Identification of Steps in the Pathway of Arsenosugar Biosynthesis

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Environmental Processes

Identification of steps in the pathway of arsenosugar biosynthesis Xi-Mei Xue, Jun Ye, Georg Raber, Barry P. Rosen, Kevin A. Francesconi, Chan Xiong, Zhe Zhu, Christopher Rensing, and Yong-Guan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04389 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Environmental Science & Technology

1

Identification of steps in the pathway of arsenosugar

2

biosynthesis

3

Xi-Mei Xue,1 Jun Ye,1 Georg Raber,2 Barry P. Rosen,3 Kevin Francesconi,2 Chan

4

Xiong,2 Zhe Zhu,4 Christopher Rensing,5 Yong-Guan Zhu 1,6*

5 6

1

7

Chinese Academy of Sciences, Xiamen 361021, China

8

2 Institute

9

3

Key Laboratory of Urban Environment and Health, Institute of Urban Environment,

of Chemistry, NAWI Graz, University of Graz, Graz 8010, Austria

Department of Cellular Biology and Pharmacology, Herbert Wertheim College of

10

Medicine, Florida International University, Miami, FL 33199

11

4

12

Engineering, University of Nottingham, Ningbo 315100, China

13

5

14

Fuzhou 350002, China

15

6

16

Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

Department of Chemical and Environmental Engineering, Faculty of Science and

College of Resources and Environment, Fujian Agriculture and Forestry University,

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

17 18 19

*Address Correspondence to Yong-Guan Zhu,

20

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

21

Road, Xiamen 361021, China

1

ACS Paragon Plus Environment


22

Phone number: +86(0)592 6190997

23

Fax number: +86(0)592 6190977

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Email address: [email protected]

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Abstract

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Arsenosugars are arsenic-containing ribosides that play a substantial role in arsenic

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biogeochemical cycles. Arsenosugars were identified more than thirty years ago, and

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yet their mechanism of biosynthesis remains unknown. In this study we report

68

identification of the arsS gene from the cyanobacterium Synechocystis sp. PCC 6803

69

and show that it is involved in arsenosugar biosynthesis. In the Synechocystis sp. PCC

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6803 ars operon, arsS is adjacent to the arsM gene that encodes an As(III) S-

71

adenosylmethionine (SAM) methyltransferase. The gene product, ArsS, contains a

72

characteristic CX3CX2C motif which is typical for the radical SAM superfamily. The

73

function of ArsS was identified from a combination of arsS disruption in Synechocystis

74

sp. PCC 6803 and heterologous expression of arsM and arsS in Escherichia coli. Both

75

genes are necessary, indicating a multi-step pathway of arsenosugar biosynthesis. In

76

addition, we demonstrate that ArsS orthologs from three other freshwater cyanobacteria

77

and one picocyanobacterium are involved in arsenosugar biosynthesis in those

78

microbes. This study represents the identification of the first two steps in the pathway

79

of arsenosugar biosynthesis. Our discovery expands the catalytic repertoire of the

80

diverse radical SAM enzyme superfamily and provides a basis for studying the

81

biogeochemistry of complex organoarsenicals.

82 83 84 85 86 87 3



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Introduction

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Arsenoribosides, commonly known as arsenosugars, were first isolated from the brown

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kelp, Ecklonia radiata, and chemically identified in 1981.1 More than 20 arsenosugars

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consisting of dimethylated arsenosugars, dimethylated thio-arsenosugars, and

93

trimethylated arsenosugars are known to occur primarily in marine organisms but also

94

in some terrestrial organisms (Figure S1).2, 3 They are natural constituents of marine

95

organisms and not toxic to marine plants or animals. Thus the synthesis of arsenosguars

96

represents a mechanism of arsenic detoxification. Arsenosugars also appear to play a

97

central role in the pathway of arsenic biotransformation in marine systems. First, they

98

are likely intermediates in the biosynthesis of the osmolyte arsenobetaine, which is the

99

dominant arsenical in marine organisms and is nontoxic in humans.1 Edmonds &

100

Francesconi proposed that arsenosugars can be converted to arsenocholine by the

101

cleavage of the C3-C4 bond of the sugar residue and methylation of the arsenic atom.

102

Further oxidation at the C-4 of the sugar residue results in the production of

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arsenobetaine.1, 4 The enzymes for glycine betaine synthesis (GbsB/GbsA) have been

104

shown to be involved in the biosynthesis of arsenobetaine from arsenocholine.5 Second,

105

arsenosugars are also the likely precursors of arsenosugar phospholipids,6, 7 which may

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be components of the algal cell membrane.8 Incorporation of arsenolipids increases

107

under low phosphate conditions and may represent a phosphate-sparing mechanism

108

under those conditions.9

109

Dimethylated arsenosugars consist of a dimethylarsinoyl moiety, in which 4


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pentavalent arsenic is bound to one oxygen, to two methyl groups and to a 5'-

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deoxyriboside (Figure S1). The dimethylated arsenosugars differ from one another only

112

in structure of the side chains at the 5'-deoxyriboside C1 position. A proposed pathway

113

for arsenic transformation in the marine environment suggests that SAM is the source

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of both the methyl groups and the 5'-deoxyribose group of arsenosugars.4

115

In this study, the arsS (hereafter referred to as SsarsS) and arsM (hereafter referred

116

to as SsarsM) genes from Synechocystis were disrupted to examine their function in

117

arsenosugar biosynthesis. Either disruption mutant was unable to synthesize

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arsenosugars, indicating that both genes are involved in the biosynthetic pathway. The

119

SsarsM and SsarsS genes were heterologously expressed in E. coli. Cells expressing

120

SsarsM could synthesize dimethylarsenate but not arsenosugar, while cells expressing

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both genes produced dimethylarsenoriboside derivatives. Our results elucidate the first

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two steps in the pathway of arsenosugar biosynthesis. The first step is addition of

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methyl groups from SAM to As(III) by the SAM methyltranserase SsArsM. The second

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step is addition of the deoxyribose moiety by the radical SAM enzyme SsArsS. This

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study demonstrates the first two steps in the pathway of arsenosugars and puts the

126

synthesis of complex organoarsenical on a firm biochemical basis.

127

Materials and Methods

128

Strains and plasmids

129

Unless otherwise indicated, E. coli was cultured aerobically in Luria − Bertani (LB)

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medium at 37°C containing 100 μg mL-1 ampicillin, 50 μg mL-1 kanamycin, or 30 μg

131

mL-1 chloramphenicol, as required. E. coli AW3110 (DE3) (ΔarsRBC) 10 was used for 5



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resistance assays. Plasmids pET-28a-SsarsS, pET-22b-SsarsS, pETDuet-SsarsS or/and

133

pET-28a-SsarsM were transformed into E. coli BL21 (DE3) or Rosetta (DE3) for

134

expression of SsArsM and SsArsS and for production of arsenical products. All strains

135

and plasmids are shown in Table S1.

136

Disruption of SsarsS and SsarsM in Synechocystis sp. PCC 6803

137

A mutant of Synechocystis with a disruption of the SsarsM gene has been described.11

138

Mutagenesis of SsarsS (Synechocystis ΔarsS) was performed (Figure S3A) as follows:

139

(a) SsarsS was amplified with primers SsarsSF and SsarsSR (Table S2); (b) the purified

140

SsarsS fragment after PCR was cloned into pMD19T simple vector (TaKaRa, Dalian,

141

China) to yield plasmid pMD-arsS; (c) a kanamycin resistance (KanR) cassette cut from

142

pKW1188 12 with EcoRI was inserted into the EcoRV site of pMD-arsS to create gene-

143

disruption KanR-insertion cassette, pMDarsSkan; (d) the plasmid was transformed into

144

Synechocystis WT according to a previous description;13 (e) Synechocystis ΔarsS was

145

obtained after three serial isolation of single colonies on BG11 agar plates containing

146

30 μg mL-1 kanamycin; (f) PCR with diagnostic primers (Table S2) was used to confirm

147

that the SsarsS gene was completely replaced. Synechocystis ΔarsS was cultivated in

148

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

149

Complementation of SsarsS in Synechocystis sp. PCC 6803

150

Complementation of the Synechocystis ΔarsS mutant (Synechocystis ΔarsS::arsS)

151

(Figure S3A) was achieved by “knock-in” of intact SsarsS containing the rbcL2A

152

promoter with a chloramphenicol resistance (CamR) gene. An intact SsarsS was

153

amplified using primers arsSc-F and arsSc-R (Table S2). The rbcL2A promoter, PrbcL2A, 6


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was produced with primers FP_CO2noEcoRI and RP_BBRBS.14 The above PCR

155

products were cloned into pMD19T simple vector to produce plasmid pMarsSc and

156

pMpromoter respectively, sequenced to confirm the integrity of the cloned flanking

157

DNA. The plasmid containing SsarsS with the promoter was constructed by inserting

158

PrbcL2A into the EcoRI and SpeI sites of the plasmid pMarsSc. A CamR gene amplified

159

from pACYCDuet-1 (Novagen, Madison, WI) using primers cmr-F and cmr-R was

160

digested with NheI and HindIII, cloned into the upstream of PrbcL2A. Finally, the

161

fragment consisting of a CamR gene, PrbcL2A, and SsarsS was linked with a plasmid

162

pKW1188 without KanR by T4 DNA ligase (MBI Fermentas, Flamborough, ON,

163

Canada) to prepare plasmid pKcmrparsS. This plasmid was used to transform

164

Synechocystis ΔarsS, and the above fragment was inserted into the neutral site slr0168.

165

In all cases, Synechocystis ΔarsS::arsS was selected on BG11 agar plates containing 30

166

μg mL-1 kanamycin and 15 μg mL-1 chloramphenicol. PCR amplification with the

167

genomic DNA from Synechocystis ΔarsS::arsS as templates and the primers gp267-9

168

and gp267-10

169

Synechocystis ΔarsS::arsS (Figure S3B). The resulting Synechocystis ΔarsS::arsS

170

strain was cultivated in 100 mL of BG11 medium with 50 μg mL-1 kanamycin and 30

171

μg mL-1 chloramphenicol.

172

As(III) transformation assays

173

After one or two weeks of continuous illumination culture, 50 or 100 mL of

174

cyanobacteria (Synechocystis WT, Synechocystis ΔarsS, and Synechocystis ΔarsS::arsS)

175

were collected by centrifuging and washing twice with cold MES buffer. 16 About 20

15

was carried out to confirm that an intact SsarsS was present in

7



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mg of lyophilized cells were added into 1.5 mL Eppendorf tubes with 1 mL of a mixture

177

of CH3OH(MeOH)/H2O (1+1, v/v). The cells were extracted by placing the tubes in an

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ultrasonic bath for 20 minutes followed by rotating on a rotary wheel overnight. The

179

extracts were centrifuged for 10 min at 13520 g. The supernatant was purged with N2

180

to remove MeOH, and then filtered through a 0.22 µm membrane filter into 1 mL

181

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

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arsenic species analysis.

183

The transformation of As(III) under the control of ArsS or/and ArsM was assayed

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in vivo. The assays were performed with Rosetta bearing pET-28a-SsarsM, Rosetta

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bearing pETDuet-SsarsS, Rosetta bearing empty pETDuet-1 and pET-28a, or Rosetta

186

bearing pET-28a-SsarsM and pETDuet-SsarsS. Cells were grown overnight at 37oC

187

with shaking (200 rpm) in LB medium containing corresponding antibiotic, diluted

188

100-fold into 5 mL of fresh LB medium. After incubation at an OD

189

carried out with 0.5 mM IPTG for 3 h, cells were treated with 1 µM, 10 µM, 100 µM,

190

1 mM or 3 µM of either As(III) or As(V). At the indicated times, the cells and culture

191

medium were collected.

192

Arsenic species analysis

193

High performance liquid chromatography - inductively coupled plasma mass

194

spectrometry (HPLC-ICP-MS) measurements on arsenic species were performed using

195

an Agilent 7500cx ICP-MS (Agilent Technologies, California, USA) for element

196

detection coupled with an Agilent 1200 HPLC system. Anion-exchange

197

chromatography separation was performed using PRP-X100 with a guard column

600 nm

of 0.5 was

8


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(Hamilton, Reno, NV, USA). A Shiseido CAPCELL PAK C18 MGII (Shiseido, Tokyo)

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with a matching guard column was employed for ion-pair reversed-phase

200

chromatographic separation. All chromatography parameters are shown in Table S3.

201

Unknown arsenic compounds were identified using HPLC-ICP-MS/electrospray

202

ionization tandem mass spectrometry (ESI-MS-MS, Agilent 6460, Agilent

203

Technologies, Waldbronn, Germany), and higher solution accurate mass (HPLC-ESI-

204

HR-MS-MS) measurements which were performed in positive mode on a Q-Exactive

205

Hybrid Quadrupole-Orbitrap MS after arsenic species were separated on a Dionex

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Ultimate 3000 series instrument (Thermo Fischer Scientific, Erlangen, Germany).

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Results

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The arsS gene is involved in arsenosugar biosynthesis.

209

Our search for candidate genes involved in arsenosugar biosynthesis was guided by the

210

hypothesis that oxidation of DMAs(III) by addition of the deoxyadenosyl radical group

211

from SAM and enzymatic hydrolytic removal of adenine would result in the formation

212

of arsenosugars in marine algae.4 Synechocystis produces two species of arsenosugars,

213

indicating that this microbe has the biosynthetic enzymes for arsenosugar synthesis.11,

214

17

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methyltransferase in Synechocystis.16 The SsarsM gene is adjacent to two other open

216

reading frames, slr0304 and slr0305 (Figure 1). A protein BLAST search using

217

SLR0304, which we termed as SsArsS, as a query revealed the presence of regions

218

conserved in the radical SAM superfamily, in particular iron-sulfur cluster (FeS) and

219

SAM binding sites. The SsarsM and SsarsS genes comprise an ars operon (Figure S2).

We previously identified the enzyme SsArsM as an As(III) S-adenosylmethionine

9



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SLR0305 is related to the family of SNARE-associated Golgi membrane proteins, and

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this gene is co-transcribed with the other two genes, but it is not clear if it has an arsenic-

222

related function. Nostoc sp. PCC 7120, which also produces arsenosugars,18 has a

223

putative arsMarsS operon. The Nostoc operon lacks a gene corresponding to SLR0305,

224

indicating that the latter does not have an arsenic-related function. We hypothesize from

225

the linkage of the arsM and arsS genes in more than one arsenosugar producers that

226

these two genes have related functions encoding a biosynthetic pathway for arsenosugar

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biosynthesis (Figure 1). We previously demonstrated that a strain in which SsarsM was

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disrupted cannot synthesize arsenosugars, supporting our hypothesis that DMAs

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production by ArsM is the first step in arsenosugar synthesis.11

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We hypothesize that ArsS catalyzes the second step in arsenosugar biosynthesis by

231

transfer of a deoxyadenosyl group from SAM to DMAs. This could be the first

232

committed step in the biosynthetic pathway because the product of ArsM, DMAs can

233

have other biosynthetic fates. ArsS is a member of the radical SAM superfamily.

234

Radical SAM enzymes are characterized by a CX3CX2C motif that provides co-

235

ordinations for binding of a unique four iron-four sulfur ([4Fe-4S]) cluster, although

236

the spacing of the cysteines in this motif can vary.19 Both the alignment of SsArsS with

237

those of functionally characterized radical SAM enzymes and construction of a

238

molecular phylogenetic tree indicate that SsArsS is closely related to MoaA (Figure S4),

239

which harbors an N-terminal [4Fe-4S] cluster involved in a 5’-deoxyadenosyl radical

240

generation and a C-terminal [4Fe-4S] cluster involved in substrate binding and/or

241

activation in conversion of 5’-GTP to an oxygen-sensitive tetrahydropyranopterin.20 10


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We have used UV/visible spectroscopy to examine a visible absorption band at 410 nm

243

for the [4Fe-4S]2+ protein (Figure S5).21

244

SsArsS is involved in the biosynthesis of arsenosugars

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To examine the involvement of ArsS in arsenosugar biosynthesis, we disrupted the

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SsarsS gene of Synechocystis by inserting a KanR cassette inside SsarsS. We evaluated

247

the ability of the parental wild type strains and its derivatives to grow. No difference in

248

growth rate in the absence of As(III) was observed among wild type, Synechocystis

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ΔarsS and Synechocystis ΔarsS::arsS strains (Figure S3C), indicating that ArsS is not

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required for viability under these growth conditions. The production of arsenosugars by

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wild type and mutant strains in medium containing different arsenic species was

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determined by HPLC-ICP-MS analysis. Consistent with earlier studies,11, 17 wild type

253

Synechocystis exposed to As(III) produces As(V) as the predominant soluble arsenic

254

species, and small amounts of MAs(V), DMAs(V) and arsenosugars (Figure 2a). Again,

255

it is not possible to deduce whether the products of the reaction were trivalent species

256

that oxidized in air to their pentavalent forms. In addition, wild type Synechocystis

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transforms MAs(V) or DMAs(V) into arsenosugars (Figure 2 c, e). Synechocystis ΔarsS

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was unable to generate arsenosugars in medium containing either As(III) or pentavalent

259

methylated arsenic species (Figure 2b, d, f). The Synechocystis ΔarsS::arsS strain

260

regained the ability to produce arsenosugars (Figure S3D). These results clearly showed

261

that SsArsS is likely involved in arsenosugar biosynthesis.

262

Heterologous expression of SsarsS and SsarsM

263

To demonstrate the linked function of SsarsM and SsarsS in arsenosugar production, 11



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the SsarsS and SsarsM genes were heterologously expressed alone or in combination

265

in E. coli Rosetta (DE3), which has neither in its genome (Figure 3A and Figure S6A).

266

Heterologous expression of arsM genes in E. coli strain AW3110 (DE3), in which the

267

chromosomal ars operon was deleted 10 has been shown to confer As(III) resistance.22,

268

23

269

resistance to As(III) (Figure S7). Surprisingly, expression of SsarsS alone conferred

270

As(III) resistance (Figure S7). SsArsS has 11 cysteine residues that might bind

271

sufficient As(III) to confer moderate resistance or some free radicals generated by the

272

process of As(III) oxidation to As(V) by SsArsS when the gene is expressed at high

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levels.

Similarly, expression of SsarsM alone in E. coli strain AW3110 (DE3) conferred

274

ICP-MS was also used to quantify total arsenic in E. coli Rosetta cells (Figure S8A)

275

treated with 0.5 mM IPTG and 1 μM As(III) for 20 h. Rosetta cells expressing SsarsS

276

accumulated the most arsenic, up to 83 mg kg-1. Cells expressing SsarsM alone

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accumulated less (44 mg As kg-1), which was similar to cells expressing both genes (39

278

mg As kg-1). The arsenic content of cells expressing both SsarsM and SsarsS peaked

279

after exposure for 3 h, and subsequently declined, suggesting that they expelled their

280

arsenic compounds from the cells (Figure S8B).

281

The arsenic species in medium were analyzed by HPLC-ICP-MS after E. coli

282

Rosetta cells were exposed to 3 μM As(III) for 12 h. No arsenosugars were identified

283

in Rosetta expressing both SsarsM and SsarsS. The major arsenic species found in

284

medium of Rosetta cells expressing both SsarsM and SsarsS or SsarsM alone was

285

DMAs(V) (Figure 3). As(III) dominated in medium of Rosetta expressing SsarsS alone. 12


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Comparative analysis of the aqueous fraction of Rosetta cells expressing SsarsM alone,

287

or SsarsS alone, or both SsarsM and SsarsS revealed that Rosetta cells expressing both

288

SsarsM and SsarsS generated unknown arsenic compounds (Figure 3B), indicating that

289

biosynthesis of these species requires the concerted action of ArsM and ArsS. These

290

unknown compounds were identified by HPLC-ESI-HR-MS-MS after Rosetta cells

291

expressing both SsarsM and SsarsS were incubated with 3 μM arsenic for 72 h, and

292

they were a group of dimethylarsinoyl-hydroxycarboxylic acids (DMA-HA, Figure S9

293

and S10).

294

None of the unknown arsenic compounds that cells expressing SsarsM and SsarsS

295

together produced eluted at the same position as 5'-deoxy-5'-dimethylarsinoyl-

296

adenosine, which is a predicted intermediate in the arsenosugar synthesis pathway

297

proposed by Edmonds and Francesconi 4 (Table 1). The results of HPLC-ICP-MS/ESI-

298

MS-MS and HPLC-ESI-HR-MS-MS measurements suggested the presence of

299

additional dimethylarsinoyl-hydroxycarboxylic acids such as 3′-dimethylarsinoyl-2′-

300

hydroxypropionic

301

dimethylarsinoyl-2′-hydroxypentanonic acid and 5′-dimethylarsinoyl-2′, 3′, 4′-

302

trihydroxypentanonic acid, as well as two unidentified arsenic compounds which could

303

not be characterized due to low concentrations (Table 1 and Figure S10). A compound

304

with the predicted composition C7H16O5As (m/z 255.02079) was also detected by

305

HPLC-ESI-HR-MS-MS, but the structure could not be confirmed (Figure S10). Figure

306

S10 shows that in addition to the four dimethylarsinoyl-hydroxycarboxylic acids

307

(except 5-dimethylarsinoyl-2 ′ -hydroxypentanonic acid) identified in incubation

acid,

4′-dimethylarsinoyl-2′,

3′-dihydroxybutyric

acid,

5′-

13



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medium, cells expressing SsarsM and SsarsS also contained the four corresponding

309

thio-arsenic compounds. There are several possible reasons why dimethylarsinoyl-

310

hydroxycarboxylic acids were produced rather than 5'-deoxy-5'-dimethylarsinoyl-

311

adenosine in E. coli expressing both SsarsM and SsarsS. First, there might be additional

312

steps catalyzed by as-yet unidentified Synechocystis enzymes not present in E. coli.

313

Second, most of the ArsS produced in E. coli may be partly active, consistent with our

314

observation that purified ArsS is extremely oxygen sensitive. Oxygen induces the

315

switch from [4Fe-4S]+ to [4Fe-4S]2+, [3Fe-4S]2+, or [2Fe-2S]2+ with loss of biological

316

activity. A future goal is to express the genes under anaerobic growth conditions.

317

The roles of cysteine residues in SsarsS

318

Assuming that the production of dimethylarsinoyl-hydroxycarboxylic acids reflects a

319

partial activity of SsArsS, the role of cysteine residues in that activity was examined.

320

Thiol chemistry plays an important role in arsenic metabolism

321

processes

322

proteins. Each of the eleven cysteine residues in SsArsS protein, Cys41, Cys45, Cys48,

323

Cys65, Cys129, Cys142, Cys264, Cys279, Cys320, Cys323, Cys331, were individually changed

324

to serine residues. Single mutants in SsarsS and SsarsM were co-transformed into E.

325

coli strain Rosetta (DE3) which were treated with As(III) (Figure S11). Three mutant

326

proteins, C65S, C129S, C142S, were able to produce unknown organoarsenicals. Cells

327

expressing the other eight mutants and SsarsM lost the ability to produce the

328

unidentified arsenic compounds. The three cysteine residues, Cys41, Cys45, Cys48, in the

329

N-terminal region of SsArsS correspond to the canonical radical SAM cysteine motif

26

24, 25

and transport

through formation of metalloid-sulfur bonds between arsenic and related

14


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CX3CX2C, while other three cysteine residues, Cys320, Cys323, Cys331, in the C-terminal

331

region of SsArsS are conserved among ArsSs.

332

The function of microbial arsS genes in arsenic transformation.

333

We identified putative ArsS orthologs with eight conserved cysteine residues and a

334

radical SAM domain in other microbes. Genes encoding for ArsS from four other

335

cyanobacteria and one eukaryotic green alga (Table S4) were co-expressed with SsarsM

336

in E. coli. The cyanobacteria Nostoc sp. PCC 7120

337

Chlorella

338

was identified in the picocyanobacterium P. marinus str. MIT 9313,29 although it has

339

not been demonstrated if this species generates arsenosugars. Cyanobacteria A.

340

platensis NIES-39 and S. elongatus PCC 6301, which have genes encoding ArsS

341

orthologs were chosen as representative cyanobacteria. Arsenic transformation was

342

analyzed in cells of E. coli co-expressing genes for orthologs of SsArsS in pETDuet-

343

arsS together with pET-28a-SsarsM. E. coli co-expressing SsarsM and NsarsS from

344

Nostoc sp. PCC 7120 produced the dimethylarsinoyl-hydroxycarboxylic acids observed

345

in E. coli expressing SsarsM and SsarsS (Figure 4 and Figure S6B). ArsSs from

346

cyanobacteria A. platensis NIES-39, S. elongatus PCC 6301, and P. marinus str. MIT

347

9313, organisms that have not been previously reported to generate arsenosugars, also

348

synthesized dimethylarsinoyl-hydroxycarboxylic acids (Figure 4). Only SsArsM and

349

CvArsS from the eukaryotic algae C. variabilis did not synthesize 5'-

350

dimethylarsenoriboside derivatives when co-expressed in E. coli. The reason for this

351

negative result is unknown, but perhaps CvArsS, which undergoes extensive post-

28

18, 27

and the eukaryotic alga

have been reported to produce arsenosugars. Recently, a putative ArsM

15



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translational processing in C. variabilis, is not modified to an active enzyme in E. coli.,

353

or perhaps those gene products did not fold properly in E. coli.

354

Discussion

355

The most abundant arsenic compounds identified in marine algae are arsenosugars.30

356

Prokaryotic cyanobacteria,

357

reported to produce arsenosugars.32 Arsenosugars have also been found in underwater

358

plants as well;33 their source, however, is likely to be epiphytic algae or bacteria because

359

plants cannot methylate arsenic

360

immediate precursor of arsenosugars.11 Moreover, the physiological role of

361

arsenosugars is still unclear. For the oxo-arsenosguars, it’s known to be non-toxic to

362

humans, one arsenosugar (Oxo-Gly) was shown to be nontoxic to murine peritoneal

363

macrophages and alveolar macrophages at micromolar levels.35 The toxicity of thio-

364

arsenosugars whose bioaccessibility are much higher than the oxo-analogs, and

365

trimethylated arsenosugars haven’t been proved yet. Arsenosugar biosynthesis may

366

serve both as a detoxification process for the organisms which generate them, and as a

367

precursor for more complex organoarsenicals. Unraveling the mechanism of

368

arsenosugar biosynthesis will help determine toxicity of arsenosugars to human and the

369

physiological role of arsenosugars in arsenic biogeochemical cycling.

27

eukaryotic green algae

34

28, 31

and fungi all have been

and dimethylarsenic has been postulated to be the

370

Until our study, the pathway of arsenosugar biosynthesis was unknown. Edmonds

371

and Francesconi 4 proposed a pathway in marine alga in which DMAs(V) is reduced to

372

DMAs(III), which is then oxidized by addition of the deoxyadenosyl group from SAM

373

to yield the intermediate 5'-deoxy-5'-dimethylarsinoyl-adenosine. Enzymatic, 16


Page 17 of 33


374

hydrolytic removal of adenine would be followed by formation of arsenosugars. This

375

proposal is supported by the identification of the proposed intermediate 5'-deoxy-5'-

376

dimethylarsinoyl-adenosine in the kidney of the giant clam Tridacna maxima,36 which

377

contains large quantities of arsenosugars owing to symbiotic algae growing in the

378

mantle of the clam. However, the product of arsenic methylation by ArsM enzymes is

379

almost certainly DMAs(III) and not DMAs(V),37, 38 so reduction of DMAs(V) is not

380

required. The ArsM reaction is required for producing the substrate of ArsS, but DMAs

381

has other fates such as further methylation to TMAs, so it is not a committed step in the

382

reaction pathway. Moreover, we show that co-expression of ArsM and ArsS in E. coli

383

produced many dimethylarsinoyl-hydroxycarboxylic acids but not 5'-deoxy-5'-

384

dimethylarsinoyl-adenosine. We are considering two possible pathways for

385

arsenosugar biosynthesis to account for the compounds produced by E. coli. Regardless

386

of the actual pathway, our data strongly support our hypothesis that the ArsS reaction

387

catalyzes the first committed step in arsenosugar biosynthesis.

17



Page 18 of 33

388 389

The likely reason that arsenosugars are not generated by E. coli co-expressing

390

SsarsS and SsarsM is that the intermediate of arsenosugar biosynthesis cannot be

391

further transformed into arsenosugars because E. coli does not have the genes involved

392

in arsenosugar biosynthesis. An alternative fate for this intermediate, however, could

393

be that it is rapidly degraded in vivo to small arsenic-containing compounds such as

394

those that we found produced by the transgenic E. coli constructed for this study.

395

It is notable that the Synechocystis ars operon containing arsM and arsS is not

396

induced by As(III) or As(V), and the operon does not contain an arsR gene for an

397

As(III)-responsive transcription factor.39 Consistent with our observation of

398

constitutive expression of the arsM and arsS genes, the amount of arsenosugars and 18


Page 19 of 33


399

arsenosugar phospholipids in Synechocystis treated with different concentrations of

400

As(V) did not significantly increase with increasing arsenic concentations.17 The

401

greater accumulation in Synechocystis of arsenosugar phospholipid compared with

402

arsenosugars suggests that arsenosugars are the precursors of arsenosugar

403

phospholipids and not the end products. The absence of arsenosugar intermediates in

404

Synechocystis suggests that DMAs is rapidly transformed into arsenosugars in those

405

cells, and that the arsenosugar intermediates, DMAs(III) and MAs(III), are probably

406

transient intermediates that do not accumulate in the cells.

407

Thus, we hypothesize that SsArsS, a putative radical SAM enzyme, adds a 5'-

408

deoxyadenosyl radical (dAdo·) to DMAs(III) and hydrolyzes the adenine from 5'-

409

deoxyadenosine simultaneously to generate 5'-deoxy -5'-dimethylarsinoyl-ribose.

410

SsArsS has a CX3CX2C motif similar to the assembly site for the [4Fe-4S] cluster found

411

in radical SAM enzymes. In addition, SsArsS has the other three cysteine residues in

412

the C-terminal region of SsArsS, Cys320, Cys323, Cys331, a CX2CX7C motif that

413

binds another putative [4Fe-4S] cluster (Figure S12), which would put it in a subgroup

414

of radical SAM enzymes that bind two [4Fe-4S] clusters, including MoaA,20 BioB,40

415

FbiC,41 LipA,42 TYW1.43 However, this CX2CX7C motif in the C-terminal region of

416

the SsArsS protein, which is not conserved in all radical SAM enzymes, is 5'-deoxy-5'-

417

not similar to other proteins that contained two or more iron-sulfur clusters.19 Mutations

418

in any of the cysteine residues in the CX2CX7C motif resulted in the loss of the function

419

for arsenosugar synthesis, consistent with a role for that motif in ArsS function.

420

Moreover, none of the cysteine mutants in either motif produced 5'-deoxy-5'19



(Figure

S11),

indicating

Page 20 of 33

421

dimethylarsinoyl-adenosine

that

the

5'-deoxy-5'-

422

dimethylarsinoyl-riboside derivative was formed before 5'-deoxy-5'dimethylarsinoyl-

423

adenosine.

424

In summary, ArsS is the only member of the radical SAM superfamily shown to

425

be involved in arsenosugar synthesis. This study adds a new function to the repertoire

426

of enzymes of organoarsenical biotransformations and the radical SAM superfamily.

427

Further biochemical analysis of complex arsenic species and their molecular

428

mechanisms of biosynthesis and degradation will enable a better understanding for the

429

ecological roles of these complex organoarsenicals.

430 431

Acknowledgments

432

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

433

(41877422 and 21507125), Crossing-Group Project of KLUEH (KLUEH-201802), the

434

Austrian Science Fund (FWF) project number I2412-B21, and National Institutes of

435

Health Grants R01 GM55425 and ES023779 to bpr.

436 437

References

438

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439 440

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(2)

Nischwitz, V.; Pergantis, S. A. Mapping of Arsenic Species and Identification of a Novel

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Arsenosugar in Giant Clams Tridacna Maxima and Tridacna Derasa Using Advanced Mass

442

Spectrometric Techniques. Environ. Chem .2007, 4 (3), 187–196.

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to the Cycling of Arsenic in Marine Ecosystems. Environ. Sci. Technol. 2015, 49 (1), 33–50. (4)

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Duncan, E. G.; Maher, W. A.; Foster, S. D. Contribution of Arsenic Species in Unicellular Algae Edmonds, J. S.; Francesconi, K. A. Transformation of Arsenic in the Marine Environment. Experienta 1987, 43, 553–557.

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Hoffmann, T.; Warmbold, B.; Smits, S. H. J.; Tschapek, B.; Ronzheimer, S.; Bashir, A.; Chen, C.; Rolbetzki, A.; Pittelkow, M.; Jebbar, M.; Seubert, A.; Schmitt, L.; Bremer, E. Arsenobetaine: 20


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Zhu, Y.; Xue, X.; Kappler, A.; Rosen, B. P.; Meharg, A. A. Linking Genes to Microbial Biogeochemical Cycling : Lessons from Arsenic. Environ. Sci. Technol. 2017, 51, 7326–7339.

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Garcıi´a-Salgadoı, S.; Raber, G.; Raml, R.; Magnes, C.; Francesconi, K. A. Arsenosugar Phospholipids and Arsenic Hydrocarbons in Two Species of Brown Macroalgae. Environ. Chem.

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Oxo-Arsenosugars by a Freshwater Unicellular Green Alga, Chlamydomonas Reinhardtii. Biosci.

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Arsenic Species in Plants from Yellowknife, Northwest Territories, Canada. Environ. Sci.

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Giant Clam Tridacna Maxima: Isolation and Identification of an Arsenic-Containing Nucleoside.

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Sa´nchez-Riego, A. M.; Pez-Maury, L. L.; Florencio, F. J. Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803. PLoS One 2014, 9 (5), e96826.

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Graham, D. E.; Xu, H.; White, R. H. Identification of the 7,8-Didemethyl-8-Hydroxy-5-

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Deazariboflavin Synthase Required for Coenzyme F420 Biosynthesis. Arch. Microbiol. 2003,

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Urban, O. F.; Morris, T. W.; Reed, K. E.; Al, M. E. T. Lipoic Acid Metabolism in Escherichia

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Coli: The LplA and LipB Genes Define Redundant Pathways for Ligation of Lipoyl Groups to

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Apoprotein. J. Bacteriol. 1995, 177 (1), 1–10.

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Perche-Letuvée, P.; Kathirvelu, V.; Berggren, G.; Clemancey, M.; Latour, J. M.; Maurel, V.;

551

Douki, T.; Armengaud, J.; Mulliez, E.; Fontecave, M.; Garcia-Serres, R.; Gambarelli, S.; Atta,

552

M.. 4-Demethylwyosine Synthase from Pyrococcus Abyssi Is a Radical-S-Adenosyl- L-

553

Methionine Enzyme with an Additional [4FE-4S]+2 Cluster That Interacts with the Pyruvate Co-

554

substrate. J. Biol. Chem. 2012, 287 (49), 41174–41185.

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 23



Page 24 of 33

581 582 583

The TOC was created by Dr Xi-Mei Xue

584

TOC artwork

585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 24


Page 25 of 33


618 619

Legends

620 621

Table 1 Arsenic species in E. coli cells expressing SsarsM and SsarsS and medium were

622

determined by HPLC-ICP-MS/HR-ES-MS analysis using PRP-X100 when E. coli cells

623

expressing SsarsM and SsarsS were induced with 0.5 mM IPTG and 3 μΜ As(III) or

624

As(V).

625

A

m/z

Formula

Delta m [ppm]

138.97346

C2H8O2As

0.02

Molecular structure C H3 O

As

OH

C H3

210.99459

C5H12O4As

OH

-0.41

C H3

B

O

O

As OH

C H3 C H3

C

239.02589

C7H16O4As

-0.99

O

O

As OH

C H3 OH

241.00514

C6H14O5As

OH

-1.00

D

O

OH

As C H3

271.0157

C7H16O6As

-0.87

E

O

C H3

OH

C H3 O

OH

O

As OH

C H3 OH

OH

626 627 628 629 25



Page 26 of 33

630 631

Figure 1. Cyanobacterial ars operons. Shown are the putative operons of Synechocystis

632

sp. PCC 6803 and Nostoc sp. PCC 7120. The arsM (slr0303, alr3095) genes encode

633

As(III) S-adenosylmethionine methyltransferases. Slr0305 from Synechocystis sp. PCC

634

6803 belongs to SNARE-associated superfamily, alr3096 of Nostoc sp. PCC 7120

635

encodes PitA (the low affinity inorganic phosphate transporter), arsS (slr0304, alr3097)

636

encodes a radical SAM superfamily enzyme shown in this report to be involved in

637

arsenosugar biosynthesis.

638

Figure 2. Anion-exchange HPLC-ICP-MS chromatograms of arsenic species from (a)

639

wild type Synechocystis and (b) Synechocystis ΔarsS after exposure to 1 µM As(III) for

640

two weeks, (c) wild type Synechocystis and (d) Synechocystis ΔarsS incubated with 10

641

µM MAs(V) for two weeks, (e) wild type Synechocystis and (f) Synechocystis ΔarsS

642

treated with 100 µM DMAs(V) for one week.

643

Figure 3 A. Western blot analysis of SsArsM and/or SsArsS expression in E. coli strain

644

Rosetta bearing the indicated expression plasmid constructs. MSR, MR, SR, or CK

645

containing pETDuet-SsarsS and pET-28a-SsarsM, pET-28a-SsarsM, pETDuet-SsarsS,

646

or pETDuet and pET-28a, respectively, induced by IPTG. B. Typical chromatograms

647

of arsenic species in E. coli strain Rosetta medium incubated with 3 μM As(III) by ion-

648

pairing

649

dimethylarsinoyl-hydroxycarboxylic acids.

650

Figure 4. Arsenic species transformed by different combination of ArsS orthologs and

651

SsArsM. A. Western blot analysis of SsArsM and different ArsS orthologs expression

652

in Rosetta bearing the indicated expression plasmid constructs. MNsS, MSeS, MPmS,

reverse-phase

column

HPLC-ICP-MS.

DMA-HA

indicates

the

26


Page 27 of 33


653

MApS, MCvS indicate E. coli strain Rosetta cells containing pET-28a-SsarsM and

654

pETDuet-NsarsS, pETDuet-SearsS, pETDuet-PmarsS, pETDuet-AparsS, or pETDuet-

655

CvarsS induced by IPTG respectively. B. Typical chromatograms of arsenic analysis in

656

E. coli strain Rosetta medium treated with As(III) by ion-pairing reversed-phase

657

column HPLC-ICP-MS of SsArsM and different ArsS orthologs expressed in E. coli

658

strain Rosetta. DMA-HA indicates the dimethylarsinoyl-hydroxycarboxylic acids. The

659

mix consists of As(III), MAs(V), DMAs(V), As(V), and TMAO.

660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 27



Page 28 of 33

690

Associated content

691

Supporting Information: Supporting methods, Reagents and solutions (pS5),

692

Confirmation of arsMarsS operon (pS5), Phylogenetic analyses of SsArsS (pS6),

693

Comparative growth curves for WT, disruption strains and complementary strains

694

(pS6), Cloning arsM and arsS from cyanobacteria (pS7), Resistance assays (pS8),

695

Nucleotide sequence synthesis of ArsS orthologs from other sequenced organisms

696

(pS8), Construction of SsarsS cysteine mutants (pS9), Immunological detection of ArsS

697

and ArsM (pS10), The structures of several common arsenic compounds found in

698

nature (Figure S1), bacterial strains and plasmids (Table S1), Primer sequences used

699

for PCR amplification in the disruption, complementation, gene expression, site-mutant,

700

and ars operon identification strategies (Table S2), ICP-MS and chromatographic

701

conditions used for arsenic species analysis (Table S3), Confirmation of arsMarsS

702

operon (Figure S2), Gene deletion and complementation (Figure S3), Phylogram of

703

SsArsS and representative enzymes of the radical SAM superfamily with different

704

functions (Figure S4), The UV/visible spectroscopy of SsArsS shows a shoulder

705

centered at 410 nm for the [4Fe-4S]2+ protein (Figure S5), SDS-PAGE analysis (12%,

706

Coomassie blue-stained) of SsArsM and/or ArsS expression in Rosetta bearing the

707

indicated plasmid (Figure S6), SsArsS or /and SsArsM confers resistance to As(III) in

708

E. coli (Figure S7), Arsenic concentrations of different E. coli Rosetta cells or Rosetta

709

cells expressing SsarsM and SsarsS (Figure S8), ICP-MS and HR-ESMS measurements

710

of unknown arsenic species in E. coli medium (Figure S9), HPLC-ICP-MS (A) and HR-

711

ESMS measurements (B) of unknown arsenic species in E. coli cells (Figure S10),

712

Arsenic species transformed by different combination of SsarsS mutants and SsarsM 28


Page 29 of 33


713

(Figure S11), The information of ArsS orthologs studied in this study (Table S4),

714

Multiple alignment of SsArsS and other five ArsS orthologs (Figure S12).

715 716 717 718 719 720 721 722

29



Cyanobacterial ars operons. Shown are the putative operons of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120. The arsM (slr0303, alr3095) genes encode As(III) S-adenosylmethionine methyltransferases. Slr0305 from Synechocystis sp. PCC 6803 belongs to SNARE-associated superfamily, alr3096 of Nostoc sp. PCC 7120 encodes PitA (the low affinity inorganic phosphate transporter), arsS (slr0304, alr3097) encodes a radical SAM superfamily enzyme shown in this report to be involved in arsenosugar biosynthesis. 283x122mm (96 x 96 DPI)


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Anion-exchange HPLC-ICP-MS chromatograms of arsenic species from (a) wild type Synechocystis and (b) Synechocystis ΔarsS after exposure to 1 µM As(III) for two weeks, (c) wild type Synechocystis and (d) Synechocystis ΔarsS incubated with 10 µM MAs(V) for two weeks, (e) wild type Synechocystis and (f) Synechocystis ΔarsS treated with 100 µM DMAs(V) for one week. 330x322mm (96 x 96 DPI)



A. Western blot analysis of SsArsM and/or SsArsS expression in E. coli strain Rosetta bearing the indicated expression plasmid constructs. MSR, MR, SR, or CK containing pETDuet-SsarsS and pET-28a-SsarsM, pET28a-SsarsM, pETDuet-SsarsS, or pETDuet and pET-28a, respectively, induced by IPTG. B. Typical chromatograms of arsenic species in E. coli strain Rosetta medium incubated with 3 μM As(III) by ionpairing reverse-phase column HPLC-ICP-MS. DMA-HA indicates the dimethylarsinoyl-hydroxycarboxylic acids. 407x197mm (96 x 96 DPI)


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Arsenic species transformed by different combination of ArsS orthologs and SsArsM. A. Western blot analysis of SsArsM and different ArsS orthologs expression in Rosetta bearing the indicated expression plasmid constructs. MNsS, MSeS, MPmS, MApS, MCvS indicate E. coli strain Rosetta cells containing pET-28a-SsarsM and pETDuet-NsarsS, pETDuet-SearsS, pETDuet-PmarsS, pETDuet-AparsS, or pETDuet-CvarsS induced by IPTG respectively. B. Typical chromatograms of arsenic analysis in E. coli strain Rosetta medium treated with As(III) by ion-pairing reversed-phase column HPLC-ICP-MS of SsArsM and different ArsS orthologs expressed in E. coli strain Rosetta. DMA-HA indicates the dimethylarsinoyl-hydroxycarboxylic acids. The mix consists of As(III), MAs(V), DMAs(V), As(V), and TMAO. 333x157mm (96 x 96 DPI)


Identification of Steps in the Pathway of Arsenosugar Biosynthesis

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