mm, 10 µm) with a

490

Hamilton PEEK pre-column (11.2 mm, 12–20 µm) at 1.0 mL min-1 .

491

Fig. 2 Arsenic speciation in Synechocystis sp. PCC 6803 cells (WT and ∆arsM) after

492

two weeks exposure to 1 µM As(III) (a), 10 µM MMA(V) (b), or 100 µM DMA(V)

493

(c). The cells were not treated with H2O2 before analysis by anion-exchange

494

HPLC/ICPMS.

495

with NH3·H2O) was pumped through a Hamilton PRP-X100 anion-exchange column

496

(4.1×250 mm, 10 µm) with a Hamilton PEEK pre-column (11.2 mm, 12–20 µm) at

497

1.5 mL min-1. The column temperature was 40oC.

498

Fig. 3 Growth curves for Synechocystis WT and Synechocystis ∆arsM grown in

499

arsenic-free, 1 mM As(V), or 1 mM As(III) containing BG11 medium with shaking

500

96 rpm at 30oC under continuous light. Growth was monitored by measuring the

501

optical density at 730 nm (n=3)

The mobile phase containing 15 mM NH4H2PO4 (pH 5.6, adjusted

23



Page 24 of 27

502 503

Associated content

504

Supporting Information: Supporting methods, the method of construction of

505

complementation mutant synechocystis sp. PCC 6803 ∆arsM::arsM (pS2), the

506

primers were used in this experiments (Table S1), the four most common arsenosugars

507

found in nature, these compounds are most abundant in marine algae, and in animals

508

consuming marine algae (Fig. S1), diagram of resistance gene insertion and primer

509

positioning for construction of the arsM deletion and complement in Synechocystis sp.

510

PCC 6803, and PCR analysis of Synechocystis sp. PCC 6803 after transformation (Fig.

511

S2), anion-exchange HPLC/ICPMS Chromatograms of arsenic speciation in medium

512

of Synechocystis WT and Synechocystis ∆arsM were exposed to 1 µM As(III) (Fig.

513

S3),

514

Synechocystis ∆arsM::arsM incubated with As(III), MMA(V), and DMA(V) for two

515

weeks (Fig. S4), HPLC/ICPMS chromatograms of extracts from Synechocystis ∆arsM

516

showing the absence of arsenosugars 1 (Fig. S5), HPLC/ICPMS and HPLC/ESIMS

517

chromatograms of extracts from Synechocystis ∆arsM and Synechocystis WT showing

518

the presence of arsenosugars 1 & 2 (Fig. S6), arsenic concentration of acid digests of

519

cells exposed to different arsenic species and the proportions of different arsenic

520

species in the cells (Fig. S7).

anion-exchange

HPLC/ICPMS

Chromatograms

of

arsenic

speciation

24


Page 25 of 27


322x251mm (96 x 96 DPI)



241x178mm (96 x 96 DPI)


Page 26 of 27

Page 27 of 27


317x243mm (96 x 96 DPI)


Arsenic Methyltransferase is Involved in Arsenosugar Biosynthesis by

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