Arsenic biotransformation potential of microbial arsH responses in the biogeochemical cycling of arsenic-contaminated groundwater

Accepted Manuscript Arsenic biotransformation potential of microbial arsH responses in the biogeochemical cycling of arsenic-contaminated groundwater Jin-Soo Chang, In-Ho Yoon, Kyoung-Woong Kim PII:

S0045-6535(17)31623-5

DOI:

10.1016/j.chemosphere.2017.10.044

Reference:

CHEM 20066

To appear in:

ECSN

Received Date: 9 February 2017 Revised Date:

5 October 2017

Accepted Date: 7 October 2017

Please cite this article as: Chang, J.-S., Yoon, I.-H., Kim, K.-W., Arsenic biotransformation potential of microbial arsH responses in the biogeochemical cycling of arsenic-contaminated groundwater, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.10.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Running Head: Microbial arsH Response in Extremely Arsenic-Contaminated Groundwater

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Arsenic biotransformation potential of microbial arsH responses in the

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biogeochemical cycling of arsenic-contaminated groundwater

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Molecular Biogeochemistry Laboratory, Biological & Genetic Resources Institute (BGRI), Hannam University (Jeonmin Campus), 505 Inno-Biz Park, 1646 Yuseong-daero, Yeseong-gu, Daejeon 34054, Republic of Korea 2 Decommissioning Technology Research Division, Korea Atomic Energy Research Institute (KAERI), 111 Daedeok-daero, 989 beon-gil, Yuseong-gu, Daejeon 34057, Republic of Korea 3,* School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

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* Corresponding author: Phone: +82-62-715-2442, Fax: +82-62-715-2434, E-mail: [email protected]

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Jin-Soo Chang1·In-Ho Yoon2·Kyoung-Woong Kim 3,*

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Manuscript submitted to Chemosphere

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Abstract ArsH encodes an oxidoreductase, an NAD(P)H-dependent mononucleotide reductase, with an

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unknown function, frequently within an ars operon, and is widely distributed in bacteria. Novel arsenite-

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oxidizing bacteria have been isolated from arsenic-contaminated groundwater and surface soil in Vietnam. We

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found that ArsH gene activity, with arsenite oxidase in the periplasm; it revealed arsenic oxidation potential of

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the arsH system. Batch experiment results revealed Citrobacter freundii strain VTan4 (DQ481466) and

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Pseudomonas putida strain VTw33 (DQ481482) completely oxidized 1 mM of arsenite to arsenate within 30-

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50h. High concentrations of arsenic were detected in groundwater and surrounding soil obtained from Vinh Tru

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village in Ha Nam province (groundwater: 11.0 µg/L to 37.0 µg/L ; and soil: 2.5 mg/kg, 390.1 mg/kg),

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respectively. An arsH gene encoding an organoarsenical oxidase protein was observed in arsenite-oxidizing

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Citrobacter freundii strain VTan4 (DQ481466), whereas arsB, arsH, and arsH were detected in Pseudomonas

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putida strain VTw33 (DQ481482). arsH gene in bacteria was first reported from Vietnam for resistance and

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arsenite oxidase. We proposed that residues, Ser 43, Arg 45, Ser 48, and Tyr 49 are required for arsenic binding

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and activation of arsH. The ars-mediated biotransformation strongly influenced potential arsenite oxidase

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enzyme of the operon encoding a homogeneous arsH. Results suggest that the further study of arsenite-

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oxidizing bacteria may lead to a better understanding of arsenite oxidase responses, such as those of arsH, that

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may be applied to control biochemical properties; for example, speciation, detoxification, bioremediation,

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biotransformation, and mobilization of arsenic in contaminated groundwater.

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Key words: molecular biogeochemistry, arsH, ArsH, arsenite oxidase, groundwater, Vietnam

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1. Introduction

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(0.0001%) (Nriagu, 2002). Bacteria can play an important role in mediating redox transformations in extreme

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environments, and arsenic-resistance (ars) operons are commonly found in terrestrial microorganisms (Oremland

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and Stolz, 2003). Arsenic exists in a variety of forms-such as As(-III), As(0), As(III), As(V), monomethylarsonic

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acid (MMA; V), dimethylarsinic acid (DMA; V), and trimethylarsine oxide, and bacteria are involved in

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biochemical degradation, detoxification mechanisms, and redox transformation in the natural environment (Pous

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et al., 2015). At present, operons known to contain arsenic resistance system (ars) genes (arsR, arsD, arsA,

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arsB, arsC, arsH) (Silver and Phung, 1996; Mo et al., 2011), include the As-(III)-resistant aox, arx, and aro

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system group and As(V)-resistant arr system group (Saltikov and Newman, 2003; Hèry et al., 2003; Kumari and

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Jagadevan, 2016). In an arsenic resistance system, arsR encodes a leader gene, a helix-turn-helix repressor of an

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operator-binding region, and first operates as an arsenite-binding dimer (Wu and Rosen, 1991; Silver and Phung,

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1996). The arsD is a secondary leader gene for transcription repressors (Chen and Rosen, 1945; Wu and Rosen,

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1993). In contrast, the ars operons of the Bacillus subtilis chromosome, Staphylococcus aureus pI258,

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Staphylococcus xylosus pSX268, Escherichia coli R773, E. coli R46, E. coli chromosome, Yersinia yop

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plasmids, Acidithiobacitlus ferrooxidans chromosome, and Pseudomonas putida chromosome encode additional

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proteins; ArsA encodes an arsenite-stimulated ATPase, and ArsB encodes an inner membrane protein, ArsD

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encodes another metal-regulatory gene, and ArsC encodes a reductase that transforms arsenate to arsenite (Silver

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and Phung, 1996). Note that the products of arsA and arsB or arsAB genes are two subunits of an ATP-coupled

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efflux/influx pump (Bröer et al., 1993). The ArsA protein induces the ATPase activity of the catalytic subunit,

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whereas the ArsB protein induces the function of the inner membrane pump of the arsenic channel (Tisa and

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Rosen, 1990; San Francisco et al., 1989).

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Arsenic is a ubiquitous toxic element in the Earth’s crust, in which it is generally present at a low level

Arsenate reductase (glutaredoxin) is an enzyme that catalyzes the chemical reaction: arsenate + glutaredoxin ⥩ arsenite + glutaredoxin disulfide + H2O

The two substrates of this enzyme are arsenate and glutaredoxin, with the three products being

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arsenite, glutaredoxin disulfide, and water. ArsH operons are widely distributed in bacteria, and are primarily

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involved in biochemical processes in the transfer of electrons to an electron acceptor. This is consistent with

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efficient NADPH-dependent mononucleotide reductases, such as cyanobacterium Synechocystis sp. PCC 6803

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(Hervás et al., 2012), and FMN reductase activity such as Shigella flexneri (Vorontsov et al., 2007).

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Synechocystis sp. PCC 6803, ArsH protein has a 74% sequence identity; it crystallizes as a tetramer. Shigella

ACCEPTED MANUSCRIPT flexneri ArsH protein has also been shown to be structurally homologous to NADPH-dependent FMN

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reductases having N- and C-terminal extensions in the crystal structure of an apo protein that has an α/β/α-fold

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typical of a conserved (T/SXRXXSX(T/S) fingerprint motif. The arsenic resistance protein ArsH purified from

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heterogeneously expressed Escherichia coli BL21 has a flavoprotein that binds NADPH in vitro (Wu et al.,

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2010). This ArsH arsenic resistance system has been reported in Bacillus sp. (FJ607354) from mine water,

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Serratia marcescens (DQ112331) obtained from mine tailings, which acts as an azoreductase, and Pseudomonas

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putida (AY866406) from mine tailings, which functions in arsenite oxidase response; however

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Acidothiobacillus ferrooxidans did not appear to confer arsenic resistance on Escherichia coli (Dave et al.,

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2008). The arsenic resistance system of Yersinia enterocolitica has an unknown ArsH gene that is involved in

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arsenic redox mechanisms (Neyt et al., 1997; Chen et al., 2015). Therefore, though arsenic resistance systems

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clearly involve ArsH function, the details remain unclear; frequently, ars is operon-encoded and is widely

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distributed in bacteria. The flavoprotein ArsH from Sinorhizobium meliloti catalyzes exhibits the

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NADPH:FMN-dependent reduction of molecular O2 to hydrogen peroxide and the reduction of azo dyes in

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arsenic detoxification (Ye et al., 2007). However, the purification and ars-aro-aox gene group having a

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heterogeneous-type arsenic resistance protein ArsH have mainly been investigated in highly As-contaminated

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soils.

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The objectives of this study are (i) to examine the effect of arsenic contamination on human health in

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extreme environments with respect to different levels of As-rich drinking water, surface water, tube well water,

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rain water, ground water, rice exposure, sediment exposure, and hair exposure in Vietnam through a literature

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review; (ii) to identify specific genes that, together with ArsH, are required for arsenite oxidase, and evaluate the

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phylogenetic relationship of ars genotype characteristics; and (iii) to investigate arsH-mediated

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biotransformation, which posited to play an important role in the response, by exploring its arsenite-oxidase

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function in relation to its biogeochemical environment, detoxification, bioremediation, and molecular

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biogeochemistry.

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2. Materials and methods

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2.1.

Field sampling and arsenic determination

118 Ha Nam province in Vietnam was selected as the site for collecting soil samples (5 g of surrounding soil

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from six sites, and 5 g of surrounding soil (1 m) from seven sites) and groundwater at six sites having arsenic-

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contaminated soil produced from four villages (Bo De: latitude 20° 30′ N, longitude 106° 05′ E, Hoa Hau:

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latitude 20° 28′ N, longitude 106° 10′ E, Nhan Dao: latitude 20° 34′ N, longitude 106° 07′ E, and Vinh Tru:

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latitude 20° 33′ N, longitude 106° 01′ E) in October 2004 (Fig. 1). Groundwater samples were shaken, followed

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by filtration of the groundwater phase through a PTFE membrane filter (Whatman 0.45 µm pore size, 13 mm),

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and the filtrate was stored at 4 °C until analysis. The samples from study areas in Vietnam were to the method

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reported by Nguyen, et al. (2009). The pH and Eh (mV) of water samples were measured in the field using an

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Orion electrode (Orion model 290A portable meter fitted with an Orion model 9107 electrode, USA). Soil

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samples were air-dried, passed through a 10-mesh sieve, and then extracted by shaking for one hour after

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distilled water (25 mL) was added, to evaluate water-soluble contents (1:5, soil: solution); after shaking, the

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solution was filtered through a membrane filter (Whatman 0.45 µm, 13 mm, USA). For total digestion, each soil

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sample (0.25 g) was mixed with 1 mL of HNO3 (65%, Merck, USA) and 3 mL of HCl (37 %, Merck, USA). The

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mixture was heated to 70°C, shaken for one hour, and then diluted with 6 mL of distilled water (1:20, soil:

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solution) (Ure and Alloway, 1995). The sample was then filtered through a membrane filter (Whatman 0.45 µm,

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13 mm, USA), and arsenic concentration was measured using inductively coupled plasma-mass spectrometry

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(ICP-MS; Agilent 7500, USA) and a hydride-generation atomic absorption spectrophotometer (HG-AAS, Perkin

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Elmer 5100, Waltham, USA) having an arsenic detection limit of 1 µg/L. All analytical measurements were

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conducted in duplicate.

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2.2.

Cultivation of arsenite-oxidizing bacteria decision

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For isolation of arsenic-resistant microorganisms, each groundwater sample (100 µL) and soil sample

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(1 g) was supplemented with 1 mM of sodium arsenite (NaAs(III)O2) (Sigma, USA) or 1 mM of sodium

ACCEPTED MANUSCRIPT arsenate (Na2HAs(V)O4) (Aldrich, USA) and then inoculated into Stainer’s basal medium (MSB) (Sambrook

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and Russel, 2012). Pure culture condition and genomic DNA isolation are described in the Supplementary Data.

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Sequences of primers used for PCR of 16S rDNA and arsenic-resistance system genes are presented in Table S1.

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Genomic DNA was isolated from pure-culture bacteria. The PCR amplification of the 16S rDNA gene was

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conducted using a universal primer set comprised of the following primers: BGRI-09 sense (5’-ATC ATG GCT

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CAG ATT GAA CGC -3’) and BGRI-1588 antisense (5’-T ACC TTG TTA CGA CTT CTA CCT-3’). Arsenic-

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resistant system genes and phylogenic analyses are described in the Supplementary Material. Batch tests were

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conducted independently in triplicate using 250 mL glass flasks that contained 60 mL of MSB medium, 1 mM

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sodium arsenite NaAsO2 (Sigma, USA), and 1 mM D (+)-glucose. For batch tests, drinking water-isolated

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bacteria (107/CFU) were inoculated aerobically at 28°C for 50 hours while shaking (180 rpm). Redox assays of

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arsenite-oxidizing bacteria are also described in the Supplementary Material. All analytical measurements were

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conducted in duplicate. Representative sequences reported in this study have been deposited in GenBank under

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the accession numbers DQ481464 to DQ481483 for PCR products of 16S rRNA genes.

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3. Results and discussion

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3.1.

Arsenic contamination in Vietnam

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(Fig. 1). High concentrations of arsenic were found in groundwater and surrounding soil obtained from Vinh Tru

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village in Ha Nam province (groundwater : 420.7 µg/L and 637.0 µg/L ; and soil: 280.7 mg/kg, 390.1 mg/kg,

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respectively; Table 1). The mobilization of arsenic from groundwater to the surface soil areas used as drinking

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water sources in Ha Nam province was likely induced by both biogeochemical activity and hydrological

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migration. High concentrations of arsenic are seen in the following locations: groundwater of Hoa Hau village,

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Ha Nam province > 884.0 µg/L (Kim et al., 2009; Pham et al., 2017), shallow-well water of Dong Thap 321.0

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µg/L (Shinkai et al., 2007), surface water of Phu Tan > 24.0 µg/L (Hanh et al., 2011), drinking water of Hanoi >

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150.0 µg/L (Nitzsche et al., 2015), rain water of Thanh Tri > 0.58 µg/L (Agusa et al., 2006), sediment exposure

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of Haiphong Harbor > 188.0 mg/kg (Ho et al., 2012), rice exposure of Phu Tan > 22.0 mg/kg (Hahn et al., 2011),

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and hair exposure of Phu Tan > 21.0 mg/kg (Hahn et al., 2011) (Fig. 1). Recent study revealed arsenic

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concentrations in groundwater ranged of 12.8-884 µg/L with mean values from Chuyen Ngoai and Chau Giang

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in Vietnam (Pham, et al., 2017). Nguyen, et al. (2009) reported 100% for groundwater and 42% for treated water

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but 57%-64% for groundwater for treatment water. Also, about 80% of groundwater in these areas revealed

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arsenic concentration higher than the World Health Organization drinking water limit of 10 µg/L.

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Arsenic may to also accumulated in the human body through the consumption of water and/or food in

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Vietnam. In particular, arsenic contamination has been deemed severe according to regional reports in Ha Nam

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(Hanh et al., 2011; Huy et al., 2014), Hanoi (Ahusa et al., 2014; Winkel et al., 2011), Long An (Hoang et al.,

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2010), Chau Ciang (Pham et al., 2017), Van Phuc (Al Lawati et al., 2012; Pham et al., 2017; Nitzsch et al., 2015;

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Eiche et al., 2017), Dong Anh (Al Lawati et al., 2012), the Mekong River Delta (Berg et al., 2007; Buschmann et

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al., 2008; Eiche et al., 2008; Hug et al., 2008; Buschmann and Berg, 2009; Nguyen and Itoi, 2009; Stuckey et al.,

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2016), the Red River (Postma et al., 2007; Phuong et al., 2008; Postma et al., 2010; Postma et al., 2012; Postma

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et al., 2016), and Ly Nhan (Agusa et al., 2014), and Nam Du (Norrmann et al., 2008) (Fig. 1). According to

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Nguyen, et al. (2009), local people had stopped using contaminated groundwater as drinking water after arsenic

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contamination was detected. Arsenic is present in the ecosystems, and a large number of people are exposed to

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ACCEPTED MANUSCRIPT its toxicity under an extreme geochemical environment. Exposure to high levels of arsenic in drinking water

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commonly occurs through various pathways; subsequently, arsenic becomes enriched and has reached chronic

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damage levels in hair, rice, sediment, rain water, surface water, tube well water, and ground water in Vietnam.

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Postma, et al. (2016) reported modeling groundwater arsenic content over a 6,000-year period in a 20-m thick

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aquifer revealed an increase in As during the first 1,200 years when it reached a maximum of about 600 µg/L,

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moreover after 6,000 years arsenic content decreased to 33 µg/L. The Vietnamese government must formulate

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positive solutions and policy practices; presence of arsenic in drinking water and the food cycle must be

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addressed. Various methods may be used to remove arsenic; detoxification by biological processing is one

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method of biological treatment. Failure to mitigate arsenic levels in the environment may cause a threat to

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public health from poisoning. Arsenic contamination is present in ecosystems, and many people are exposed

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daily to its toxicity and extreme geochemical environments.

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Environmental ars genotype of microbial arsenic transformations

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Rresence of environmental ars genotypes displaying microbial arsenic transformations due to bacteria

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from surface soil and groundwater was evaluated using PCR (Table 1). Table 1 shows the transcription repressor

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protein arsR (Rosenstein, et al., 1994), and arsD (Wu and Rosen, 1993), a second regulatory gene whose

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product functions as a “throttle” by setting an upper limit on operon function in Pseudomonas putida strain

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VTs34 (DQ481477), with arsB efflux/influx and aoxB arsenite oxidase activity coding together in surface

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arsenic-contaminated soil. When the arsD gene type was determined, Aquaspirillum sp. strain VTs22

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(DQ481472), Pantoea agglomerans VTs24 (D481474), Pseudomonas putida strain VTs32 (DQ481475), and

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Citrobacter freundii strain VTw32 (DQ481481), in the surface water environment were also investigated. As

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such, the arsR-arsD pair provides a sensitive mechanism for sensing environmental arsenic (Kruger, et al.,

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2013). ArsR and ArsD have also evolved binding sites for interaction with other ars genotypes, and leader genes

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that were not detected in activities of the oxidation and reduction genes have been investigated. Results

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highlighted the relationship between the leader gene and other ars genotype activities in arsenic oxidation and

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reduction in extreme environments. However, ars genotype activity, appearing with As (III) and/or As (V)

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phenotypes, determined using various methods, display leader gene activity under aerobic and anaerobic

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conditions (Oremland and Stolz, 2003).

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arsR and arsD were detected, as were arsenite oxidase genes aoxR, aoxA, aoxB and arsenate reduction genes

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arrB, aroA, aroB, and arsC. All were ubiquitous in surface soil samples obtained from areas with groundwater

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contaminated from arsenic in surface soil. An ArsR member is involved in regulation of arsenic via the arsenite

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oxidase operon in Thiomonas arsenitoxydans (Molnler, et al., 2014). Microorganisms present in groundwater or

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surface soil are involved in arsenic redox reactions in a natural state, and so may also be useful for arsenic

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detoxification, bioremediation, and metabolism (Sarkar and Paul, 2016; Kumari and Jagadevan, 2016). Arsenite

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oxidase is critical in the biogeochemical cycle of As species changes that highlights potential application of such

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geomicrobiology for the removal of arsenic from surface soil and groundwater. Arsenic resistance system (ars)

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of arsenite-oxidizing bacteria is critical, and this affects arsenic species changes and detoxification in an

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ecological role, because it allows arsenite-oxidase bacteria to control the biological cycle in groundwater

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ecosystems.

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3.3.

Arsenite oxidase responses of microbial arsH geochemistry

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Arsenite oxidase responses of microbial arsenic resistance system operons are encoded by arsB and

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aoxR in the Citrobacter freundii strain VTan4 (DQ481466) from surface soil, but Pseudomonas putida strain

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VTw33 (DQ481482) from groundwater had only arsH (420 bp) (Fig. 2.B). In Fig. 2A, arsH arsenite oxidase

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responses in bacterium Pseudomonas putida strain VTw33 (DQ481482) were resistant a relatively growing dead

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time under aerobic (3 mM; 8 days) and anaerobic (6 mM; 7 days) conditions. Doubling time of Pseudomonas

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putida strain VTw33 (DQ481482) under these experimental conditions was 20 h (phenotype As (III) and As (V),

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G+C content 53 mol%, γ-Proteobacteria, and physiological properties (Table S2) from arsenic-contaminated

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groundwater (420.7 µg/L) (Table 1).

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Batch test results revealed that Pseudomonas putida strain VTw33 (DQ481482) completely oxidized 1

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mM of arsenite to arsenate within 45-50 hours (Fig. 2.D), and arsenite oxidase responses of only arsH (Fig. 2.B).

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Rresults suggests studying arsenite-oxidizing bacteria may lead to a better understanding of the molecular

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geomicrobiology of biogeochemical cycle activity, that may then be applied to bioremediation of arsenic-

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contaminated groundwater via surface soil. Previously, Neyt, et al. (1997) reported that the chromosomal arsH

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gene was of unknown function, and Lopex-Maury, et al. (2003) demonstrated arsenic sensing in cyanobacterium

ACCEPTED MANUSCRIPT Synechocystis sp. that is likely required for arsenic resistance; no definitive role for ArsH has been reported to

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date. In other studies, a comparison of a soil-rhizosphere-plant system with arsH has been useful for uncultured

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Acinotobacter sp. strain MRI67(DQ539027) and uncultured Serratia sp. strain MRI-64 (DQ539023) from

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arsenite-oxidizing bacteria. ArsH operon may be present in some strains, as Thiomonas arsenitoxidans 3As; it

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may also be supplemented by other ars genes related to arsenic resistance, such as arsH (Paez-Espino, et al.,

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2009). However, other arsenic metabolism-like genes, including arsH were not expressed as there was no cDNA

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hit (Paez-Espino, et al., 2009). Arsenic methylation by arsH is significant, and the effect on As species changes

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and the mobility in groundwater.

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Phylogenetic analysis of the almost-complete 16S rRNA sequence of arsenic-resistant bacteria with

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bacterial ars, aro, arr, and aox genes revealed that they had members of the α-, β-, and γ-Proteobacteria and that

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they had an arsenic redox capacity (Fig. 3). Results suggest that the bacterial arsenite oxidase reaction could

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play a biogeochemical role in highly arsenic-contaminated groundwater in extreme environments investigated in

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this study. Overall, analysis of 1,447 unambiguous nucleotide positions of γ-Proteobacteria revealed that the

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arsH gene of Pseudomonas putida strain VTw33 (DQ481482), isolated from highly arsenic-contaminated

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groundwater obtained from Vihn Tru village, was most closely related to the P. putida group (sequence

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similarity of up to 98%). These groups have arsenite-oxidase ability, as follows; P. putida strain OS-5

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(AY952321) arsB gene, P. putida strain RW-28 (DQ112331) arsD, arsB gene, P. putida strain OW-16

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(DQ112328) arsC, arsH gene, and P. putida strain OS-19 (AY866406) arsB, arsH, arrA gene. The γ-

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Proteobacteria with sequence similarities with regard to arsenic oxidase capacity revealed a phylogenetic

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similarity of 75% to the reported arsenite-oxidizing bacterium NT-26 (AF159453) aroS, aroR, aroA, aroB genes

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of the ars system. However, the β-Proteobacteria group of arsenite-oxidizing bacteria has been reported (Muller,

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et al. 2006), though the product of arsH was more efficient than arsenite oxidase efficacy of arsenite-oxidizing

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bacterium NT-14 (AY027497) aoxB, Alcaligenes faecalis SRR-11 (EF446888) aoxR, and the Herminiimonas

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arsenicoxydans strain ULPAs1 (AY728038) aoxA, aoxB, aoxC, aoxD gene system.

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This study isolated unknown bacterium belonging to β-Proteobacteria that exhibited an arsH methylation

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arsenite oxidase response. However, this response was confirmed only in the α-, and γ-Proteobacteria arsH gene

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group, and is expected to affect the oxidation of arsenic by the products of the aox gene group. Microbial arsH

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in a geochemical environment in groundwater mechanisms having mine water may suggest, ecologically, that

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the bacterial arsenite-oxidase response transformations shown in this study play an important role in the

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biogeochemical cycle. Influence of arsenite oxidase on arsH enzymes of arsenite-oxidizing bacteria was

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investigated using groundwater contaminated by arsenic in the surface soil in Vietnam. Results may improve

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our understanding of molecular geomicrobiology, that can then be applied to self-control measures, such as

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environmental science and technology. ArsH protein sequence alignments of the arsH family from arsenite-oxidizing bacteria were conducted

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using various arsenic-resistant bacteria based on a BLAST search (National Center for Biotechnology

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Information, www.ncbi.nlm.nih.gov, and PDB; Protein Data Bank, http://www.rcsb.org/pdb.home/home.do)

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(Fig. S1). The figure shows the ArsH family bacteria that were most closely related to strains identified in this

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study were arsenite-oxidizing bacterium Citrobacter freundii strain VTw22 (DQ481478), Citrobacter freundii

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strain VTw32 (DQ481481), Pseudomonas putida strain VTw33 (DQ481482), Ochrobacterium tritici strain

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SCII24 (DQ490090), uncultured bacterium pMT3 (EF618730), Sinorhizobium meliloti (NP_385180) (Ye, et al.

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2007), Acidithiobacillus ferrooxidans (EF584662) (Wu, et al., 2010), Acidithiobacillus ferrooxidans DX5

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(CSU208059) (Wu, et al., 2010), Yersinia enterocolitica (WP_013749490), and Yersinia enterocolitica Tn2502

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(YEU58366) (Neyt, et al., 1997), that had up to a 42.12% sequence identity. According to prior research, ArsH

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from Shigella flexneri (Vorontsov, et al., 2007), Sinorhizobium meliloti (Ye, et al., 2007), and Synechocysits sp.

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strain PCC 6803 (Hervás, et al., 2012; Zhang, et al., 2014; Xue, et al., 2014) exhibited strong NADPH-

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dependent FMN reductase activity. It has also been proposed that the structure may provide insight into the role

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of ArsH in arsenic detoxification (Ye, et al., 2007). In the figure, triangles are used to indicate residues

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putatively involved in arsenic binding: Ser 43, Arg 45, Ser 48, and Tyr 49 in arsenic binding (Wu, et al., 2010;

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Ye, et al., 2007). The arsH mutants sensitive to oxidation by arsenite may be involved in coordinating oxidation

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generated by arsenic (Hervás, et al., 2012; Xue, et al., 2014). In addition, Pseudomonas putida strain VTw33

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(DQ481482) ArsH plays a role that may also be involved in arsenite oxidase response, and is involved in

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coordinating arsenic species changes that influence mobilization in natural environments with high arsenic

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contamination in groundwater contaminated through arsenic in surface soil, that is interesting in the context of

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this study.

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Presence of microbial arsH in a geochemical environment in groundwater ecosystems with mine

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water may suggest that bacterial arsenite-oxidase response transformations revealed in this study play an

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significant role in the biogeochemical cycle. This finding may also improve our understanding of molecular

303

geomicrobiology, that may be applied to self-control measures such as environmental microbiology research.

304

We revealed here ArsH gene activity, with arsenite oxidase in the periplasm; elucidating arsenic oxidation

305

potential of the arsH system. We identified the possible mechanism of arsenate oxidase ArsH reaction microbial

ACCEPTED MANUSCRIPT response, and provide a graphical abstract overview of membrane-associated uptake and efflux/influx genes

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associated with arsenite oxidase activity in the biogeochemical cycling of extremely contaminated groundwater.

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Arsenic is given more emphasis here, due to its involvement of enzymes in the periplasm and cytoplasm as well

309

as in ArsB/YqcL transport and the aoxS efflux/influx pump. The arsB efflux transport systems extend from the

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cytoplasm across the outer membrane of gram-negative bacteria ; however, how substrates affect the

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efflux/influx system, that are not associated with outer membrane protein function, is unclear.

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Based on this study, further discussion are as follows. This study demonstrated for the first time natural

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state arsH genes could be involved in arsenic biogeochemical cycling through detoxification and changes in

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arsenic in the groundwater ecosystem. The arsB efflux transport systems extend from the cytoplasm across the

315

outer membrane of gram-negative bacteria. Natural state arsH genes may be involved in arsenic biogeochemical

316

cycling as indicated by the potential arsenic detoxification process and change in arsenic species in the

317

groundwater ecosystem. Therefore, arsenite oxidase is significant in the biogeochemical cycle of arsenic species,

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that highlights potential application of such geomicrobiology for the removal of arsenic from surface soil and

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groundwater. Isolated arsenic bacteria completely oxidize 1mM of arsenite to arsenate within 30-50 h, indicating

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arsenite oxidizing bacteria facilitates lesser arsenic release, thereby highlighting the possible scope for

321

controlling soil and groundwater contamination and/or developing a bioremediation system. We further believe

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that research into the extreme environment of highly contaminated groundwater caused by surface soil

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communities may facilitate development of arsenite-oxidase response biotransformation processes using

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arsenite-oxidizing bacteria and arsH gene system activity under various natural conditions.

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4. Conclusions

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High arsenic exposure occurs through various pathways from drinking water and becomes enriched

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and reaches chronic damage levels in hair, rice, sediment, and contaminated rain, surface, tube well, and ground

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water in Vietnam. In this study, we revealed ArsH gene activity, with arsenite oxidase in the periplasm; it

331

revealed arsenic oxidation potential of the arsH system. However, this was only confirmed in the α-, and γ-

332

Proteobacteria arsH gene group, and can be expected to play a critical role in impacting the aox gene group

333

regarding oxidation of arsenic. Possible mechanisms of microbial arsH response of arsenite oxidase was

334

characterized for Pseudomonas putida strain VTw33. arsH gene in bacteria was first report from Vietnam for

335

resistance and arsenite oxidase. This suggests studying arsenite-oxidizing bacteria may lead to better

ACCEPTED MANUSCRIPT understanding of molecular geomicrobiology of biogeochemical cycle activity, that may be applied to

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bioremediation of arsenic-contaminated groundwater via surface soil. We believe development of research into

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extreme environments of highly arsenic-contaminated groundwater and surface soil communities may provide

339

the key to arsenite-oxidase response transformations processes with arsenite-oxidizing bacteria and arsH gene

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system activity in various natural conditions. Natural state arsH genes could be involved in arsenic

341

biogeochemical cycling with highlight of potential arsenic detoxification process and species change in the

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groundwater ecosystem.

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Acknowledgments

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This study was partly supported by the International Environmental Research Center (IERC) at the Gwangju

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Institute of Science and Technology (GIST) and by the Start-Up growth program (S2411470, Development of

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microbe agent for purification of arsenic contaminated water) from the Small and Medium Business

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Administration (SMBA, Korea) in Republic of Korea.

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Supplemental data related to this article can be found at http://

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Supplemental Material

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List of Figure Captions and Table Legends

504 Fig. 1. Geographical representation of the study sites, showing the sampling stations at Vinh Tru, Bo De, Hoa

506

Hau, and Nhan Dao in Vietnam. The arsenic concentrations in water-source areas of drinking, surface, tube well,

507

and ground water, and the levels in rice, nail, hair, and soil are shown from both previous reports and this study.

508

(Note: based on 1: Nguyen and Itoi 2009; 2: Kim et al. 2009; 3: Huy et al. 2014; 4: Hanh et al. 2011; 5: Nitzsche

509

et al. 2015; 6: Agusa et al. 2006; 7: Winkel et al. 2011; 8: Agusa et al., 2006; 9: Shinkai et al. 2007; 10: Hoang et

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al. 2010; 11: Shinkai et al. 2007; 12: Hanh et al. 2011; 13: Ho et al. 2012; 14: Winkel et al. 2011; 15: Al Lawati et al.

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2012; 16: Buschmann and Berg 2009; 17: Eiche et al. 2011; 18: Buschmann et al. 2008; 19: Hug et al. 2008; 20:

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Nguyen et al. 2009; 21: Berg et al. 2001; 22: Postma et al. 2010; 23: Postma et al. 2007; 24: Berg et al. 2007; 25:

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Phuong et al. 2008; 26: Agusa et al. 2014; 27: Berg et al. 2007; 28: Postma et al. 2016; 29: Pham et al. 2017; 30:

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Nitzsche et al. 2015; 31: Phung et al. 2017; 32: Eiche et al. 2017; 33: Stuckey et al. 2016; 34).

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Fig. 2. Ethidium-bromide-stained agarose gels of PCR-amplified ars genes from Citrobacter freundii strain

517

VTan4 (DQ481466): (A) Pseudomonas putida strain VTw33 (DQ481482), (B) Line: M, Lambda φX174

518

DNA/HaeIII size markers (Promega, USA). The time-course variation in culture turbidity (OD600) and

519

arsenite/arsenate concentrations in cultures of Citrobacter freundii strain VTan4 (DQ481466), and (C)

520

Pseudomonas putida strain VTw33 (DQ481482). (D) In MSB medium containing arsenite (1 mM). The

521

experiment was performed independently in triplicate in batch mode using a working volume of 60 mL at 28 °C.

522

Each data point represents the average of the readings for each experiment (■ concentration of arsenite;

523

concentration of arsenate; ● concentration of arsenite without bacterial inoculation; ∆ concentration of arsenate

524

without bacteriall inoculation; + culture turbidity (OD600)).

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Fig. 3. Phylogenetic tree based on the 16S rRNA sequences of arsenic-resistance systems showing the position

527

of the arsenite-oxidase isolate, Citrobacter freundii strain VTan4 (DQ481466), and Pseudomonas putida strain

528

VTw33 (DQ481482). The arsR, arsD, arsA, arsB, arsC, arsH, arrA, arrB, aroA, aroB, aoxS, aoxR, aoxA,

529

aoxB, aoxC, and aoxD genes of the arsenic-redox mechanisms in bacteria of the same genera and species and

530

sequences in this study are shown in bold type. The tree was constructed from a matrix of pair-wise genetic

531

distances using the neighbor-joining method. The phylogenetic data were obtained by aligning the various

532

arsenic-resistant bacterial sequences using standard parameters. The scale bar represents 0.05 substitutions per

533

100 nucleotides within the 16S rRNA sequence.

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Table 1. Identification, arsenic-resistant system genotype, phenotype, doubling time, and survival of bacterial strains during chronic exposure to sodium arsenite under aerobic and anaerobic conditions. Using PCR, each strain was analyzed for the presence within their genomic DNA of the arsR, arsD, arsA, arsB, arsC, arsH, arrA, arrB, aroA, aroB, aoxS, aoxR, aoxA, aoxB, aoxC, and aoxD genes, as described in the Experimental section. In Ha Nam province (Bo De villages, Hoa Hau villages, Nhan Dao villages, Vinh Dao villages), arsenic was present in the ground water and soil samples; the concentrations of arsenic species were determined.

540 541 542 543 544

a b c e h

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Dead time: mM; day

ars genotype d

Phenotype c

(aerobic/anaerobic)b

pH / Eh(mV)

Groundwater: As(III)/As(total) (µg/L)

arsB+ arsH+

DQ279764

95/Escherichia coli

24

DQ481464 DQ481465 DQ481472 DQ481478

97/Averyella dalhousiensis 98/Citrobacter freundii 97/Aquaspirillum sp. 99/Citrobacter freundii

28 24 40 24

DQ481466 DQ481467 DQ481473 DQ481474 DQ481479 DQ481480

98/Citrobacter freundii 97/Serratia grimesii 98/Pseudomonas putida 99/Pantoea agglomerans 98/Citrobacter freundii 98/Enterobacter sp.

20 24 24 24 28 20

DQ481468 DQ481469 DQ481475 DQ481481

99/Buttiauxella agrestis 99/Citrobacter freundii 99/ Pseudomonas putida 99/Citrobacter freundii

20 28 24 28

DQ481470 DQ481471 DQ481476 DQ481477 DQ481482 DQ481483

99/Citrobacter freundii 98/Enterobacter amigenus 98/Enterobacter sp. 99/Pseudomonas putida 99/Pseudomonas putida 99/Pantoea agglomerans

1;4 0;4 1;3 0; 10 5; 4 2;10

As(III) r,As(V) r

aoxS+ aoxR+

1; 4 1; 10 1; 4 8; 4

As(V) r As(III) r, As(V) r As(III) r, As(V) r As(III) r, As(V) r

aoxR+ arrB+ arsD+ arsB+ aoxR + arsH+ aoxR+

7.1 7.1 7.1 6.5

60.9 88.4 72.9 92.3

6; 14 1; 0 1; 2 8; 7 5; 10

As(III) r, As(V) r As(V) r As(III) r As(III) r, As(V) r As(V) r As(V) r

arsB+ aoxR+ arsH+ arsB+ aoxB+ arsD+ arsB+ arsC+ arsR+ aroA+ aoxB+ arsH+ aoxB+ arsH+

7.1 6.9 6.9 7.0 6.8 6.8

50.2 71.2 98.6 91.3 82.5 117.8

0;4 1;1 8;14 0;4

7; 10 4; 10 0; 5 2; 10

As(V) r As(III) r, As(V) r As(III) r As(III) r, As(V) r

arsR+ arsH+ aroA+ arsC+ arsH+ arsD+ arsB+ arsD+ arsH + aoxR+

7.0 7.0 7.1 6.9

151.1 94.5 128.4 119.2

24 24 18 20 20 24

0;4

2; 10 8; 10 1; 14 8; 5 6; 7 1; 1

As(III) r, As(V) r As(III) r As(III) r, As(V) r As(III) r As(III) r, As(V) r As(V) r

aroA+ arrB+ aoxA+ nd arsB+ arsC+ arsR+ arsD+ arsB+ aoxB+ arsH+ aroB+ arsH

7.0 6.9 7.0 6.9 7.0 7.1

168.9 82.1 83.7 59.5 103.0 81.0

6;14 8;7 3;8

40.7

11.1 nd

nd

11.7 12.7

d

I

Isolate site: from groundwater.

nd: not determined.

f

g

nd nd 2.5 nd

44.0 24.2 230.0 110.7

nd 0.7 1.7

184.0 110.4 23.7

9.5 1.9 30.2 nd

214.5 280.7 390.1 140.6

201.2

420.7 637.2

ars resistance relationship analyzed for the ars, arr, aro,aox genotype: +, positive PCR product generated..

Control strain: Korean Collection for Type Cultures (KCTC338, KCTC 1636); American Type Culture Collection (ATCC 3178, ATCC 15522).

232.2 200.2 55.7

388.0 178.0

Arsenic concentration mM test: Aerobic tests - Arsenic conc.; survival (days) / Anaerobic tests - Arsenic conc.; survival (days). As(III)r, resistance to 1g of sodium arsenite per liter; As(V)r, resistance to 1g of sodium arsenate per liter.

67.9 60.2 10.1 120.9

GenBank number at NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ ).

Isolate site: from surrounding soil (1m).

Surface soil: As(III)/As(total) (mg/kg)

arsR+ arsD+ arsC+ arsB+ arsH+

KCTC338;ATCC3178 KCTC1636;ATCC15522

SC

Bo De VTan2 h VTan3 h VTs22 g VTw22 I Hoa Hau VTan4 h VTan6 h VTs23 g VTs24 g VTw23 I VTw24 I Nhan Dao VTan7 h VTan8 h VTs32 g VTw32 I Vinh Tru VTan9 h VTan10 h VTs33 g VTs34 g VTw33 I I VTw34

Doubling time (h)

M AN U

RW-29

f

16S rDNA Similarly (%) to known bacteria

TE D

E. coli DH5 α Alcaligenes sp.e Saccharomyces castellii e

Isolate accession no.a

EP

Strain or isolate (site)

AC C

536 537 538 539

Control strain Isolate site: from surrounding soil. The third replicate analyses were performed for each sample; average.

547

Fig. 1

EP

546

AC C

545

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

550 551

Fig. 2

AC C

549

EP

TE D

M AN U

SC

RI PT

548

ACCEPTED MANUSCRIPT 552 553

AC C

EP

TE D

M AN U

SC

RI PT

554

555 556 557 558 559 560

Fig. 3

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

561

ACCEPTED MANUSCRIPT

Highlights


Natural state arsH genes could be involved in arsenic biogeochemical cycling,

RI PT

highlighting the potential for arsenic bioremediation.
arsH gene in bacteria was firstly report from Vietnam form resistance and arsenite oxidase.

SC


ars genotype displaying microbial arsenic transformation in bacteria is evaluated from arsenic contaminated soil and groundwater in Vietnam.

M AN U


Pseudomonas putida strain VTw33 completely oxidized 1 mM of As (III) to As (V)

AC C

EP

TE D

within 30 h.