SSRN Id4688123

1 Construction of Tetrahymena strains with highly active arsenic methyltransferase genes
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2 for arsenic detoxification in aquatic environments
3 Wenjun Xionga,b, Wei Weia, Man Hec, Bin Huc, Jun Mena, Jiawei Tud*, Wei Miaoa,b,e*
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5 a Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
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6 b University of Chinese Academy of Sciences, Beijing 100049, China
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7 c College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China
8 d School of Resource and Environmental Science, Wuhan University, Wuhan 430072,
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10 e Key laboratory of Lake and Watershed Science for Water Security, Chinese Academy of
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11 Sciences, Nanjing 210008, China
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14 *To whom correspondence may be addressed:

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15 Wei Miao, Email: miaowei@ihb.ac.cn
16 Jiawei Tu, Email: tujiawei@whu.edu.cn

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4688123
20 Abstract
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21 Biomethylation is an effective means of arsenic detoxification by organisms living in
22 aquatic environments. Ciliated protozoa (including Tetrahymena species) play an important
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23 role in the biochemical cycles of aquatic ecosystems and have a potential application in
24 arsenic biotransformation. This study compared arsenic tolerance, accumulation, methylation,
25 and efflux in 11 Tetrahymena species. 19 arsenite (As(III)) S-adenosylmethionine (SAM)
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26 methyltransferase (arsM) genes, of which 12 are new discoveries, were identified, and protein
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27 sequences were studied. We then constructed recombinant cell lines based on the
28 Tetrahymena thermophila (T. thermophila) wild-type SB210 strain and expressed each of the
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29 19 arsM genes under the control of the metal-responsive the MTT1 promoter. In the presence
30 of Cd2+ and As(V), expression of the arsM genes in the recombinant cell lines was much
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31 higher than in the donor species. Evaluation of the recombinant cell line identified one with
32 ultra-high arsenic methylation enzyme activity, significantly higher arsenic methylation

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33 capacity and much faster methylation speed than other reported arsenic methylated organisms,
34 which methylated 89% of arsenic within 6.5 h and had an excellent capacity for the arsenic
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35 detoxification of lake water containing As(V). This study has made a significant contribution
36 to our knowledge on arsenic metabolism in protozoa and demonstrates the great potential to
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37 use Tetrahymena species in the arsenic biotransformation of aquatic environments.
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39 Keywords: Tetrahymena, Arsenate, Biomethylation, Methyltransferase, Detoxification,
Biotransformation.
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41 1. Introduction
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42 In recent years, arsenic (As) pollution has become a global problem that poses a serious
43 threat to both the environment and public health (He and Charlet, 2013; Li et al., 2021).
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44 Arsenic is a highly toxic carcinogen that occurs widely in nature and accumulates in the
45 human body. Long-term exposure to arsenic increases the risk of several cancers (including
46 skin, lung cancer, and bladder cancers), as well as other diseases (Chen et al., 1992; Khairul et
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47 al., 2017; Smith et al., 1992). Therefore, efficient methods of environmental remediation are
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48 needed to address arsenic pollution.
49 In contrast to traditional physical and chemical arsenic treatment techniques, arsenic

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50 biotransformation is a natural process and, therefore, is more potent and sustainable
51 (Dabrowska et al., 2021; Irshad et al., 2021; Mallick et al., 2014; Rahman et al., 2014). To
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52 detoxify environmental arsenic, organisms have evolved a series of metabolic strategies
53 including active extrusion, intracellular chelation, and transformation of arsenic into less-toxic
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54 organic forms (Stolz et al., 2002; Tsai et al., 2009). Among these strategies, reduction of
55 inorganic arsenicals to As(III) and further methylation is a major arsenic detoxification

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56 pathway (Bhattacharjee and Rosen, 2007; Ye et al., 2012). Through reduction and
57 biomethylation, various organisms can transform highly toxic inorganic arsenicals into
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58 monomethylarsenite (MMA(III)), dimethylarsenite (DMA(III)), and trimethylarsenite
59 (TMA(III)), which can be further oxidized to less-toxic molecules such as MMA(V),

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60 DMA(V), and trimethylarsenic oxide (Qin et al., 2006; Rahman and Hassler, 2014). Although
61 DMA(III) and MMA(III) are more toxic than inorganic arsenicals, they are transient
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62 intermediates that do not accumulate in cells and are excreted into the environment (Chen et
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63 al., 2014; Qin et al., 2006). Consequently, arsenic biomethylation is considered an efficient
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64 bioremediation method for environmental arsenic pollution (Ye et al., 2012). As(III)
65 methylation is catalyzed by specific arsenite (As(III)) S-adenosylmethionine (SAM)
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66 methyltransferase (ArsM) enzymes that use S-adenosylmethionine as a methyl group donor
67 (Chen et al., 2020; Chen et al., 2022; Viacava et al., 2020). The genes responsible for arsenic
68 methylation (arsM) are ubiquitously present in organisms living in a wide range of
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69 environments and their protein products have been characterized in a variety of species.
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70 Previous studies on arsenic biomethylation by bacteria, algae, fungi, plants, and animals have
71 demonstrated that arsenic methylation capacity differs significantly among species (Nies and
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72 Silver, 2007; Yan et al., 2019). Therefore, arsenic biotransformation could be developed by
73 identifying highly active arsM genes in species with highly efficient arsenic metabolism.
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74 Ciliated protozoa are ubiquitous in aquatic ecosystems (Corliss, 2002). Of these, model
75 Tetrahymena species can be grown in culture and have a basic eukaryotic life cycle (Collins
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76 and Gorovsky, 2005). They also contain heavy metal sensing mechanisms, for example, the T.
77 thermophila MTT1 gene is induced by trace concentrations of heavy metal ions (Diaz et al.,
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78 2007). Therefore, model Tetrahymena species are widely used as efficient gene expression
79 systems, including in ecotoxicology (Persoone and Dive, 1978; Schramm et al., 2011).
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80 Previous studies found that the free-living species Tetrahymena pyriformis (T. pyriformis) has
81 a high arsenic tolerance and can rapidly methylate arsenic (Yin et al., 2011; Zhang et al.,
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82 2012). In both T. pyriformis and T. corlissi, arsenic methylation is mediated by highly active
83 forms of ArsM (Wei et al., 2016; Ye et al., 2014). However, despite the high arsenic tolerance
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84 and abundant genetic resources related to arsenic methylation in Tetrahymena, no in-depth

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85 research has been conducted to identify the key arsM genes and analyze ArsM enzyme
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86 activity in these species. Given the ubiquitous presence of arsenic and the important role of
87 protozoa in aquatic ecosystems, there is an urgent need to determine arsenic tolerance and
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88 stress response across different Tetrahymena species and identify high-activity ArsM
89 enzymes with a potential application in arsenic biotransformation.
90 In this study, we compared arsenic tolerance and evaluated arsenic accumulation,
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91 methylation, and efflux in 11 Tetrahymena species. Similarity searches identified a total of 19
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92 arsM genes in these and other four sequenced species. We constructed metal-responsive
93 recombinant cell lines by expressing each of the 19 arsM genes under the control of the MTT1
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94 promoter in the T. thermophila wild-type SB210 strain and established a system to evaluate
95 their arsenic methylation ability. Thus, we identified a recombinant cell line with ultra-high
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96 arsenic methylation capacity and evaluated its ability to methylate As(V) in samples of lake
97 water containing different arsenic concentrations. In response to increasing arsenic

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98 concentrations, the arsenic methylation capacity of this recombinant cell line increased,
99 demonstrating an excellent arsenic detoxification ability. These results improve our

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100 knowledge of arsenic metabolism in protozoa and demonstrate the great potential to use
101 Tetrahymena species in the arsenic biotransformation of aquatic environments.

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103 2. Materials and Methods

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104 2.1. Materials
105 Reagents were obtained from the following suppliers: super proteose peptone (Becton,
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106 Dickinson and Company, USA); ferric citrate, glucose, and Na2HAsO4·7H2O (Sigma, USA);
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107 yeast extract (Oxoid, UK); standard solutions of As(V), As(III), MMA(V), and DMA(V)
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108 (National Standards Substance Center, China); NH4NO3 (Prin-Cen Scientific, China); and
109 CdCl2 ·2.5H2O (Aladdin, China).
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111 2.2. Tetrahymena species and culture conditions
112 11 Tetrahymena species were obtained from the American Type Culture Collection, the
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113 Tetrahymena Stock Center, and the National Aquatic Biological Resource Center (Table S1).
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114 Separate cultures of the 11 Tetrahymena species were maintained in super proteose peptone
115 (SPP) medium containing 2% proteose peptone, 0.2% glucose, 0.1% yeast extract, and
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116 0.003% ferric citrate at their optimum culture temperature, with shaking at 135 rpm (Orias et
117 al., 1999). For all species, the cell density was recorded using a Beckman counter (Beckman
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118 Coulter, USA). The morphology of T. thermophila was recorded by a compound microscope
119 (Nikon Eclipse Ni, Japan).

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120 2.3. Comparison of As(V) resistance among Tetrahymena species
121 The arsenic tolerance of 11 Tetrahymena species was determined by measuring the
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122 median effect concentration (EC50) at 24 h after As(V) exposure. This metric indicates the
123 As(V) concentration needed to inhibit cell growth by 50%. Arsenic exposure concentrations
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124 for Tetrahymena species are described in Text S1 of the Supplementary materials. Cell
125 density was measured at 0 h and 24 h using a Beckman counter and relative growth rates were
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126 calculated by comparison to the untreated control.
127
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128 2.4. High performance liquid chromatography-inductively coupled plasma mass
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129 spectrometry (HPLC-ICP-MS) analysis of arsenic species
130 Arsenic species were analyzed by HPLC-ICP-MS (Nan et al., 2018). For this, a NexION
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131 300X ICP-MS (Perkin Elmer, USA) was connected to a HPLC unit (Prin-Cen ELSpe-2
132 HPLC, Prin-Cen Scientific, China). A Prin-Cen Specia Fast Column (4.0 × 50 mm, Prin-Cen
133 Scientific, China) and dual mobile phases of ammonium nitrate solution (phase A, 8 mmol/L
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134 NH4NO3; phase B, 20 mmol/L NH4NO3) were used. Sample collection and pretreatment were
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135 described in Text S2 of the Supplementary materials. Arsenic species present in the samples
136 were identified by comparing the retention times with those of standards (including As(V),
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137 As(III), MMA(V), and DMA(V)) and were quantified based on the peak areas of external
138 calibration curves.

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140 2.5. Identification of ArsM enzymes in 15 Tetrahymena species

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141 Based on single-cell/single-nucleus amplification technology, the genomes of 11
142 Tetrahymena species and four others (T. empidokyrea, T. glochidiophila, T. paravorax and T.
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143 vorax) were sequenced, assembled, and annotated using Illumina Sequencing and Nanopore
144 Sequencing technology (Xiong et al., 2019). The ArsM protein sequences in T. borealis,
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145 T. canadensis, T. empidokyrea, T. paravorax, T. pyriformis, T. shanghaiensis, and T. vorax
146 were used as seed sequences for similarity searching (TBLASTN) against the assembled
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147 scaffold sequences of the other Tetrahymena species. Open Reading Frames (ORFs) in the
148 Tetrahymena species were predicted using ORFfinder

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149 (https://www.ncbi.nlm.nih.gov/orffinder/) and verified by PCR (polymerase chain reaction); a

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150 profile-based Pfam (http://pfam-legacy.xfam.org/) domain search showed that all ORFs
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151 belong to the Methyltransf_31 conserved protein domain family (Pfam: PF13847).
152 The amino acid sequences of the ArsM/AS3MT in different organisms were obtained
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153 from the GenBank (https://www.ncbi.nlm.nih.gov/). Amino acid sequences of the arsenic
154 enzymes were analyzed by BioEdit software and a Maximum Likelihood (ML) tree was
155 constructed using MEGA 11.
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156
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157 2.6. Construction of recombinant T. thermophila SB210 strains containing different arsM
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159 The plasmid pMTT1-GFP-Bsr was obtained from the Tetrahymena Stock Center
160 (Cornell University, USA). Original GFP (Green Fluorescent Protein) or Bsr (Blasticidin
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161 resistance) of pMTT1-GFP-Bsr were substituted by arsM or Pmr (Paromomycin resistance)
162 via PCR and ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co.,Ltd, China).
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163 Correct substitutions were verified by sanger sequencing. Recombinant T. thermophila SB210
164 cell lines containing different arsM genes in the somatic macronucleus were constructed by
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165 particle bombardment. For this, gold particles were coated with DNA constructs containing
166 the 5ʹ flanking region of MTT1, arsM, a Pmr or Bsr cassette, and the 3ʹ flanking region of
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167 MTT1, and then bombarded into pre-starved wild-type T. thermophila SB210 cells using a
168 biolistic particle delivery system (CassidyHanley et al., 1997). Through homologous
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169 recombination, the endogenous MTT1 coding region was replaced with the arsM cassette,
170 conferring cadmium-inducible ArsM expression (Mochizuki, 2008; Vogt and Mochizuki,
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171 2013). Bombarded cells were incubated at 30 °C for 3 h, and positive recombinants were
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172 selected by adding paromomycin or blasticidin to the medium at an initial concentration of
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173 100 μg/mL and gradually increasing to 192 mg/mL (for Pmr selection) or 5 mg/mL (for Bsr
174 selection). All copies of the MTT1 coding region in the macronucleus were substituted by
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175 arsM cassette in transformants were confirmed by PCR. Sequences of all the primers used for
176 PCR were in Table S2. Fluorescence analysis in recombinant cell line SB210-GFP were
177 described in Text S3 of the Supplementary materials.
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179 2.7. Evaluation of recombinant cell lines arsenic methylation capacity in lake water
180 The SB210-Tmob2ArsM recombinant cell line was tested for arsenic accumulation and
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181 methylation in lake water sample. Samples of lake water (East Lake in Wuhan, Southeast
182 China) were filtered through a gauze-covered funnel to remove large sand grains and then
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183 through a polyether sulfone membrane (0.22 μm), and stored at 4 °C. Samples containing
184 different As(V) concentrations (100, 250, 500, 2500 and 5000 μg/L) were prepared using
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185 As(V) stock solution. SB210-Tmob2ArsM cells were grown to mid-logarithmic growth stage
186 in SPP medium (cell density, 150×104 cells/mL) and then collected by centrifugation at 395 ×
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187 g for 3 min (room temperature) and washed twice with ultrapure water. The cells were then
188 added to the mixture of lake water plus As(V) solution and incubated at 30 °C and 60 rpm.
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189 Cells and aqueous were removed after 48 h for the analysis of arsenic species and cell growth.
190
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191 2.8. Statistical analysis
192 Statistical analysis was described in Text S3 of the Supplementary materials.

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194 3. Results and Discussion
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195 3.1. Arsenic tolerance of 11 Tetrahymena species
196 To construct Tetrahymena strains suitable for arsenic detoxification, we first determined
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197 the arsenic tolerance and stress responses of 11 Tetrahymena species. Growth analysis
198 showed that the maximum cell density and growth rate varied across species (Figure 1A). The
199 cell density at the start of the logarithmic growth phase, maximum cell density, and
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200 generation time were determined for each species (Table S3). These data indicate the most
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201 appropriate initial cell density for arsenic exposure for each species. EC50 values for 24 h
202 As(V) exposure were used to determine the arsenic tolerance of each Tetrahymena species.
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203 The large variation in EC50 values indicates that arsenic tolerance varies greatly among the 11
204 Tetrahymena species (Figure 1B): T. shanghaiensis had the highest arsenic tolerance
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205 (25.86 μg/mL) and T. canadensis the lowest (1.48 μg/mL). Among the 11 Tetrahymena
206 species, 8 of them had a 24-h EC50 value exceeding 5 μg/mL. Therefore, to ensure the highest
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207 levels of arsenic methylation while maintaining optimum cell growth, a concentration of
208 5 μg/mL As(V) was selected for the comparative analysis of ArsM capacity.
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209 There are previous works on EC50 values for As(V) exposure in T. pyriformis (18-h
210 EC50=40 μM; 24-h EC50=33.27-36.72 μM) and T. thermophila (36-h EC50=1.79 mg/L) (Ye et
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211 al., 2014; Zhang et al., 2012; Zhang et al., 2015). Our datas are similar to previous works,
212 with minor differences due to different experimental conditions or Tetrahymena cell lines.
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213 Comparing with different organisms (Table S4), all 11 Tetrahymena species were
214 significantly more As(V) resistant than Lemna disperma, Bosmina longirostris and some
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215 algae (Passino and Novak, 1984; Rahman et al., 2014; Vocke et al., 1980), less As(V)
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216 resistant than other species of bacteria, especially those living in environments that are
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217 heavily contaminated with arsenic, such as Rhodobacter capsulatus (72-h EC50 =
218 152.25 μg/mL) (Lin et al., 2014), and T. shanghaiensis was more As(V) resistant than the
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219 aquatic plant Spirodela polyrhiza L ( 7-d EC50 = 13.62 μg/mL) (Zhang et al., 2011).
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221 Figure 1. (A) Growth curves of the 11 Tetrahymena species and a micrographic image
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222 of T. the (bar, 50 μm); (B) 24-h EC50 values of the 11 Tetrahymena species. Three replicates
223 were used for all species. (T. sha): T. shanghaiensis; (T. ame): T. americanis; (T. mob): T.
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224 mobilis; (T. cor): T. corlissi; (T. bor): T. borealis; (T. tro): T. tropicalis; (T. the): T.
225 thermophila; (T. mim): T. mimbres; (T. pig): T. pigmentosa; (T. pyr): T. pyriformis; (T. can): T.
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226 canadensis.
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228 3.2. Evaluation of dynamic changes in arsenic accumulation and methylation in 11
229 Tetrahymena species
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230 The uptake and metabolism of inorganic arsenic are important for arsenic detoxification
231 by Tetrahymena. In response to inorganic arsenic exposure, arsenic is taken up and
232 accumulates in the cells; it is then metabolized via the following reactions:
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233 As(V)→As(Ⅲ)→MMA(V)→DMA(V). In the detoxification process, As(V) is first reduced
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234 to As(III) by glutathione and then methylated to the less-toxic pentavalent methylarsenic
235 (MetAs) species (including DMA(V) and MMA(V)) (Rahman and Hassler, 2014). To
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236 evaluate arsenic metabolism by the 11 Tetrahymena species, arsenic accumulation,
237 transformation and efflux were determined by measuring the levels of arsenic species
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238 ((As(V), As(III), MMA(V), and DMA(V)) in both Tetrahymena cells and culture medium
239 using HPLC-ICP-MS (Zhu et al., 2008).

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240 Arsenic species present in both cells and culture medium were analyzed at 0.5, 6.5, 12.5,
241 18.5, and 24.5 h after exposure to 5 μg/mL As(V). Over the 24.5 h incubation period, the total
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242 arsenic content of cells varied from 59.18 μg/g to 366.34 μg/g (dry weight; Figure 2A).
243 Intracellular concentrations of As(V), As(III), MMA(V), and DMA(V) varied significantly
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244 among species. T. pyriformis had the highest intracellular content of methylated arsenic
245 187.65 μg/g, corresponding to methylation of 95% of the total intracellular arsenic; this was
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246 3.7 times of that in T. tropicalis (in which 26% of the total intracellular arsenic was
247 methylated). In T. thermophila, neither MMA(V) nor DMA(V) were detected at any time
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248 point. The concentrations of As(III), MMA(V), and DMA(V) in the culture medium also
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249 varied significantly among species, indicating a wide variation in arsenic efflux (Figure S1).
250 To clearly reflect the differences in arsenic methylation ability among the 11
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251 Tetrahymena species, the primary methylation index (PMI; ratio of MMA + DMA to total As)
252 and secondary methylation index (SMI; ratio of DMA to MMA + DMA) were used to
253 evaluate the percentage of methylated to total arsenic and the overall level of arsenic
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254 methylation, respectively (Cui et al., 2020; Liu et al., 2017). These indexes were determined
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255 in Tetrahymena cells only (PMIc and SMIc) and in the system containing cells and culture
256 medium (PMIs and SMIs). Values for PMIc and SMIc are shown in Figure 2B. For
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257 T. pyriformis, T. shanghaiensis, T. canadensis, T. mobilis, T. mimbres, and T. borealis, the
258 PMIc and SMIc values increased rapidly to 85% within 18.5 h; in contrast, the rate of arsenic
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259 methylation was slower in T. americanis, T. pigmentosa, T. corlissi and T. tropicalis. SMIs
260 values and trends were similar to those of SMIc. In contrast, PMIs values were smaller and
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261 trends were different compared with PMIc values (Figure 2B and Figure S2), indicating lower
262 overall arsenic uptake and significant differences in uptake among the species. For those
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263 species with similar PMIc and SMIc values, the order of PMIs values was
264 T. shanghaiensis﹥T. canadensis, T. pyriformis, and T. borealis﹥T. mobilis and T. mimbres.

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265 Despite relatively low PMIc and SMIc values, owing to their higher arsenic uptake, T.
266 americanis and T. pigmentosa had the highest PMIs values of all 11 Tetrahymena species
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267 (Figure 2B and Figure S2).

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269 Figure 2. (A) Intracellular concentrations of four arsenic species; (B) PMIc and SMIc. 11
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270 Tetrahymena species were exposed to 5 μg/mL As(V) for 0.5–24.5 h. Data are the mean ± SD
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271 (n = 3). SD, standard deviation.
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273 As previously reported, ArsM levels are related to arsenic methylation capacity in two
274 Tetrahymena species (T. pyriformis and T. corlissi) (Wei et al., 2016; Ye et al., 2014). To
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275 expand this analysis, we used homology searching and protein domain analysis to identify the
276 arsM genes in Tetrahymena species. The 19 predicted arsM genes are shown in Table S5. 19
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277 arsM genes identified, 12 had not been previously reported. Of the 15 Tetrahymena species,
278 10 contain one arsM gene, three (T. mimbres, T. mobilis, and T. tropicalis) contain two, one
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279 (T. glochidiophila) contains three, and one (T. thermophila SB210) contains none. Multiple
280 sequence alignment of the ArsM enzymes from the 15 Tetrahymena species showed that they
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281 are members of the Methyltransf_31 family which appears to have methyltransferase activity
282 and similar to related proteins in other organisms, contain three conserved motifs (motifs I–
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283 III) and five conserved cysteine sites (Cys59, Cys60, Cys100, Cys188 and Cys238, cysteines
284 are numbered according to TborArsM; Figure S3). In addition, the 19 predicted ArsM
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285 proteins had some unique characteristics, including three incompletely conserved cysteines
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286 (Cys66, Cys76 and Cys81), two absolutely conserved cysteines (Cys126 and Cys193), and
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287 Cys–Cys pairs at the C-terminus (Figure S3).
288 In summary, we have established a method to evaluate dynamic changes in arsenic
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289 accumulation and methylation in Tetrahymena. Although several of the 11 Tetrahymena
290 species had an excellent capacity for arsenic accumulation or arsenic methylation, this
291 property varied greatly among species, as did the number of arsM genes.
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293 3.3. Comparison of ArsM catalytic activity by establishing T. thermophila SB210
294 recombinant cell lines er

295 Owing to differences in the growth rate, arsenic tolerance and number of arsM genes
296 among the Tetrahymena species, we decided to express ArsM enzymes from the different
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297 species in the same genetic background and compare the arsenic methylation capacity of the
298 recombinant cell lines. Due to the high growth rate, good arsenic tolerance (Figure 1), and the
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299 lack of arsM gene (Table S5), the T. thermophila SB210 strain was, therefore, used as a
300 chassis species to compare the expression and activity levels of arsM genes from the 15
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301 Tetrahymena species and associated arsenic methylase activity. The 19 arsM genes, as well as
302 representative algae, bacterial, fungal, and human arsM genes from 9 outgroups, were
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303 individually transferred into T. thermophila SB210 cells (Table S5 and Table S6) and a total
304 of 23 T. thermophila SB210 recombinant cell lines were obtained (Table S7 and Figure S4).
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305 The ORF of the arsM (algae, bacterial, fungal, and human) adapted to the genetic code of
306 Tetrahymena and can be correctly expressed.

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307 In each recombinant cell line, the endogenous MTT1 coding region was replaced by an
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308 arsM gene (Figure 3A). In Tetrahymena, the MTT1 gene is a highly efficient heavy metal
309 sensing element with a highly efficient metal-responsive promoter that is induced by trace
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310 concentrations of heavy metals such as As, Cd, Cu, and Hg (Amaro et al., 2011, 2014; Díaz et
311 al., 2007; Rodriguez-Martin et al., 2022; Wang et al., 2011; Yu et al., 2005). To detect
312 recombinant protein expression in T. thermophila SB210 cells, an SB210-GFP cell line
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313 expressing GFP was used. To obtain high levels of protein expression, recombinant
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314 SB210-GFP cell lines were exposed to As plus Cd in the culture medium. In the presence of
315 5 μg/mL As(V), Cd2+ concentrations of 0, 0.05, 0.1, and 0.2 μg/mL induced protein
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316 expression, as shown by flow cytometry (Figure 3B and C). Co-treatment with 5 μg/mL
317 As(V) and 0.1 or 0.2 μg/mL Cd2+ induced similar high protein expression levels. Thus,
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318 co-treatment with 0.1 μg/mL Cd2+ and 5 μg/mL As(V) was used for all further experiments.
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319
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320 Figure 3. (A) Schematic diagram of target gene homologous recombination; (B)
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321 Fluorescence micrographs (bar, 50 μm); (C) GFP protein expression induced by 0, 0.05, 0.1,
322 and 0.2 μg/mL Cd2+ combined with 5 μg/mL As(V), a.u., arbitrary units; The significance
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323 difference analysis is marked with “a, b”, no common superscript has significance difference
324 (P < 0.001).
325
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326 As controls, wild-type T. thermophila SB210 cells were exposed to 0.1 μg/mL Cd2+ plus
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327 5 μg/mL As(V) (control group 1) or 5 μg/mL As(V) only (control group 2). For the 23
328 T. thermophila SB210 recombinant cell lines and two control groups, the amount of different
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329 arsenic species present in both cells and culture medium (Figure S5A and B) and the arsenic
330 methylation capacity (Figure S5C) were determined after treatment with Cd2+ plus As(V) for
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331 0.5, 2.5, 6.5 and 12.5 h. Intracellular arsenic accumulation was similar in all recombinant cell
332 lines and control groups, whereas the level and type of arsenic species present in cells and
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333 culture medium varied among the recombinant cell lines (Figure S5A and B). Of these, 16
334 cell lines (tro1, mob2, sha, ame, vor, cor, glo1, glo2, pyr, can, par, mim2, mim1, bor, pig and
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335 emp) could convert >53% of the intracellular arsenic into pentavalent MetAs species within
336 12.5 h, demonstrating a strong arsenic methylation capacity. Whereas, 3 cell lines (Hs, Cs and
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337 tro2) had weak methylation capacity. Neither MMA(V) nor DMA(V) were detectable in the
338 cells or culture medium for four cell lines and two control groups (mob1, glo3, Af, Rp, CG1
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339 and CG2).
340 Significant differences in the PMIc, SMIc, PMIs, and SMIs values were found among the
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341 23 recombinant cell lines (Figure S5C and Figure S6), confirming that this is an effective
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342 system to measure the catalytic activity of different ArsM enzymes. Despite the intracellular
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343 methylarsenic content of some recombinant cell lines (e.g., SB210-TshaArsM and
344 SB210-TameArsM) reaching saturation at or before 6.5 h, the methylarsenic content in
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345 culture medium and PMIs continued to increase up to 12.5 h, indicating sustained arsenic
346 uptake and methylarsenic efflux (Figure S5A, B and Figure S6). Moreover, due to similar
347 levels of total arsenic uptake, differences in PMIc among the recombinant cell lines were
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348 similar to the differences in PMIs (Figure S5C and Figure S6). These results show that the
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349 most of the 23 recombinant cell lines can methylate arsenic.
350 Nine representative recombinant cell lines were selected to analyze the relationship
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351 between arsenic methylation capacity and ArsM protein sequence. The amount of different
352 arsenic species present in cells and the arsenic methylation capacity were shown in Figure 4A
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353 and B. ArsM activity of the recombinant cell lines followed the general rule that the closer the
354 genetic distance between the gene source species and the chassis species, the better the
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355 activity of the recombinant enzyme. Since red algae, humans, bacteria and fungi are distantly
356 related to Tetrahymena, recombinant ArsM proteins from these species, such as HsArsM, had
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357 the weakest arsenic methylation capacity (Figure 4B and C).
358 Most of the Tetrahymena species with multiple arsM genes had one or two ArsM
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359 enzymes lacking arsenic methylase activity, for example, Tglo3ArsM from T. glochidiophila
360 (which has three ArsM genes) and Tmob1ArsM from T. mobilis (which has two arsM genes)
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361 (Figure 4B and C). Phylogenetic analysis showed that the two arsM genes in T. mimbres and
362 three arsM genes in T. glochidiophila were clustered together, suggesting the recent
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363 duplication of arsM genes during the evolution of these Tetrahymena species. In contrast, the
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364 two arsM genes of T. mobilis and T. tropicalis did not cluster together, indicating that gene
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365 duplication had occurred before species differentiation.
366 Sequence alignment of the ArsM enzymes suggests that mutation of three incompletely
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367 conserved cysteines (Cys66, Cys76 and Cys81) are responsible for loss of the arsenic
368 methylation function of Tetrahymena ArsM enzymes (Figure 4B and D). The T. thermophila
369 SB210 cell line lacking methylation ability could produce MMA(V) and DMA(V) after
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370 transfection with arsM genes, indicating that arsenic methylation is solely determined by
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371 ArsM in Tetrahymena. Lastly, two recombinant cell lines, SB210-Tmob2ArsM and
372 SB210-Ttro1ArsM, had the highest PMIc and SMIc values, indicating that they had the
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373 highest intracellular catalytic activity.
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374
375 Figure 4. (A) Intracellular concentrations of four arsenic species; (B) PMIc and SMIc.
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376 Recombinant Tetrahymena cell lines were exposed to 0.1 μg/mL Cd2+ plus 5 μg/mL As(V)
377 for 0.5, 2.5, 6.5 or 12.5 h. Data are the mean ± SD (n = 3). CG1, control group 1; (C)
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378 Sequence alignment of arsenic methylase genes in Tetrahymena and outgroups; Red
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379 represents strong arsenic methylation capacity, blue represents weak arsenic methylation
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380 capacity, black indicates no methylation capacity, gray indicates that five genes from the
381 outgroups were not transferred into T. thermophila SB210 strain, and bold text represents the
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382 T. thermophila SB210 recombinant cell lines used in this analysis; (D) Sequence alignment of
383 ArsM proteins.
384
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385 We next compared PMIc and SMIc values in recombinant cells and corresponding
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386 wild-type parental cells (recombinant vs endogenous expression; SB210-Tmob2ArsM vs
387 T. mobilis, SB210-Ttro1ArsM vs erT. tropicalis). PMIc and SMIc values for
388 SB210-Tmob2ArsM cells within 2.5 h reached 85% and 95%, respectively, whereas only
389 77% and 72% respectively, were achieved in T. mobilis even after 12.5 h. Similarly, PMIc and
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390 SMIc values for SB210-Ttro1ArsM cells within 2.5 h eached 88% and 97%, respectively,
391 whereas only 17% and 26% respectively, were achieved in T. tropicalis even after 12.5 h
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392 (Table S8). This result indicates that the MTT1 promoter amplifies arsM gene expression and
393 increases the arsenic methylation capacity of recombinant cells.

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394 The comparison of arsenic methylation ability of SB210-Tmob2ArsM and
395 SB210-Ttro1ArsM with the previously reported organisms was listed in Table 1.
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396 SB210-Tmob2ArsM and SB210-Ttro1ArsM had significantly higher arsenic methylation
397 capacity and much faster methylation speed than other organisms, such as the copepod
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398 Paracyclopina nana, freshwater fish Carassius auratus, the two axenic freshwater
399 phytoplankton species Closterium aciculare and Pediastrum duplex. Based on its high
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400 sensitivity to arsenic stress, strong arsenic uptake, and efficient arsenic methylation ability,
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401 the SB210-Tmob2ArsM cell line was used for all subsequent experiments.
402
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403 Table 1. Comparison of PMIC values in different organisms.
Species PMIc (%) Time Reference
Ostreococcus tauri 6 8-d Zhang et al., (2013)
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Closterium aciculare 30 14-d Papry et al., (2021)
Paracyclopina nana 32 24-h Byeon et al., (2020)
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Pediastrum duplex 42 14-d Papry et al., (2021)
Carassius auratus 43 10-d Cui et al., (2020)
Shewanella oneidensis MR-1 47 5-d Wang et al., (2016)
SB210-Tmob2ArsM
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89 6.5 h this study
SB210-Ttro1ArsM 90 6.5 h this study
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404
405 Since the MTT1 promoter can be induced by arsenic alone (Díaz et al., 2007;
406 Rodriguez-Martin et al., 2022), arsenic methylation following exposure to 5 μg/mL As(V)
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407 only or 0.1 μg/mL Cd2+ plus 5 μg/mL As(V) was compared in two recombinant cell lines:
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408 SB210-Tmob2ArsM with a higher methylation capacity, and SB210-TcanArsM with a lower
409 methylation capacity. Figure S7 shows concentrations of arsenic species in cells and culture,
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410 along with the proportion of each species, PMIc, SMIc, PMIs, SMIs, cell density, and arsenic
411 uptake. Compared with the condition with Cd2+ addition, in the presence of As(V) only, the
412 rate of intracellular methylarsenic accumulation in SB210-Tmob2ArsM and

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413 SB210-TcanArsM was slower, because arsM gene expression was lower. However, the PMIc
414 values in SB210-Tmob2ArsM and SB210-TcanArsM at 12.5 h still reached 65% and 45%
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415 respectively (Figure S7C), the PMIs values of SB210-Tmob2ArsM and SB210-TcanArsM at
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416 12.5 h were both 5% higher, possibly due to the higher cell density and higher total arsenic
417 uptake (Figure S7E, F, H). Notably, for SB210-TcanArsM in the absence of Cd2+, the SMIc at
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418 12.5 h reached 75% but the SMIs was only 34%. However, in SB210-Tmob2ArsM, the SMIc
419 and SMIs reached nearly 100% whether or not Cd2+ was present, indicating a wide variation in
420 the activity of ArsM enzymes from the different Tetrahymena species (Figure S7D and G).
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421 Although the methylarsenic accumulation rate was slower after exposure to As(V) only, a
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422 similar level of arsenic methylation and greater accumulation of methylarsenic were achieved
423 in SB210-Tmob2ArsM compared with a longer exposure to Cd2+ plus As(V); this was
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424 attributed to the ultra-high catalytic activity of the T. mobilis ArsM. This result shows that
425 recombinant cell lines may have important applications in the efficient biotransformation of
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426 inorganic arsenic pollution in natural water environment.
427
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428 3.4. Biotransformation of arsenic pollution in lake water with recombinant Tetrahymena cell
429 line
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430 The SB210-Tmob2ArsM cell line was used to assess the ability of methylating As(V) in
431 lake water. The As(V) concentration in lake water with high levels of arsenic pollution is
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432 usually 50–5000 μg/L (Pfeiffer et al., 2015). Therefore, As(V) was added to water samples
433 collected from East Lake in Wuhan to a final concentration of 100, 250, 500, 2500, or 5000
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434 μg/L (Hussain et al., 2021; Podgorski and Berg, 2020). A cell density of 150×104 cells/mL
435 and incubation time of 48 h were used to enable the system to convert as much As(V) as
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436 possible. The arsenic species in culture medium and SB210-Tmob2ArsM cells were
22
437 investigated after exposure to As(V) only or to Cd2+ plus As(V) (Figure 5). In the absence of
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438 Cd2+, DMA(V) was the main intracellular arsenic species at all As(V) concentrations
439 (Figure 5A, C, D). After exposure to Cd2+ plus As(V), the induction of arsM genes in all cells
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440 was higher compared with As(V) only for all As(V) concentrations and the methylarsenic
441 concentration in the culture medium increased with increasing As(V) concentration. In
442 contrast, the PMIs decreased (Figure 5B and F). After exposure to As(V) only, the PMIs was
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443 only 11% for 100 μg/L As(V) but increased significantly to 56% at 250 μg/L As(V). This
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444 indicates that the arsM gene is weakly induced at low As(V) concentrations and moderately
445 induced at higher As(V) concentrations, thereby increasing the arsenic methylation capacity
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446 of SB210-Tmob2ArsM cells (Figure 5F). At higher As(V) concentrations (2500 and
447 5000 μg/L), the toxicity of Cd2+ is lower than As(V), therefore, the addition of Cd2+ had an
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448 insignificant effect, similar PMIs values for As(V) treatment were obtained in the presence or
449 absence of Cd2+ (~22% and 12%, respectively; Figure 5F). However, at 250 and 500 μg/L
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450 As(V), As(V) toxicity is was much lower than Cd2+ toxicity, as demonstrated by the higher
451 cell density and increased arsenic uptake of Tetrahymena cells (Figure 5E and H), resulting in
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452 a higher PMIs in the absence of Cd2+ (Figure 5F).

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453
454
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Figure 5. Concentrations of arsenic species in recombinant SB210-Tmob2ArsM cells
455 and culture medium after exposure to lake water containing different As(V) concentrations.
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456 (A) Intracellular concentrations and respective proportions of four arsenic species; (B)
457 concentrations and respective proportions of three new arsenic species (As(III), MMA(V),
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458 DMA(V)) in culture medium; (C) PMIc values; (D) SMIc values; (E) final cell density; (F)
459 PMIs values; (G) SMIs values; and (H) arsenic uptake. 0.1, SB210-Tmob2ArsM exposed to
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460 As(V) plus 0.1 μg/mL Cd2+; 0, exposed to As(V) only.
461
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462 Methylarsenic efflux from SB210-Tmob2ArsM cells was next investigated by
463 calculating the percentage of methylarsenic to total arsenic in both cells and culture medium.
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464 The culture medium contained most methylarsenic after 48 h (Table S9). At high As(V)
465 concentrations (2500 or 5000 μg/L), the percentage of methylarsenic in cell was extremely
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466 low in the presence or absence of Cd2+. This result might be attributed to cell death, as
24
467 evidenced by the low final cell density (Figure 5E). However, at low As(V) concentrations
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468 (250 and 500 μg/L) without Cd2+, the final cell density remained high, the levels of
469 methylarsenic in cell and culture medium were high, and >62% of methylarsenic was
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470 discharged into the culture medium (Figure 5E and Table S9). This result indicates that
471 SB210-Tmob2ArsM cells can continuously take up and methylate As(V) from the culture
472 medium, and then discharge methylarsenic into the culture medium.
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473 Based on the results of this study, the construction of T. thermophila SB210 recombinant
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474 cell lines, their response to metal ions in aqueous solution, and the proposed process of
475 converting inorganic As(V) into methylarsenic were illustrated in Figure 6. Using
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476 T. thermophila SB210 cells with strong arsenic reduction activity and no arsenic methylation
477 ability as the chassis organism, recombinant cell lines were constructed by replacing the
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478 endogenous MTT1 coding region with each of the 19 arsM genes identified from the 15
479 Tetrahymena species. Due to the ultra-sensitive response of MTT1 promoter to metal ions
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480 (especially Cd2+), the recombinant cell lines responded to Cd2+ and As(V) in the culture
481 medium by overexpressing the arsM genes. After uptake by cells, As(V) was first reduced to
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482 As(III), then transformed to MMA(V) and DMA(V) through methylation by the
483 overexpressed ArsM enzyme, and eventually effluxed into the culture medium.
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25
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484
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485 Figure 6. Schematic representation of construction of the recombinant cell lines by
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486 combining the MTT1 endogenous promoter with an ectopic arsM gene, the response of the
487 recombinant gene to extracellular metal ions in the surrounding water, and the conversion of
488
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inorganic As(V) into methylarsenic. pMTT1-ArsM: arsM gene driven by the MTT1 promoter.
489
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490 This work demonstrates that arsM genes can be overexpressed in recombinant cell lines
491 in response to extracellular Cd2+ and As(V), and that the ArsM enzymes have a greater
492 capacity for arsenic methylation in the expression system than in the corresponding wild-type
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493 species due to the high expression efficiency of the MTT1 promoter. Moreover, comparative
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494 analysis of the methylation capacity of different recombinant cell lines identified the
495 recombinant SB210-Tmob2ArsM cell line with ultra-high methylation capacity. To achieve
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496 maximum As(V) detoxification of aqueous environments, Cd2+ induction might be required at
497 very low As(V) concentrations. However, SB210-Tmob2ArsM can be directly used for As(V)
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498 pollution remediation without Cd2+ induction at As(V) concentrations of 250–5000 μg/L.
499 Therefore, the SB210-Tmob2ArsM could efficiently methylate As(V) in lake water samples
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500 and is expected to be useful for the future biotransformation of arsenic-contaminated water
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501 bodies.
502
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503 4. Conclusions
504 In this study, we compared arsenic tolerance, arsenic accumulation, methylation, and
505 efflux in 11 Tetrahymena species. Similarity searches identified a total of 19 arsM genes in
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506 these and other four sequenced species, and 12 of them were new discoveries.
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507 Metal-responsive recombinant cell lines were further constructed by expressing each of the
508 arsM genes under the control of the MTT1 promoter in the T. thermophila SB210 strain. The
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509 highly effective methylation ability of the recombinant SB210-Tmob2ArsM cell line was
510 identified, which methylated 89% of arsenic within 6.5 h, showing higher arsenic methylation
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511 ability than currently reported arsenic methylation species. It also has an excellent capacity
512 for the arsenic detoxification of lake water containing As(V), 56% of arsenic was methylated
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513 at 250 μg/L As(V) in 48 h. Overall, the Tetrahymena strain with highly active arsenic
514 methyltransferase gene is a promising candidate for the treatment of As(V) in aquatic
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515 environments, providing a feasible approach to promote arsenic detoxification and reduce
516 As(V) contamination.

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517
518 CRediT authorship contribution statement

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519 Wenjun Xiong: Conceptualization, Methodology, Data curation, Formal analysis, Writing –
520 original-draft. Wei Wei: Conceptualization, Methodology, Software. Man He: Methodology,
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521 Supervision. Bin Hu: Methodology, Supervision. Jun Men: Methodology, Software.
27
522 Jiawei Tu: Methodology, Supervision, Funding acquisition, Writing − review & editing. Wei
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523 Miao: Methodology, Supervision, Resources, Funding acquisition, Writing − review &
524 editing.
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525
526 Declaration of Competing Interest
527 The authors declare that they have no known competing financial interests or personal
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528 relationships that could have appeared to influence the work reported in this paper.
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529
530 Data availability er

531 Data will be made available on request.
532
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533 Acknowledgments
534 This research was supported by the National Key R&D Program of China (No.
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535 2020YFA0907400), the Natural Science Foundation of China (No. 21976208 and 32300414),
536 the Bureau of Science and Technology of Wuhan (No. 2019020701011483), the Strategic
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537 Priority Research Program of the Chinese Academy of Sciences (No. XDPB18), and the
538 China Postdoctoral Science Foundation (No. 2023M732706).

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539
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1 Construction of Tetrahymena strains with highly active arsenic methyltransferase genes

5 a Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

8 d School of Resource and Environmental Science, Wuhan University, Wuhan 430072,

14 *To whom correspondence may be addressed:

15 Wei Miao, Email: miaowei@ihb.ac.cn

16 Jiawei Tu, Email: tujiawei@whu.edu.cn

22 aquatic environments. Ciliated protozoa (including Tetrahymena species) play an important

24 arsenic biotransformation. This study compared arsenic tolerance, accumulation, methylation,

25 and efflux in 11 Tetrahymena species. 19 arsenite (As(III)) S-adenosylmethionine (SAM)

32 ultra-high arsenic methylation enzyme activity, significantly higher arsenic methylation

37 use Tetrahymena species in the arsenic biotransformation of aquatic environments.

39 Keywords: Tetrahymena, Arsenate, Biomethylation, Methyltransferase, Detoxification,

49 In contrast to traditional physical and chemical arsenic treatment techniques, arsenic

55 inorganic arsenicals to As(III) and further methylation is a major arsenic detoxification

58 monomethylarsenite (MMA(III)), dimethylarsenite (DMA(III)), and trimethylarsenite

59 (TMA(III)), which can be further oxidized to less-toxic molecules such as MMA(V),

65 methylation is catalyzed by specific arsenite (As(III)) S-adenosylmethionine (SAM)

68 methylation (arsM) are ubiquitously present in organisms living in a wide range of

84 and abundant genetic resources related to arsenic methylation in Tetrahymena, no in-depth

89 enzymes with a potential application in arsenic biotransformation.

90 In this study, we compared arsenic tolerance and evaluated arsenic accumulation,

97 water containing different arsenic concentrations. In response to increasing arsenic

99 demonstrating an excellent arsenic detoxification ability. These results improve our

101 Tetrahymena species in the arsenic biotransformation of aquatic environments.

103 2. Materials and Methods

104 2.1. Materials

109 CdCl2 ·2.5H2O (Aladdin, China).

111 2.2. Tetrahymena species and culture conditions

119 (Nikon Eclipse Ni, Japan).

120 2.3. Comparison of As(V) resistance among Tetrahymena species

126 calculated by comparison to the untreated control.

138 calibration curves.

140 2.5. Identification of ArsM enzymes in 15 Tetrahymena species

141 Based on single-cell/single-nucleus amplification technology, the genomes of 11

145 T. canadensis, T. empidokyrea, T. paravorax, T. pyriformis, T. shanghaiensis, and T. vorax

148 Tetrahymena species were predicted using ORFfinder

149 (https://www.ncbi.nlm.nih.gov/orffinder/) and verified by PCR (polymerase chain reaction); a

155 constructed using MEGA 11.

177 described in Text S3 of the Supplementary materials.

191 2.8. Statistical analysis

192 Statistical analysis was described in Text S3 of the Supplementary materials.

229 Tetrahymena species

231 by Tetrahymena. In response to inorganic arsenic exposure, arsenic is taken up and

239 using HPLC-ICP-MS (Zhu et al., 2008).

264 T. shanghaiensis﹥T. canadensis, T. pyriformis, and T. borealis﹥T. mobilis and T. mimbres.

267 (Figure 2B and Figure S2).

288 In summary, we have established a method to evaluate dynamic changes in arsenic

294 recombinant cell lines er

306 Tetrahymena and can be correctly expressed.

324 (P < 0.001).

339 and CG2).

344 SB210-TameArsM) reaching saturation at or before 6.5 h, the methylarsenic content in

357 the weakest arsenic methylation capacity (Figure 4B and C).

383 ArsM proteins.

393 increases the arsenic methylation capacity of recombinant cells.

394 The comparison of arsenic methylation ability of SB210-Tmob2ArsM and

396 SB210-Tmob2ArsM and SB210-Ttro1ArsM had significantly higher arsenic methylation

Species PMIc (%) Time Reference

Ostreococcus tauri 6 8-d Zhang et al., (2013)

412 rate of intracellular methylarsenic accumulation in SB210-Tmob2ArsM and

452 a higher PMIs in the absence of Cd2+ (Figure 5F).

460 As(V) plus 0.1 μg/mL Cd2+; 0, exposed to As(V) only.

462 Methylarsenic efflux from SB210-Tmob2ArsM cells was next investigated by