NmtA, a novel metallothionein of Anabaena sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress

Accepted Manuscript Title: NmtA, a novel metallothionein of Anabaena sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress Authors: Divya T.V., Pallavi Chandwadkar, Celin Acharya PII: DOI: Reference:

S0166-445X(18)30025-0 https://doi.org/10.1016/j.aquatox.2018.03.035 AQTOX 4907

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Aquatic Toxicology

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17-1-2018 12-3-2018 29-3-2018

Please cite this article as: T.V., Divya, Chandwadkar, Pallavi, Acharya, Celin, NmtA, a novel metallothionein of Anabaena sp.strain PCC 7120 imparts protection against cadmium stress but not oxidative stress.Aquatic Toxicology https://doi.org/10.1016/j.aquatox.2018.03.035 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.

NmtA, a novel metallothionein of Anabaena sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress

Divya T V1, Pallavi Chandwadkar1 and Celin Acharya1*, 2 Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400085,

India

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Homi Bhabha National Institute, Anushakti Nagar, Mumbai, 400094, India

Author for correspondence

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*

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Mailing address: Molecular Biology Division,

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Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.

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Phone: + (91) 22 25592256, E-mail: [email protected]

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Fax: + (91) 22 25505326

Highlights

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Metallated Anabaena metallothionein (NmtA) showed insensitivity towards proteolysis.

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nmtA expression was induced in the presence of metals but not under oxidative stress.

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A pronounced role of NmtA in protecting Anabaena against cadmium toxicity was observed.

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Abstract Metallothioneins (MTs) are low molecular weight, sulfhydryl-containing, cysteine-rich, metalbinding proteins. Eukaryotes have multiple metallothionein genes; however, there is dearth of

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reports on prokaryotic metallothioneins. Bacterial MTs with SmtA from Synechococcus PCC 7942 as prototype have been studied in the context of cadmium detoxification. In this study, a

smtA related ORF, namely nmtA, was identified in the heterocystous, nitrogen-fixing

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cyanobacterium, Anabaena PCC 7120. A recombinant N-terminal histidine-tagged Anabaena NmtA protein was overexpressed in Escherichia coli and purified. The protein was identified

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by peptide mass fingerprinting using MALDI-TOF Mass Spectrometry as putative

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metallothionein of Anabaena PCC 7120 with a calculated mass of ~6.1 kDa. While the native

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metallated NmtA exhibited resistance against proteolysis, metal free apo-NmtA resulting from

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acid and dithiothreitol (DTT) treatment could be digested by proteinase K revealing a metal dependent proteolytic protection of NmtA. Expression of nmtA in Anabaena PCC 7120 was

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induced evidently by cadmium, zinc and copper but not by uranium or hydrogen peroxide. Recombinant Anabaena PCC 7120 overexpressing NmtA protein revealed superior cadmium

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tolerance but showed limited influence against oxidative stress tolerance as compared with the

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strain carrying vector alone. In contrast, a mutant of Synechococcus PCC 7942 deficient in MT locus was found to be highly susceptible to H2O2 indicating a likely involvement of cyanobacterial MT in protection against oxidative damage. Overall, the study improved our

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understanding of metal tolerance mechanisms in Anabaena PCC 7120 by demonstrating a key role of NmtA in cadmium tolerance.

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Keywords: metallothionein, Anabaena, cadmium, tolerance

1. Introduction Cyanobacteria represent a morphologically diverse group of oxygenic, gram-negative

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photosynthetic prokaryotes which are thought to have contributed for the initial oxygenation of earth’s atmosphere (Schopf, 1975). They are widely distributed in fresh water, marine and

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terrestrial environments and are reportedly abundant in metal-contaminated environments

(Kanamaru et al., 1994). These organisms are confronted with an “assortment’ of transition metal ions in the aquatic environments. While they require these metals to perform oxygenic

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photosynthesis and nitrogen fixation, the same metal cations may cause oxidative stress, as

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shown for iron and copper (Baptista and Vasconcelos, 2006). The toxicity of the transition

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metals is deleterious but so is the lack of these metals in the cyanobacteria. In all cases, the

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cyanobacterial cells need to tightly regulate the intracellular metal ion concentrations below

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toxic levels which eventually has led to the development of efficacious metal homeostasis systems in them. Various strategies adopted by them for alleviation of metal toxicity include

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sequestration of the metal on the anionic cell surface and its associated components, extracellular or intracellular precipitation of metal, reduction of metal to its less toxic oxidation

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state, active transport of metal by metal efflux pumps and/or accumulation of metal inside the cytosol by sequestration with polyphosphate bodies or metal binding proteins like metallothioneins (Acharya et al. 2009; Nies, 2003; Cavet et al., 2003; Baptista and

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Vasconcelos, 2006; Acharya and Apte, 2013). Metallothioneins (MT) are low molecular weight, cysteine-rich stress response

proteins which bind metal ions in metal-thiolate clusters and their synthesis is induced in response to elevated concentrations of various metals including zinc, cadmium and copper. A lack of a rigid structure allows MTs to coordinate several metals including essential metals like 3

zinc and copper (Gui et al., 1996), toxic metals like cadmium (Piccinni et al., 1994) and mercury (Leiva-Presa et al., 2004), and radionuclides like uranium (Acharya and Blindauer, 2016) and technetium (Lecina et al., 2015). They are found largely in eukaryotes but prokaryotic metallothioneins appear to be far from ubiquitous, with the majority documented in cyanobacteria and pseudomonads (Blindauer, 2011). Bacterial metallothioneins were first

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identified in the marine cyanobacterium Synechococcus sp. (strain RRIMP N1) (Olafson et al.,

1979), a fresh water cyanobacterium Synechococcus TX-20 (Olafson et al., 1988) and a

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gammaproteobacterium, Pseudomonas putida (Higham et al., 1984) wherein all these organisms were grown in presence of elevated cadmium concentrations. Majority of the

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experimentally confirmed bacterial metallothioneins belong to the BmtA family, with SmtA

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isolated from the freshwater cyanobacterium Synechococcus PCC 7942 as the prototype

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(Blindauer and Leszczyszyn, 2010). SmtA is the first prokaryotic MT to be fully characterized

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(Olafson et al., 1986; Shi et al., 1992). A three-dimensional solution NMR structure of the recombinantly expressed SmtA revealed a single Zn4Cys9His2 cluster (Blindauer et al., 2001).

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SmtA-like ORFs have been found in other prokaryotes like Anabaena PCC 7120, Pseudomonas putida, Pseudomonas aeruginosa and E. coli among others (Blindauer, 2011).

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However, Anabaena PCC 7120 ORF is present within the sequence of a larger ORF (asr3266) but in opposite orientation and is not identifiable within its annotated genome sequence (Cavet

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et al., 2003). Ambiguity arises with naming of the metallothionein protein in Anabaena 7120. In vitro characterizations led to the inclusion of Anabaena metallothionein into bacterial MT

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family and it is referred to as BmtA (Blindauer et al., 2002). Presently, it shows as putative metallothionein in UniProt and NCBI databases and is designated as NmtA (encoded by nmtA gene) indicated by its metal-binding function (UniProt). nmtA is said to be located between positions 3938083→3937925 within the Nostoc (Anabaena) 7120 genome sequence (Bose et al., 2006). Interestingly, genes encoding for metal-transporting P1-type ATPases in

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Mycobacterium tuberculosis and Streptomyces coelicolor have also been designated as NmtA in protein databases (Cavet et al., 2002; Kim et al. 2015). We hereafter refer Anabaena PCC 7120 metallothionein as NmtA to avoid any confusion and ambiguity associated with its name in various databases. Although NmtA protein shares only 54% identity with SmtA, all the cysteine (nine) and histidine (two) residues involved in metal binding are conserved in both the

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proteins. While eukaryotic MTs are implicated in a number of physiological processes

including metal ion homeostasis and protection against oxidative and physical stress (Palmiter,

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1998; Klaassen et al., 1999), functions beyond metal binding and detoxification have not been

reported so far for prokaryotic MTs (Blindauer, 2011; Shi et al., 1992; Blindauer et al., 2001).

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Nitrogen-fixing strains of cyanobacteria such as Anabaena are commonly used as

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biofertilizers and therefore are important components of paddy fields. The environmental

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contamination through prolonged persistence of potentially toxic metals generated as a result

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of anthropogenic activities adversely affects these cyanobacteria which are abound in such contaminated areas. These toxic metals facilitate the generation of reactive oxygen species

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(ROS). Induction of enzymatic antioxidants in response to metal exposure in Anabaena has been investigated in detail in the past (Singh et al., 2012; Panda et al., 2017). However, the

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physiological role of non-enzymatic antioxidant like metallothionein in Anabaena 7120 is not clear and has not been explored in context of metal detoxification or oxidative stress alleviation

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so far. The present study was undertaken to assess the role of NmtA protein in Anabaena PCC 7120 (hereafter Anabaena 7120) in metal and oxidative stress tolerance. The results

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demonstrate a protective role of NmtA against cadmium toxicity in Anabaena 7120. 2. Materials and methods 2.1. Organism and growth conditions Strains, plasmids and primers used in this study are listed in Table 1. Axenic cultures of Anabaena 7120 were grown in BG-11 liquid medium (Allen, 1968) (0.268 µM EDTA·Na2, 5

5.46 µM citric acid, 17.4 µM K2HPO4, 30.4 µM MgSO4·7H2O, 24.4 µM CaCl2·2H2O, 16.1 µM Na2CO3·H2O, 2.73 µM FeSO4·7H2O, and Hoogland’s reagent [46.4 M µH3BO3, 0.765 µM ZnSO4·7H2O, 1.61 µM NaMoO4·2H2O, 0.316 µM CuSO4·5H2O, 0.168 µM CO(NO3)2·6H2O]), pH 7.2 with combined nitrogen (17 mM NaNO3) under continuous illumination (30 µE m-2 s-1) without or with shaking (100 rpm) at 27ºC ± 2ºC. Growth was

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assessed by measuring chlorophyll a contents as described earlier (Mackinney, 1941). E. coli

cells were grown in Luria-Bertani medium at 37ºC with shaking (120 rpm) with appropriate

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antibiotics. While neomycin (25 µg mL-1, Nm25) was used for recombinant Anabaena strains,

chloramphenicol (34 µg mL-1, Cm34), kanamycin (50 µg mL-1, Kan50) or carbenicillin (100 µg

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mL-1, Cb100) were used for recombinant E. coli strains (Table 1).

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2.2. Expression and purification of recombinant Anabaena 7120 metallothionein, NmtA

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nmtA ORF (162 bp) was PCR-amplified from Anabaena PCC 7120 chromosomal DNA

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and cloned into pET16b at NdeI-BamHI sites to obtain pETnmtA which was subsequently transformed in E. coli BL21 (DE3) pLysS strain (Table 1). Expression of recombinant NmtA

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was induced by isopropyl β-D-thiogalactoside (IPTG; 0.7 mM) in absence or presence of 0.2 mM of ZnSO4, CdCl2, CuSO4, Co(NO3)2, Pb(NO3)2, FeSO4 and UO2(CO3)2]2- separately.

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Purification of the His-tagged NmtA protein from E. coli BL21pLysS was performed by affinity chromatography using a Ni-NTA (Ni2+-nitrilotriacetate) matrix. The purified

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Anabaena 7120 NmtA protein was used to immunize rabbits for generating specific antiserum. The primary and booster immunizations and collection of the antiserum were performed at a

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commercial facility (Radiant Research Services Pvt Ltd, India). 2.3. Protein electrophoresis, Western blotting and immunodetection The protein samples were electrophoretically resolved on 15% SDS-PAGE and visualized either with Coomassie Brilliant Blue R-250 staining or silver staining. For western blotting, the purified NmtA from E.coli cells overexpressing NmtA or the cell-free extracts from 6

AnpAM (Anabaena cells carrying empty vector) and AnnmtA+ (Anabaena cells overexpressing NmtA) were prepared, proteins (30 µg) were electrophoretically resolved on SDS-PAGE (15% gel), electroblotted onto nitrocellulose membrane (Sigma) and probed with the NmtA antiserum.

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2.4. NmtA identification by MALDI-ToF-mass spectrometry The purified protein (corresponding to ~6.1 kDa) band was excised and processed for identification as described earlier (Panda et al., 2014). Following excision, the sample was

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subjected to repeated washing with 50 mM NH4HCO3/ACN, reduction with DTT (Sigma,

India), alkylation with 55 mM iodoacetamide (Sigma, India), in-gel trypsin digestion and

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elution (Panda et al., 2014). Eluted peptides were vacuum concentrated and analyzed by

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Matrix-assisted laser desorption/ionization-Time of Flight (MALDI ToF/ToF) Autoflex mass

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spectrometer which was externally calibrated using Peptide calibration mix I (Bruker

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Daltonics, Germany) as per the manufacturer’s protocol. The analysis was carried out in positive ion reflector mode and the mass spectra were acquired with standard ToF-MS protocol.

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Spectra were acquired using FlexAnalysis software 3.0 (Bruker, Daltonics). Similarity searches were carried out using MASCOT search engine (Matrix Science, UK) using the NCBI non-

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redundant database (Panda et al., 2014).

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2.5. Preparation of apo-NmtA and Proteinase K digestion, SDS-PAGE and spectroscopic analysis of apo- and metallated NmtA Apo-NmtA (metal free NmtA) was prepared as described earlier (Acharya and Blindauer,

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2016). The purified NmtA protein was incubated with dithiothreitol (DTT, 100 mM) for 1 h. Apo-NmtA was prepared by lowering the pH to 1 by addition of 1 M HCl. The metal-free apoMT was loaded onto a gel filtration column (Sephadex G-25, GE Healthcare PD-10) preequilibrated with 0.01 M HCl and purged with N2. Apo-protein was eluted from the column with 0.01 M HCl under N2 purged conditions. To the eluted protein 100 mM DTT was added 7

to prevent the formation of disulphide bridges in the demetallated protein. The native protein or apo-NmtA (15 µg) was treated with proteinase K (0.5 µg) in Tris-NaCl buffer (pH 8) at 26ºC for overnight. The resulting samples were resolved electrophoretically on 15% SDS-PAGE and visualized by silver staining as described earlier (Acharya and Blindauer, 2016). Absorption spectra of NmtA and apo-NmtA were recorded in the wavelength range of 200-300 nm using

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a PowerWaveTM XS/XS2 microplate spectrophotometer (BioTek Instruments, USA). 2.6. Northern blot hybridization and RT-PCR

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Experimental solutions (BG 11 medium) were amended separately with 10 µM CdCl2, 25 µM ZnSO4, 25 µM CuSO4, 250 µM uranyl carbonate or 1 mM H2O2 and were allowed to equilibrate for 30 min under aerobic conditions. Experiments were initiated by inoculating

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Anabaena cells (at a density of 5 µg of chlorophyll a mL-1) and incubating them under

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illumination for 24 h. Timed aliquots were withdrawn and centrifuged at 7000 rpm for 5 mins.

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RNA was isolated from these cells using TRI reagent (Sigma) as per the manufacturer’s

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protocol. The isolated RNA was treated with amplification grade DNaseI (Sigma) and checked

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for DNA contamination by PCR. 5 µg of total RNA was electrophoretically resolved on 1% denaturing formaldehyde agarose gel using MOPS buffer. RNA was transferred to nylon

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membrane by capillary blotting overnight with 10X SSC. The nmtA gene was amplified by PCR, labeled with DIG, and hybridized to the RNA in DIG Easy Hyb buffer (overnight at

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40°C). Detection with the DIG labelled probe was carried out as per manufacturer’s protocol (DIG-High Prime DNA Labeling and Detection, Roche).

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For RT-PCR, 0.5 µg RNA was used for cDNA synthesis (Thermo Scientific First strand

cDNA synthesis kit). Real time PCR (Eppendorf Realplex 4) was carried out using nmtA specific primers (Table 1). 16S rRNA was used as an internal control. The amplification products were resolved by electrophoresis on agarose gels and detected by staining with ethidium bromide.

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2.7. Overexpression of NmtA in Anabaena 7120 nmtA gene was PCR amplified using Anabaena 7120 genomic DNA as the template with the incorporation of NdeI and BamHI restriction sites in the forward and reverse primers (Table 1) respectively. nmtA was cloned downstream to the light inducible PpsbA1 promoter into pFPN vector (Table 1). nmtA along with PpsbA1 was excised out as SalI-XmaI fragment from

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pFPNnmtA and cloned into E. coli/Anabaena shuttle vector pAM1956 (Table 1) resulting in

pAMnmtA construct. pAMnmtA was maintained in E. coli DH5α and was sequenced to

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ascertain for mutations. The strong cyanobacterial psbA1 promoter co-transcribed the cloned

nmtA gene followed by downstream gfpmut2 gene (encoding green fluorescent protein).

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Incorporation of stop codon in the reverse primer of nmtA ensured independent translation of

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NmtA and GFP. The cargo plasmid pAMnmtA was conjugally transferred into Anabaena 7120 using E. coli donor HB101 (harboring methylase encoding pRL623 and conjugal plasmid

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pRL443) (Table 1). Exconjugants were selected on BG11 N+ agar plates containing neomycin

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(25 μg mL-1). The conjugants were repeatedly subcultured for segregation and maintained

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under the selective pressure of neomycin. The recombinant Anabaena strain was designated as AnnmtA+. Similarly, Anabaena strain, AnpAM (carrying empty vector) resulting from conjugal

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transfer of cargo plasmid pAM1956 into Anabaena 7120 was generated by selection of

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exconjugants on BG11 N+ agar plates containing neomycin (25 μg mL-1). 2.8. Microscopy of Anabaena strains Bright-light and fluorescence microscopy of the AnpAM and AnnmtA+ strains were done

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at 600X magnification on a Carl Zeiss Axioscop 40 microscope. The images were captured with a charge-coupled device (CCD) Axiocam MRc (Zeiss) camera. Fluorescence microscopy of Anabaena cultures were done by exciting the cells with green light (λ: 520 nm), which is absorbed by phycocyanin, and visualizing the red chlorophyll a fluorescence (λ: 680 nm) arising as a consequence of the resonant energy transfer from phycocyanin to chlorophyll a.

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Green fluorescence of GFP was visualized using the Hg-Arc lamp (excitation 470 nm, emission 508 nm). 2.9. Determination of metal tolerance and accumulation Overnight grown cultures of E. coli carrying pET16bnmtA were inoculated in fresh LB medium containing 100 µg mL-1 carbenicillin and 34 µg mL-1 chloramphenicol and were grown

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at 37 ºC. After the cells attained OD600 of 0.6, production of NmtA was induced by the addition

of IPTG (0.7 mM) and grown further for 4 h at 37 ºC. The resulting cultures were diluted to an

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OD600 of 1.0 from which appropriate dilutions were made and plated onto LB agar plates supplemented with 100-500 µM CdCl2. The plates were incubated at 37 ºC overnight and viable colony forming units (CFUs) were counted for assessing cadmium tolerance.

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In case of Anabaena, three-day-old cultures of AnpAM and AnnmtA+ were inoculated in

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fresh BG 11 medium at a chlorophyll a density of 3 µg mL-1 and grown in presence of 10 µM

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or 20 µM Cd for 7 days under illumination. Growth was monitored by chlorophyll a estimations at regular intervals. For metal accumulation studies, aliquots (3mL) were taken out at regular

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intervals from the AnpAM and AnnmtA+ cultures exposed to 10 µM of CdCl2. Following centrifugation (7000 rpm, 5 min), the pellets and supernatants were separated. The pellets were

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completely mineralized by concentrated nitric acid and were suspended in 3 mL of 0.2% HNO3. Cadmium concentration in pellets and supernatants were estimated by atomic absorption

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spectrometry (CSAAS, Contra AA-300, Germany). 2.10. Determination of oxidative stress tolerance

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Three-day-old cultures of AnpAM and AnnmtA+ were inoculated in fresh growth medium

at a chlorophyll a density of 3 µg mL-1 and treated with hydrogen peroxide (0.5-2 mM) or methyl viologen (0.5-1 mM) or uranyl carbonate (150-250 µM). Similar experiments were conducted in combined presence of hydrogen peroxide (0.5 mM) and CdCl2 (10 µM). These

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cultures were incubated under illumination without shaking. Growth was monitored by chlorophyll a estimation. We included a mutant of Synechococcus PCC 7942 deficient in MT locus, smt i.e. strain R2-PIM8 (smt) along with R2-PIM8 (a small plasmid cured derivative of Synechoccoccus PCC 7942) (both strains were gifts from N. Robinson, Durham University, Durham, UK) in our

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studies in order to establish the role of the cyanobacterial metallothionein in oxidative stress

tolerance more directly. Exponential phase cells of strain R2-PIM8 and smt mutant strain R2-

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PIM8 (smt) of Synechococcus PCC 7942 were inoculated in BG 11 medium at a chlorophyll a

density of 3 µg mL-1 and exposed to hydrogen peroxide (3-7 mM) for 7 days under illumination

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without shaking. Growth of both the strains following H2O2 exposure was monitored by

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chlorophyll a estimations.

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2.11. Statistical analysis

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For growth and cadmium accumulation studies, triplicates of each of the three transformants of each strain was considered for calculating average ± standard deviations. Average values

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with standard deviations are shown for a representative experiment. 3. Results and Discussion

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3.1. Expression and identification of recombinant NmtA protein

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On induction with IPTG (isopropyl β-D-thiogalactopyranoside), the E. coli BL21 (DE3) pLysS strain carrying the plasmid pET16bnmtA expressed a ~ 6.1 kDa protein corresponding to the His tagged NmtA, along with the diffused protein bands of higher molecular weights

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(Fig. 1A). Expression cultures have been reported to be supplemented with metals for stability of the MT proteins (Blindauer and Leszczyszyn, 2010). However, there have been unresolved issues about the recombinant heterologous expression of MTs in the presence of the metals due to the lack of our knowledge on the mechanisms for metal incorporation into the newly synthesized MT proteins. Recombinant NmtA (53 aa) purified from E. coli cultures induced 11

with IPTG in presence of various metals like Zn, Cd, Cu, Pb, Fe, U and Co showed diffused protein bands of higher molecular weights (Fig. S1). The lower molecular weight protein band of expected size (~6.1 kDa) was not observed in all the metallated preparations (Fig. S1). Similar heterologous recombinant expression of Synechococcus metallothionein, SmtA in E.coli induced with IPTG in presence of Zn had shown diffused protein bands well above the

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expected molecular mass (Acharya and Blindauer, 2016). The heterogenous migration

behavior of metallated MTs on SDS-PAGE owing to incomplete denaturation of metal-protein

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clusters (SDS sample and gel conditions are not able to cause full denaturation of the metal–

protein clusters) often resulting in diffused protein bands is well known (McCormick et al.,

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1991). Trypsin digestion of the protein band corresponding to ~6.1 kDa (Fig. 1A) followed by

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peptide MS (mass mapping) and MS/MS measurements performed on a high resolution

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MALDI-TOF/TOF mass spectrometer confirmed the identity of NmtA unambiguously.

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MASCOT search identified the protein band as the putative metallothionein of Nostoc sp. PCC 7120 with a significant score and a calculated molecular mass of 6.14 kDa (Fig. 1B).

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3.2. Demetallation of NmtA and metal-dependent proteolytic protection of NmtA

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Anabaena metallothionein has been shown to bind four Zn2+ (Blindauer et al., 2002). Treatment of purified NmtA with DTT (100 mM) followed by 0.01 M HCl resulted in its

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demetallation and yielded a single prominent band of molecular weight close to monomeric NmtA (Fig. 2A). The protein subjected to either DTT or HCl alone resulted in diffused oligomeric bands (Fig. 2A) indicating that the removal of metals coupled with the reduction of

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inter- or intramolecular disulfide bonds could successfully resolve the erratic migration of NmtA on SDS-PAGE. Similar observations are reported for metallated and demetallated recombinant Synechococcus metallothionein, SmtA (Acharya and Blindauer, 2016).

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The metals associated with metallothionein inhibit the proteases and protect it against proteolysis (Nielson et al., 1985). The native NmtA did not show any sensitivity towards proteinase K (Fig. 2B). In contrast, NmtA treated with 0.01 M HCl alone or in combination with 100 mM DTT was completely digested by proteinase K (Fig. 2B). It was interesting to observe that the NmtA treated with DTT alone exhibited resistance to proteolysis similar to

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native NmtA revealing the metal dependent protection of metallothionein against proteolysis.

Similar protection against proteolysis was observed for NmtA expressed in presence of

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cadmium. Apo- or demetallated MTs have been reported to exist as random coil (Blindauer and Leszczyszyn, 2010) thus making it susceptible for digestion by proteinase K (Ebeling et

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al., 1974.). Resistance to proteolysis has been observed for native rat liver metallothionein

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coordinated with various metals including Cd, Ag, Hg, Pb (Nielson et al., 1985).

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The demetallation reaction of NmtA was also followed by UV-Vis spectroscopy (Fig.

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2C). Metallothioneins exhibit absorption spectra characteristic to specific metal-thiolate charge transfer transitions (Vasak and Kagi, 1983). The absorption spectrum of NmtA exhibited a peak

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at ~220 nm (Fig. 2C) due to co-ordination of Zn by cysteine ligands (Zn-S charge transfer band) which is characteristic of Zn containing MTs (Vasak and Kagi, 1983). This absorption

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was eliminated following exposure of NmtA to pH 1 causing dissociation of Zn from NmtA to

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yield metal free apo-NmtA (Fig. 2C). Presence and absence of zinc in native and apo-NmtA respectively was confirmed by Atomic Absorption Spectrophotometer (AAS) measurements. Similar spectroscopic features have been reported for other metallated and metal free (apo-)

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metallothioneins (Kagi and Vallee, 1961; Neilson and Winge, 1983). It is important to note that we obtained zinc bound NmtA from extracts of E. coli cultures not supplemented with zinc (induced only with IPTG) (Fig. 2C). LB medium itself is known to contain zinc (Hughes and Poole, 1991) and apparently facilitated the metallation of the protein. This is in agreement with the observations obtained for related Synechococcus SmtA protein (Binet et al., 2003). The 13

visualization of lower molecular weight protein band (~6.1 kDa) for NmtA (expressed in absence of any metal) on SDS-PAGE is likely due to the presence of the apo-species resulting from limited availability of zinc in LB medium (Fig. 1A) which is not the case with metal supplemented NmtA preparations wherein metal loaded high molecular weight protein bands

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were observed (Fig. S1). 3.3. nmtA expression is induced in response to metals and not oxidative stress in Anabaena

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PCC 7120

Metal-induced expression of metallothionein (MT) genes is well known in eukaryotes and prokaryotes (Turner and Robinson, 1995). RNA gel blot analysis was carried out to evaluate

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the effect of metal and oxidative stress on transcript abundance in Anabaena 7120 (Fig. 3). The

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total RNA was isolated from the wild-type Anabaena PCC 7120 cells exposed to various metals

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including cadmium, zinc, copper and uranium or hydrogen peroxide and probed with the nmtA

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gene probe. All the metals except U clearly induced expression of nmtA transcripts in Anabaena

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as compared to control, metal unstressed cells wherein nmtA expression was undetectable (Figs. 3A-E). Cadmium metal was observed to be the strongest inducer of nmtA transcription

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followed by zinc and copper. A time-dependent induction of nmtA was visualized for cadmium, zinc- and copper-treated cells wherein abundance of nmtA transcripts was observed within 15

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min of exposure followed by a decline in the transcript levels by 24 h of exposure (more prominently in Zn and Cu) (Fig. 3B-D). The related bacterial metallothionein, smtA was found to be induced in the presence of several metals including zinc, copper, cadmium, cobalt, nickel,

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lead, chromium, and mercury (Huckle et al., 1993). Mammalian MTs have been shown to scavenge free radicals because of their cysteinyl thiolate groups and their expression is induced by a variety of cellular factors including the presence of metals ions and oxidative stress (Andrews, 2000). Since bacterial metallothioneins

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are also rich in cysteine residues (16%), it was assumed that they might also be induced under oxidative stress. However, no visible induction of nmtA was observed in response to uranium or H2O2 in Anabaena 7120 (Figs. 3E and F). Both H2O2 (Cabiscol et al., 2000) and uranium (Dekker et al., 2016) are known to cause oxidative stress in microbes. There are no reports on

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induction of bacterial metallothioneins in response to oxidative stress so far. 3.4. Overproduction of Anabaena NmtA

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The nmtA ORF was cloned and overexpressed from a strong light-inducible PpsbA1 promoter in Anabaena 7120. Visualization of GFP fluorescence ensured nmtA expression

(which preceded gfpmutII gene) in the recombinant Anabaena cells (Fig. 4A). Expression of

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RNA isolated using RT-PCR revealed a substantial increase in the transcript levels of nmtA in

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AnnmtA+ over AnpAM strain (Figs. 4B and C). In agreement with the transcriptional analysis,

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the NmtA protein was found to be substantially produced in AnnmtA+ cells as compared to

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AnpAM cells (Fig. 4D). The heterogenous migration profile of NmtA protein in recombinant

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Anabaena cells on SDS-PAGE was reflected in Western blot analysis which showed diffused protein bands. No such bands were observed with the cell free extracts of AnpAM confirming

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the expression of NmtA in transgenic Anabaena 7120.

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3.5. NmtA protects E. coli and Anabaena 7120 against cadmium toxicity in vivo Metallothionein was first isolated from eukaryotes and prokaryotes in cadmium-

challenged environments and represented as Cd-binding protein offering protection against

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cadmium toxicity (Margoshe and Vallee, 1957). To assess the protective function of NmtA against cadmium toxicity, E. coli cells

overexpressing nmtA were subjected to toxic concentrations of cadmium ranging from 100 µM to 500 µM on LB agar plates. NmtA-expressing cells demonstrated better growth under cadmium exposure conditions as indicated by CFU counts (Fig. 5A). Our results are in 15

agreement with the previous studies in which overexpression of Synechococcus smtA resulted in enhanced cadmium tolerance in E. coli (Seifipour et al., 2017). Similar effects were observed for Anabaena cells overexpressing NmtA. When grown in the presence of 10 µM or 20 µM cadmium, Anabaena overexpressing NmtA (AnnmtA+) cells

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showed superior tolerance to cadmium as compared to AnpAM (Anabaena carrying empty vector) cells as indicated by growth measurements in terms of chlorophyll a contents (Fig. 5B). Significant bleaching was observed for AnpAM cells in agreement with Chl a contents

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following 7 days of 20 µM cadmium exposure as compared to AnnmtA+ cells which did not show bleaching to such large extent (Fig. 5C). Extensive fragmentation of filaments and cell

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lysis could be visualized in Cd-exposed AnpaM cells (10 or 20 µM for 7 days), whereas the

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recombinant AnnmtA+ maintained long and intact healthy filaments expressing GFP following

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similar cadmium exposure (Fig. 5D). Cadmium tolerance of transgenic Anabaena cells

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expressing mouse liver metallothionein mMT-I gene or zinc tolerance of recombinant Arabidopsis expressing Synechococcus smtA has been reported previously (Ren et al., 1998;

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Guo et al., 1999, Xu et al., 2010). However, overexpression of cyanobacterial MT in their

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native hosts and its effect in terms of metal tolerance has not been investigated yet. Even though overexpression of NmtA conferred tolerance to cadmium, no significant

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difference in cadmium accumulation could be seen in AnnmtA+ as compared to AnpAM (Fig. 5E). It has been observed that coupling of MT expression to metal concentration did not always result in increased metal accumulation (Lin and Kosman, 1990).

Also, studies have

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demonstrated that bacterial cells expressing metallothionein in the cytosol are less effective in metal accumulation rather than those cells expressing the metallothionein in the periplasm or the outer membrane (Chen et al., 1997, Pazirandeh, et al., 1995). 3.6. NmtA overexpression does not confer tolerance to oxidative stress

16

Free radical scavenging ability of eukaryotic metallothioneins owing to their high content of cysteine residues has been reported earlier (Ruttkay-Nedecky et al., 2013). Since NmtA harbored high (~16%) cysteine content, it is expected that expression of NmtA could provide protection to transgenic Anabaena cells against oxidative stress. However, non-discriminatory growth behavior for AnpAM and AnnmtA+ cells was observed in presence of increasing

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concentrations of uranyl carbonate or hydrogen peroxide (Fig. 6A and B) or methyl viologen

(Fig. S2). These results are in line of agreement with the non-inducibility of nmtA transcripts

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in response to uranium or H2O2 (Figs. 3E and F). Combined effect of hydrogen peroxide and cadmium on growth behavior for AnpAM and AnnmtA+ cells revealed similar observations as

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that of hydrogen peroxide alone (Fig. 6C) emphasizing the inefficiency of NmtA

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overexpressing phenotype for protection against oxidative stress.

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To gain a better insight into the role of cyanobacterial MTs in oxidative stress, a mutant of

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Synechococcus PCC 7942 deficient in MT locus, smt, was evaluated for its growth and tolerance in presence of increasing concentrations of hydrogen peroxide. Anabaena 7120 and

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Synechococcus 7942 metallothionein contain similar proportion (~16%) of cysteine residues (Fig. 7A). It was interesting to note that growth in smt mutants i.e R2-PIM8 (smt) was strongly

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inhibited resulting in extensive bleaching in the presence of increasing concentrations of

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hydrogen peroxide as compared to wild type cells, R2-PIM8 (Figs.7B and C). Taken together, the metallothionein overexpressing (Fig. 6) and mutant (Fig. 7) phenotype of the studied cyanobacteria suggests an important but not a predominant role of cyanobacterial

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metallothioneins in oxidative stress tolerance. Reactive oxygen species (ROS) in Anabaena 7120 have been shown to be detoxified by non-enzymatic antioxidants like phytochelatins (Chaurasia et al., 2017) or specific antioxidant enzymes, such as superoxide dismutases (SODs), catalases and peroxidases (Banerjee et al., 2013). 4. Conclusion 17

Unlike eukaryotic metallothioneins, there is limited information on prokaryotic metallothioneins. Besides, the majority of the experimental data on prokaryotic metallothionein relates to metallothionein from Synechococcus PCC 7942. We isolated and characterized a metallothionein, NmtA from Anabaena PCC 7120 and demonstrated its definitive role in cadmium tolerance. It is envisaged that NmtA may be a part of a more dynamic mechanism of

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metal detoxification apparently involving donation of bound metals to efflux systems resulting

in active extrusion of metals. However, NmtA did not display a pronounced role in oxidative

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stress tolerance. It will be worth attempting the expression of NmtA outside the cytosol which might facilitate the use of non-viable cells for metal accumulation and for efficient desorption

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of the bound metal.

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Declarations of interest: none.

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Acknowledgements

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The authors wish to thank Dr. Bandita Panda for protein identification at Department of Biosciences and Bioengineering, IIT Bombay and Dr. Sanjukta A. Kumar, Analytical

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Chemistry Division, BARC, Mumbai for metal analysis by Atomic Absorption Spectrometry. We thank Dr. H.S. Misra, Head, Molecular Biology Division, BARC for his constant

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encouragement during the course of this study. We are grateful to Prof. Nigel Robinson (Durham University, Durham, UK) for R2-PIM8 and R2-PIM8(smt) strains of Synechococcus

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PCC 7942.

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Figure legends

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Figure 1. Purification and identification of NmtA protein. E. coli cells were induced with

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IPTG and His-tagged NmtA was purified by Ni-NTA affinity chromatography. (A) Proteins (30 µg) were electrophoretically resolved by 15% SDS-PAGE followed by CBB staining.

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Lane1: Molecular weight marker, lane 2: Cell-free extracts of E. coli cells carrying empty

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vector, pET16b, lane 3: Cell-free extracts of E. coli cells overexpressing NmtA, lane 4: flow through from Ni-NTA affinity column, lane 5: Purified NmtA. (B) The band corresponding to

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~ 6.1 kDa (indicated by arrow in panel A) was processed for identification by MALDI-TOF-

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MS. The corresponding MASCOT search result is shown.

Figure 2. SDS-PAGE analyses and UV absorption spectra of native and apo-NmtA. The apo-NmtA and native NmtA proteins (1.5-2 µg) were resolved on 15% SDS-PAGE and visualized by silver staining (A) before and after (B) proteinase K digestion. (C) UV absorbance spectra of NmtA and apo-NmtA.

28

Figure 3. Expression analysis of nmtA in Anabaena PCC 7120 in the presence of different stresses by Northern-blotting. Total RNA was isolated from (A) untreated (control) cells or from cells treated with (B) 10 µM CdCl2, (C) 25 µM ZnSO4,(D) 25 µM CuSO4, (E) 250 µM uranyl carbonate or (F) 1 mM H2O2. 5 µg RNA was resolved on 1 % formaldehyde-agarose gel, transferred onto a nylon membrane, and probed with the DIG-labelled nmtA. Equal loading

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of RNA was confirmed by ethidium bromide stained rRNA from various samples.

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Figure 4. Overexpression of NmtA in Anabaena PCC 7120. (A) Bright field (BF) and

Fluorescence (FM) photomicrographs under Blue light excitation (BLE) (600X magnification)

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using Hg-Arc lamp (excitation 470 nm, emission 508 nm) of AnpAM and AnnmtA+. (B)

N

Confirmation of overexpression of nmtA transcripts by RT-PCR. 500 ng of total RNA was used for cDNA synthesis which served as template for PCR performed with nmtA specific primers.

M

A

Relative fold change expression in AnpAM and AnnmtA+ obtained by ΔΔCt method with respect to wild type is shown. (C) The amplified products resulting from RT-PCR were

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resolved on 1.5 % agarose gel and visualized by ethidium bromide staining. The lower panel represents the products of 16S rRNA used as control. (D) Western blotting and

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immunodetection of NmtA in AnpAM and AnnmtA+ cells with NmtA antiserum (1:1000 dilution). Inset shows purified NmtA from recombinant E. coli cells immunodetected with

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NmtA antiserum (1:5000 dilution) as a positive control. The characteristic diffused MT protein

A

profile was observed in both the cases.

Figure 5. Effect of NmtA overexpression on cadmium tolerance. (A) CFU counts of E. coli harboring pET16b and pET16bnmtA in the presence of 0-500 µM cadmium. (B) Effect of cadmium on growth of AnpAM and AnnmtA+ as assessed by contents of chlorophyll a. (C) AnpAM and AnnmtA+ cultures were exposed to 10-20 µM cadmium for 7 days and

29

subsequently the cultures were transferred to microtitre plate and photographed. (D) Bright field (BF) and Fluorescence (FM) microphotographs (600X magnification) of AnpAM and AnnmtA+ cells after 7 days of treatment with cadmium. Yellow and red arrows indicate the fragmented cells due to cadmium exposure in AnpAM cells. (E) Cadmium accumulation in

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AnpAM and AnnmtA+cells exposed to 10 µM CdCl2.

Figure 6. Effect of NmtA overexpression on the oxidative stress tolerance of Anabaena

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7120. Growth of AnpAM and AnnmtA+ cells assessed by contents of chlorophyll a in the

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presence of (A) uranium or (B) hydrogen peroxide or (C) both hydrogen peroxide and cadmium

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Figure 7. Effect of oxidative stress on smt deficient mutants of Synechococcus PCC 7942.

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(A) Sequence alignment of NmtA with SmtA retrieved from NCBI protein database using

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ClustalW. The conserved cysteine residues are indicated by * (B) Growth of R2-PIM8 and R2PIM8 (smt) strains of Synechococcus PCC 7942 assessed by contents of chlorophyll a after 7

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days of exposure to various concentrations of H2O2. (C) The corresponding flask cultures

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mentioned in (B) are photographed.

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Table 1. Primers, plasmids and strains used in the study Description Reference GGAATTCCATATGACAACCGTAACTCAAATG This study CGCGGATCCTTAACAGCCACAGCCATTATG CACACTGGGACTGAGACAC Pinto et al. (2012) CTGCTGGCACGGAGTTAG

pAM1956

KanR, promoterless gfpmut2 reporter gene

pFPNnmtA

170 bp nmtA fragment cloned in pFPN

A

Primer nmtA Fwd nmtA Rev 16S Fwd 16S Rev Plasmid pFPN

CbR, KanR, integrative expression vector

39

Chaurasia et al. (2008) Yoon and Golden (1998) This study

Wild-type strain

Lab collection

Elhai et al. (1997)

Lab collection

Anabaena 7120 harboring pAM1956, NmR Anabaena 7120 harboring pAMnmtA, NmR

Derivative of R2-SPc with pPMI8 integrated into the metF gene, ApS SmR MetR2-PIM8 transformed with linearized pRECSU which consists of smt flanking sequences interrupted by E. coli plasmid pSU19 containing the cat gene, ApS SmR Met- CmR

A

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ED

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R2-PIM8 (smt)

Lab collection

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Anabaena strain Anabaena PCC 7120 AnpAM AnnmtA+ Synechococcus strain R2-PIM8

Lab collection

Novagen This study

SC R

HB101R2

F- recA41 endA1 gyrA96 thi-1 hsdR17 (rk- mk-) supE44 relA λ lacU169 F- ompT gal dcm lon hsdSB(rB- mB-) λ(DE3) pLysS (CmR) F- mcrB mrr hsdS20 (rB- mB-) recA13leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 (SmR) lnV44 λDonor strain carrying pRL623 (encoding methylase) and pRL443 (conjugal plasmid)

U

BL21 (DE3) pLysS HB101

This study

N

pET16b pETnmtA E. coli strain DH5α

~1 kb XmaI-SalI fragment from pFPNnmtA cloned in pAM1956 vector AmpR, expression vector 170 bp nmtA fragment cloned in pET16b

A

pAMnmtA

40

This study This study

van der Plas et al.,1990 Turner et al., 1993