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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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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
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Abstract
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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
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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
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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
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genome through homologous recombination 18. To date, ArsM homologues have been
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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
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acid (HEPES), glycerol, sodium chloride (NaCl), imidazole, sodium arsenite
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(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
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suspended in 1.5 mL fresh BG11 medium containing 30 µL pTarsMKan (about 100
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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
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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
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homogenates were subsequently centrifuged at 13680 g on a Hermle Z326K (Hermle
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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
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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
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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
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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.
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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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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
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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
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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
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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
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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
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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
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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
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Legends
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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
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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
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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
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