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Ecotoxicology and Human Environmental Health
Protective Role of Freshwater and Marine Rotifer Glutathione S-Transferase Sigma and Omega Isoforms Transformed into Heavy Metal-Exposed Escherichia coli Jin-Sol Lee, Hye-Min Kang, Chang-Bum Jeong, Jeonghoon Han, Heum Gi Park, and Jae-Seong Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01460 • Publication Date (Web): 05 Jun 2019 Downloaded from http://pubs.acs.org on June 9, 2019
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Environmental Science & Technology
Hg
Cd Cu Zn
Oxidative stress
GST
IPTG
GST
GST overexpression
pET-28a(+) vector
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-revised
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Protective Role of Freshwater and Marine Rotifer Glutathione S-
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Transferase Sigma and Omega Isoforms Transformed into Heavy
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Metal-Exposed Escherichia coli
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Jin-Sol Leea,#, Hye-Min Kanga,#, Chang-Bum Jeonga, Jeonghoon Hana,
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Heum Gi Parkb, and Jae-Seong Leea,*
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a Department
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b
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Gangneung, South Korea
of Biological Science, Sungkyunkwan University, Suwon, South Korea
Department of Marine Resource Development, Gangneung-Wonju National University,
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ABSTRACT: Glutathione S-transferases (GSTs) play an important role in phase II of
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detoxification to protect cells in response to oxidative stress generated by exogenous
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toxicants. Despite their important role in defense, studies on invertebrate GSTs have mainly
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focused on identification and characterization. Here, we isolated omega and sigma classes of
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GSTs from the freshwater rotifer Brachionus calyciflorus and the marine rotifer Brachionus
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koreanus and explored their antioxidant function in response to metal-induced oxidative
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stress. The recombinant Bc- and Bk-GSTs were successfully transformed and expressed in
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Escherichia coli. Their antioxidant potential was characterized by measuring kinetic
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properties and enzymatic activity in response to pH, temperature, and chemical inhibitor. In
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addition, a disk diffusion assay, reactive oxygen species assay, and morphological analysis
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revealed that GST transformed into E. coli significantly protected cells from oxidative stress
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induced by H2O2 and metals (Hg, Cd, Cu, and Zn). Stronger antioxidant activity was
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exhibited by GST-S compared to GST-O in both rotifers, suggesting that GST-S plays a
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prominent function as an antioxidant defense mechanism in Brachionus spp. Overall, our
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study clearly showed the antioxidant role of Bk- and Bc-GSTs in E. coli and provides a
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greater understanding of GST class-specific and interspecific detoxification in rotifer
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Brachionus spp.
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ABSTRACT ART
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■ INTRODUCTION
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Oxidative stress is generated through an imbalance in redox homeostasis and is known to
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cause oxidative damage to biomolecules.1,2 Various biological defense mechanisms occur in
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organisms to maintain redox homeostasis. Among these mechanisms, the glutathione-
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mediated antioxidant system plays a critical role in detoxification in response to oxidative
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stress by converting reactive oxygen species (ROS) into water and oxygen via a multiple step
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process.3,4
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Glutathione S-transferases (GSTs) are responsible for maintaining redox homeostasis in
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the phase II detoxification phase by catalyzing the conjugation of reduced glutathione (GSH)
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and/or reducing peroxides with electrophilic compounds and by binding toxin to make them
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more hydrophilic, which assists excretion in the detoxification process.5,6 Although GSTs
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have been widely identified across animal taxa,7,8 they seem to have evolved in a lineage-
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specific way depending on the phylogenetic placement of the organism. For instance, while
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only 16 GST genes were identified in the human genome, massive gene duplication of GST
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was observed in invertebrate genomes.9-13
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The antioxidant role of GST has been reported in several studies.15,16 For example, GST
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was up-regulated in response to a variety of metals (CdCl2, CuCl2, HgCl2, and PbCl2) in the
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bacterium Proteus mirabilis, and metal resistance was enhanced when GST was
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overexpressed.14 In the Cd-exposed polychaete Perinereis nuntia, expression level of seven
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GSTs and the total GST activity were increased.15 However, despite the critical role of GSTs
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in detoxification and lineage-specific evolution in invertebrates, only a few studies have
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characterized the function of invertebrate GST isoforms. Therefore, the aim of this study was
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to characterize the defensive function of sigma and omega GST isoforms in two rotifers (B.
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calyciflorus and B. koreanus) of the Brachionus genus in response to oxidative stress
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generated by metals (Hg, Cd, Cu, and Zn).
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Metals are one of the dominant pollutants in the environment, causing adverse effects on
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aquatic organisms. Especially, the generation of oxidative stress is one of the major
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mechanisms of toxicity from metals and has been widely reported in aquatic invertebrates.
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For example, in the copepod Tigriopus japonicus, numerous metals (e.g., As, Cd, Cu, Ag,
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and Zn) increased the levels of ROS along with the transcriptional level of heat shock
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proteins.16 In the rotifer B. koreanus, the activity of GST and its expression level of GST
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isoforms were significantly up-regulated upon increased levels of ROS in response to Cd.17
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Strong evidence indicates that oxidative stress is induced by metals; therefore, we used Cd,
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Cu, Hg, and Zn as metal toxicants to induce oxidative stress. 3 ACS Paragon Plus Environment
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Species of the rotifer Brachionus are widely distributed and found in both freshwater (e.g.,
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B. calyciflorus) and seawater (e.g., B. koreanus). They occupy an important ecological niche
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as a primary consumer with many beneficial characteristics for laboratory studies such as
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ease of maintenance, small size, high fecundity, and a short life cycle (~1 day).18,19 Moreover,
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recent studies have facilitated the use of rotifer as an experimental species for in-depth
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molecular ecotoxicological studies,20,21 and whole genome databases have been recently
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established.22,23
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In this study, to obtain a better understanding of the function of GSTs in response to
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metal-induced oxidative stress, GST-sigma (GST-S) and omega (GST-O) were first isolated
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from B. calyciflorus and B. koreanus. They were then characterized by measuring several
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biochemical properties after overexpression in E. coli and in response to four different metals
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(Hg, Cd, Cu, and Zn). Our results indicate better insight into the detoxification processes of
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aquatic organisms and a deeper understanding of interspecific tolerance in Brachionus spp.
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■ MATERIALS AND METHODS
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Rotifer Culture. The rotifers B. calyciflorus and B. koreanus were collected in
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Zwartenhoek in the Netherlands and Uljin in South Korea, respectively. B. calyciflorus was
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cultured in freshwater with Chlorella sp. (approximately 6 × 106 cells/mL), and B. koreanus
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was cultured in 15 practical salinity units (psu) artificial seawater (ASW) with Tetraselmis
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suecica (approximately 6 × 104 cells/ml) as a food supply. All cultures were maintained at
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25°C with a 12:12 h photoperiod (light:dark). Species was identified by morphological
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analysis and mitochondrial cytochrome oxidase 1 gene sequencing.24-26
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Chemicals and Reagents. H2O2, metals (HgCl2 [purity ≥99.5%], CdCl2·2.5H2O [purity
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79.5-81.0%], ZnCl2 [purity ≥98.0%], CuSO4·5H2O [purity 99.999%]), N-ethylmaleimide
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(NEM), Cibacron blue (CB), hematin, and isopropyl-β-D-thiogalactoside (IPTG) were
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obtained from Sigma-Aldrich. Restriction enzymes EcoRI and SalI were purchased from
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TaKaRa Shuzo Co.
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Acute Toxicity Testing. Ten adult rotifers were incubated in 4 mL ASW containing
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different concentrations of metals (Hg: 0 - 0.5 [1.8 nM] and 10 µg/L [36.8 nM], Cd: 0 - 0.1
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[438 nM] and 200 [876 µM], Zn: 0 - 1 [7.34 µM] and 100 [734 µM], and Cu: 0 - 0.1 [0.4
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µM] and 2.5[10 µM] mg/L for B. calyciflorus and B. koreanus, respectively) for 24 h in 12-
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well culture plates (SPL Life Sciences) under laboratory conditions without food. Rotifers 4 ACS Paragon Plus Environment
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showing no sign of movement were regarded as dead under a stereomicroscope (SZX-
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ILLK200, Olympus Co.) All experiments were performed in triplicate and the values of no
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observed effect concentration (NOEC) and median lethal concentration (LC50) were
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calculated using Probit analysis (ToxRat Ver.2.09).
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Recombinant Proteins. GSTs were amplified as described in previous studies with
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forward/reverse primers designed from open-reading frames (ORFs) (Table S1).27 After
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elution, the products were double-digested by EcoRI and SalI and inserted into pET-28a
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vector with a 6× His-tag. To express the recombinant Bk-GSTs and Bc-GSTs proteins, the
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recombinant vectors were transformed into E.coli BL21 (DE3) and incubated at 37°C in
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Luria-Bertani (LB) medium containing kanamycin (50 µg/ml). When cultures reached a 0.5
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optical density at 600 nm (OD600), 0.5 mM IPTG was added, and the cultures were incubated
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for 36 h at 37°C to induce the recombinant proteins. After incubation, the cells were
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centrifuged at 2,500 g at 4°C for 10 min, re-suspended in cold buffer (300 mM NaCl, 50 mM
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sodium phosphate, 5 mM imidazole, pH 8.0) containing a protease cocktail inhibitor (Roche),
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and subsequently sonicated three times for 10 min with a sonicator (Vibra-Cell) at a setting of
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30%. The resultant cell lysate was centrifuged at 10,000 g for 10 min at 4°C. His-tag affinity
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columns (5 ml Ni+-NTA resin; Bio-Rad) were used for purifying soluble recombinant
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proteins, and the eluted proteins were determined by 12.5% SDS-PAGE and Western
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blotting. The concentration of protein was measured by the Bradford method.28
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Enzyme Kinetics. To investigate kinetic properties of GSTs, GST activity was measured
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in response to different substrates (1-chloro-2,4-dinitrobenzene, CDNB [0.5 to 4 mM];
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glutathione, GSH [0.25 to 2 mM]) and under different pH and temperature by following the
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protocol of previous study.29 The data were plotted on a Lineweaver-Burk plot to determine
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Km and Vmax values. The enzyme inhibition assay was performed using three different
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inhibitors; CB (0.01-10 µM), hematin porcine (0.01-10 µM), and N-ethylmaleimide (NEM)
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(1-10,000 µM) according to a method by Tahir et al.30 The enzyme was pre-incubated in
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conditions described above for 30 min, and GST activity was detected. The residual enzyme
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activity was relative to that of a control.
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Reactive Oxygen Species Measurement. E. coli expressing Bc-GSTs and Bk-GSTs were
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harvested by centrifugation at 2,500 g for 10 min then washed three times with PBS. After
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washing, cells were re-suspended in PBS, and the cell number was counted using the colony
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forming unit (CFU/ml) method. The cells (2×107 CFU/mL) were mixed with 10 µM 2′,7′5 ACS Paragon Plus Environment
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dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) with a minimal
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inhibitory concentration (MIC) of metals (HgCl2, CdCl2·2.5H2O, ZnCl2, and CuSO4·5H2O)
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and then incubated for one hour in a shaking incubator at 100 rpm in the dark at room
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temperature. The intensity of fluorescence was measured by spectrophotometer (485 nm for
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excitation; 520 nm for emission) and the level of intracellular ROS was calculated relative to
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the control.
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Disk Diffusion Assay. To compare the antioxidant activities of GST-recombinant vector-
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transformed E. coli and the control vector-transformed E. coli in response to H2O2 and metals
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(HgCl2, CdCl2·2.5H2O, ZnCl2, and CuSO4·5H2O), a disk diffusion assay was performed.
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Briefly, the transformed E. coli were incubated overnight at 37°C in LB broth with
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kanamycin (50 µg/mL) and then diluted 1/100 in LB broth. When the cell density reached 0.5
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at OD600, 0.1 mM IPTG was added and incubated for 4 h at 37°C then spread on LB agar
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plates. Sterilized Whatman filter-paper disks (6 mm diameter) were placed on the plates and
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treated with different concentration of H2O2 (0 to 50 µM) and metals (0 to 296 mM). The
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treated plates were incubated overnight at 37°C, and the clear zone diameter was measured to
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assess antioxidant activity of the transformed E. coli.
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Measurement of Minimum Inhibitory Concentration in Response to Heavy Metals.
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Recombinant E. coil cells expressing either Bk-GSTs or Bc-GSTs were incubated in LB
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medium until the cell density reached 0.4 at OD600. Cells (5 μL) were then incubated at 37°C
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with 200 μL LB medium containing various concentrations of metals (HgCl2, CdCl2·2.5H2O,
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ZnCl2, and CuSO4·5H2O). After incubation, the cells were transferred into 96-well plates, and
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turbidity was measured using a spectrophotometer.
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Transmission Electron Micrography in Response to Hg. Recombinant E. coil cells
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expressing either Bk-GSTs or Bc-GSTs were exposed to HgCl2 for 24 h and cells were
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sampled according to Kang et al.21 to observe morphology of cells using transmission
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electron micrography (TEM) at 120 kV (Tecnai G2 Spirit Twin).
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Bio-concentration factor, Phylogenetic analysis, Expression of Glutathione S-
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Transferase Genes in Response to Copper, and Statistical Analysis. A detailed
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description of all materials and methods is incorporated in the Supporting Information.
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■ RESULTS 6 ACS Paragon Plus Environment
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Acute Toxicity Testing. In B. calyciflorus, the 24 h LC50 values of metals (Hg, Cd, Zn,
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and Cu) were determined as presented in Table S2. The order of toxicity was Hg, Cd, Cu,
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and Zn for B. calyciflorus and Hg, Cu, Cd, and Zn for B. koreanus. The highest toxicity was
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induced by Hg in both rotifers, while B. koreanus was more resistant to all metal exposures
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compared to B. calyciflorus. All the exposure concentrations in the following experiments
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were determined from acute toxicity values. In addition, the bioaccumulation of metals in B.
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calyciflorus and B. koreanus was shown in Table S3. BCF factors for B. calyciflorus were
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higher than B. koreanus, supporting our toxicity data.
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Phylogenetic Analysis of Bk-GSTs and Bc-GSTs. Fifteen GST-sigma and two GST-
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omega genes from B. calyciflorus and 15 GST-sigma and two GST-omega genes from B.
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koreanus were identified in the genomes of the two rotifers. Sigma- and omega-GSTs in B.
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koreanus (Bk-GST-S, GenBank no. MH189372; Bk-GST-O, GenBank no. MH189370) and
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B. calyciflorus (Bc-GST-S, GenBank no. MH189308; Bc-GST-O, GenBank no. MH189305)
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had a clear orthologous relationship (Figure 1a). The ORF length of sigma class in B.
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calyciflorus and B. koreanus was 624 bp (207 aa), and that of the omega class was 705 bp
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(234 aa). Amino acid sequence similarity between classes was 72.38% for the sigma class
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and 74.36% for the omega class (SI Figure S1a).
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Expression of GST genes in Response to Copper. Transcriptional levels of Bc-GST and
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Bk-GST genes were analyzed to investigate the transcriptional response of GSTs upon Cu
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exposure. Overall, different expression patterns were observed in B. calyciflorus and B.
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koreanus. In B. koreanus, several GST genes, including GST-S4 were significantly up-
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regulated (P
