Redox-dependent chaperone/peroxidase function of 2-Cys-Prx from the cyanobacterium Anabaena PCC7120: role in oxidative stress tolerance
© Banerjee et al.; licensee BioMed Central. 2015
Received: 20 July 2014
Accepted: 29 January 2015
Published: 21 February 2015
Cyanobacteria, progenitors of plant chloroplasts, provide a suitable model system for plants to study adaptation towards different abiotic stresses. Genome of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena PCC7120 harbours a single gene (alr4641) encoding a typical 2-Cys-Peroxiredoxins (2-Cys-Prxs). 2-Cys-Prxs are thiol-based peroxidases that also function as molecular chaperones in plants and other systems. The Alr4641 protein from Anabaena PCC7120 shows high level biochemical similarities with the plant 2-Cys-Prx. The physiological role played by the Alr4641 protein in Anabaena was addressed in this study.
In Anabaena PCC7120, alr4641 transcript /Alr4641 protein was induced in response to abiotic stresses and its promoter was active in the vegetative cells as well as heterocysts. The wild-type Alr4641 protein or Alr4641 lacking the peroxidatic cysteine (Alr4641C56S) or the resolving cysteine (Alr4641C178S) existed as higher oligomers in their native form. The wild-type or the mutant Alr4641 proteins showed similar chaperone activity, but only the wild-type protein exhibited peroxidase activity indicating that unlike peroxidase activity, chaperone activity was not dependent on cysteines. In contrast to other 2-Cys-Prxs, chaperone/peroxidase activity of Alr4641 was dependent on its redox state and not oligomerization status. Alr4641 could protect plasmid DNA from oxidative damage and physically associate with NADPH-dependent thioredoxin reductase (NTRC). Like 2-Cys-Prxs from plants (e.g. rice), Alr4641 could detoxify various peroxides using NTRC as reductant. On exposure to H2O2, recombinant Anabaena PCC7120 strain over-expressing Alr4641 (An4641+) showed reduced content of reactive oxygen species (ROS), intact photosynthetic functions and consequently better survival than the wild-type Anabaena PCC7120, indicating that Alr4641 can protect Anabaena from oxidative stress.
The peroxidase/chaperone function of Alr4641, its inherent transcriptional/translational induction under different abiotic stresses and localization in both vegetative cells and heterocysts could be an adaptive strategy to battle various oxidative stresses that Anabaena encounters during its growth. Moreover, the recombinant Anabaena strain over expressing Alr4641 showed higher resistance to oxidative stress, suggesting its potential to serve as stress-tolerant biofertilizers in rice fields.
Peroxiredoxins (Prxs) are ubiquitous peroxidases with important roles in detoxification of hydrogen peroxide, alkyl hydroperoxides and peroxynitrites [1,2]. Prxs are characterized by a conserved Alkylhydroperoxide C (AhpC) or Thiol–Specific Antioxidant (TSA) domain that contains a thioredoxin fold. Prxs have highly conserved cysteine residues, peroxidatic cysteine (Cp) and resolving cysteine (Cr), which are essential for peroxidase activity. Based on their catalytic mechanisms and the presence of conserved cysteine residues, Prxs are classified into three groups, namely, typical 2-Cys-Prx, atypical 2-Cys Prx (which are subdivided into type II Prx and PrxQ) and 1-Cys-Prx .
The typical 2-Cys-Prxs are functionally conserved across diverse organisms and form the largest group of peroxiredoxins. Recently, 2-Cys-Prx has been shown to be a conserved marker of circadian rhythms in all the three phylogenetic domains viz. Eukaryota, Bacteria and Archaea . In typical 2-Cys-Prxs, Cp is present near N-terminus while Cr is located in the vicinity of C-terminus. On reaction with a peroxide substrate, Cp (Cys-SH) is oxidized to sulfenic acid (Cys-SOH), which in turn reacts with the thiol group of the resolving cysteine from other subunit to form an intermolecular disulfide bridge . The active form of enzyme is regenerated with the help of reductants like thioredoxin. In the presence of excess substrate (e.g. H2O2), Cp of 2-Cys-Prx may undergo overoxidation to form sulfinic acid (Cys-SO3), which prevents disulfide bond formation, rendering the enzyme inactive. However, in many organisms, sulfiredoxin (Srx) reduces the overoxidized Cp to its catalytically active form [6,7]. Sensitivity to overoxidation depends on the structural motifs, GG(L/V/I)G and YF, which are believed to be present in the eukaryotic 2-Cys-Prxs, but generally absent in the corresponding prokaryotic enzymes .
The typical 2-Cys-Prx plays a vital role in detoxifying peroxides in all the kingdoms of life. Transgenic Arabidopsis with decreased 2-Cys-Prx in chloroplast showed oxidative damage of chloroplastid proteins indicating that 2-Cys-Prx protects the photosynthetic machinery from oxidative damage [9,10]. Also, Arabidopsis mutant lacking both the chloroplastid 2-Cys-Prx displayed altered redox homeostasis and showed increased H2O2 levels in leaves . Overexpression of 2-Cys-Prx has been shown to protect potato plants from oxidative stress and high temperature . In tobacco, the chloroplastid 2-Cys-Prx has been implicated in protecting cells from photoinhibition following exposure to high light, methyl viologen (MV) or t-butyl hydroperoxide . Disruption of gene encoding 2-Cys-Prx in Synechocystis as well as in Synechococcus eliminated tolerance against H2O2 [14,15]. In bacteria like Sulfolobus solfataricus and Vibrio vulnificus, 2-Cys-Prx has been proposed to detoxify endogenously generated hydrogen peroxide, thus, supporting its role as an anti-oxidative stress protein [16,17].
Interestingly, the typical 2-Cys-Prx not only defends cells from oxidative stress, but also functions as redox-regulated chaperone depending on its oligomerization status . The 2-Cys Prx from Pseudomonas aeruginosa, on exposure to H2O2, converts into a low molecular weight (LMW) form from its high molecular weight (HMW) form. This change triggers a chaperone to peroxidase functional switch . In case of 2-Cys Prx from yeast, oxidative stress and heat shock triggers conversion from LMW form to HMW structure, which shows chaperone activity . In stroma of chloroplast, under conditions of stress, the dimeric 2-Cys-Prx switches to its oligomeric form and binds reversibly to the thylakoid membrane . It is widely believed that the dimeric form of 2-Cys-Prx shows peroxidatic functions while oligomerization is essential for chaperone activity .
Cyanobacteria, progenitors of plant chloroplasts, were the first organisms to produce oxygen as a by-product of photosynthesis [22,23]. Hence, it is expected that these organisms would have developed elaborate mechanisms to overcome oxidative stress. Filamentous forms of nitrogen-fixing cyanobacteria (e.g. Anabaena) are economically important as biofertilizers during cultivation of paddy in Southeast Asia . Anabaena PCC7120, a filamentous, heterocystous, diazotrophic cyanobacterium, that tolerates abiotic stresses like radiation and desiccation, has been used as a suitable model system to study the fundamental aspects of adaptive responses to various stresses including oxidative stress in our laboratory [25-29]. Genome sequence analysis has shown Anabaena PCC7120 to possess several peroxiredoxin genes/ORFs (e.g. all1541, alr2503, all2375, all2556, alr3183, alr4404, alr4642 and alr4641) . The Alr3183, Alr2503, All2375 and All2556 belong to PrxQ-type of peroxiredoxins, All1541 is a type II Prx, Alr4404 is a 1-Cys-Prx, Alr4642 is Prx-like, whereas Alr4641 is a typical 2-Cys-Prx [26,31].
Earlier, 2-Cys-Prx from Anabaena was shown to be prone to over-oxidation  and was found to utilize NADPH-dependent thioredoxin reductase (NTRC) as reducing agent for peroxidase activity like the 2-Cys-Prx from rice . In this study, expression analysis in response to various stresses, redox dependent chaperone/peroxidase function and the role played by this enzyme in protecting Anabaena from oxidative stress were addressed. Along with oxidative stress, alr4641/Alr4641 was induced by salt/osmotic/γ-radiation stress in Anabaena and the Alr4641 protein was expressed in the vegetative cells as well as heterocysts. Alr4641 formed higher oligomeric complexes and showed peroxidase/chaperone function. Unlike peroxidase activity, chaperone activity of Alr4641 did not depend on the conserved cysteine residues. Interestingly, reduction of Alr4641 with DTT resulted in loss of chaperone activity whereas treatment with H2O2 inactivated peroxidase function. Over-expression of Alr4641 in Anabaena protected the photosynthetic machinery from H2O2-induced damage via its peroxidatic cysteine, leading to better survival than the wild-type Anabaena; thus, establishing its protective role in overcoming oxidative stress.
Abiotic stresses induce alr4641/Alr4641 expression in Anabaena
Alr4641 promoter is expressed in the vegetative cells as well as heterocysts
The alr4641 promoter and its adjacent DNA were cloned upstream of the gfp reporter gene in reporter vector, pAM1956, and transferred into Anabaena PCC7120 (An4641prom). An4641prom was grown under nitrogen-fixing conditions and subjected to microscopic analysis. Interestingly, along with the vegetative cells, GFP fluorescence was also observed in the heterocysts indicating that the alr4641 promoter was active in heterocysts as well as in the vegetative cells (Figure 2C). Moreover, the Alr4641 protein was also detected in proteins extracted from heterocysts on Western blots (Figure 2D). Expression of the Alr4641 protein was monitored in the wild-type Anabaena cultures grown under nitrogen-supplemented or nitrogen-fixing conditions. No significant difference in the production of Alr4641 was observed (Figure 2E) suggesting that the absence of combined nitrogen in the growth medium does not affect Alr4641 expression in Anabaena.
Oligomerization of Alr4641 is independent of cysteine residues
For functional characterization, the 2-Cys peroxiredoxin protein (Alr4641) from Anabaena was over-expressed in E. coli with N-terminal His-tag and purified near to homogeneity by affinity chromoatography (Figure 3B). The putative peroxidatic (Cys-56) and resolving (Cys-178) cysteines of Alr4641 were individually mutated to serine by site-specific mutagenesis and the corresponding proteins (Alr4641C56S and Alr4641C178S) were purified to near homogeneity. Gel filtration analysis revealed Alr4641 to elute in a fraction corresponding to decamer/dodecamer (Figure 3C). Native PAGE analysis showed the wild-type Alr4641 as well as the mutants to be present as higher oligomers (Figure 3D). These results suggest that peroxidatic and resolving cysteine residues are not involved in oligomer formation. On SDS-PAGE, the wild-type Alr4641 protein migrated as a monomer under reducing conditions (in presence of DTT), while in absence of DTT, a 50 kD protein, corresponding to its dimeric form was observed, indicating formation of inter subunit disulfide bond. Electrophoretic separation showed both Alr4641C56S and Alr4641C178S to be present as monomers irrespective of the presence or absence of reducing agent (DTT) on SDS PAGE (Figure 3E). Tryptophan fluorescence spectra of the wild-type Alr4641, Alr4641C56S and Alr4641C178S proteins showed no shift in peak position at 340 nm suggesting that the absence of Cys residues does not alter the compactness of their structure (Additional file 2). Reduction of the wild-type Alr4641 did not alter the oligomeric state of the protein, but on treatment with H2O2, few smaller oligomers along with the major higher oligomeric form were observed on native PAGE (Figure 3F). As the oligomeric state of Alr4641 remained unchanged, it was of particular interest to analyze the chaperone/peroxidase function of Alr4641 after exposure to oxidizing or reducing agents. Results pertaining to these activities are described in the next two sections.
Alr4641 protein loses its chaperone function on reduction
Alr4641 protects plasmid DNA from oxidative damage and shows Trx/NTRC-dependent peroxidase activity
The NTRC protein from Anabaena was over-expressed in E. coli, purified by affinity chromatography (Figure 5D). Surface Plasmon Resonance (SPR) was employed to study interaction of Alr4641 with NTRC. The Alr4641 protein was immobilized on a bare gold sensor chip while NTRC was used in the mobile phase. The interaction between the two proteins was confirmed by a concentration-dependent increase in the SPR signal (Figure 5E). Equilibrium analysis showed a good Lorentz fit with the experimental values (Additional file 3) and the equilibrium constant (KD) was observed to be 1.037x10−6 ± 4.76x10−8 M. NTRC was also employed to evaluate the peroxidase activity of Alr4641 in the presence of different peroxidase substrates. Among the three substrates tested, best activity was observed with H2O2 followed by t-butyl hydroperoxide and cumeme hydroperoxide (Additional file 4). With increasing concentrations of H2O2, a reduction in the peroxidase activity was observed, indicating that excess H2O2 inactivated the Alr4641 protein (Figure 5F).
Alr4641 forms over-oxidized monomer in vivo under conditions of oxidative stress
Over-expression of Alr4641, but not Alr4641C56S, causes reduction in intracellular ROS generation on exposure to H2O2
Earlier, the NTRC protein was shown to physically interact with the purified Alr4641 protein (Figure 5E). In addition, capability of NTRC to associate with the Alr4641 protein from An4641+ cells free extracts was assessed by pull down experiments. As shown in Figure 7F, substantial amount of Alr4641 was bound when NTRC was immobilized on NiNTA agarose. In the absence of NTRC, hardly any Alr4641 bound to the empty resin (Figure 7F).
The intracellular levels of ROS in the wild-type Anabaena, An4641+ or AnC56S+ cells exposed to H2O2 for 1 h were assessed with the fluorogenic probe DCHFDA. Under control conditions, ROS levels were very low in all types of cells. However, on exposure to 1 mM H2O2, substantially higher levels of ROS were observed in the wild-type Anabaena PCC7120 and AnC56S+ cells as compared to the An4641+ cells (Figure 7G).
Over-production of Alr4641 protects the photosynthetic machinery and enhances survival in response to oxidative stress in Anabaena
PSII activity in Anabaena cultures treated with H 2 O 2
F v /F m
0.287333 ± 0.00585
0.195 ± 0
0.286 ± 0.018358
0.28275 ± 0.01345
Prxs form a phylogenetically ancient group of enzymes with a major role in detoxification of peroxides . Generally, Prxs show a moderate catalytic activity, but their high cellular content seems to compensate for their reduced efficiency in decomposing peroxides . It is believed that the antioxidant system in chloroplasts, organelle with highest content of Prxs in a plant cell, has evolved from cyanobacteria. Anabaena bears a resemblance to plant chloroplasts in being equipped with an oxidation sensitive 2-Cys-Prx (i.e. Alr4641) along with its reducing partner NTRC  and showing a low catalase activity [8,25].
The presence of the 0.9-knt alr4641 transcript (Figure 1) indicates that in spite of their adjacent location, the alr4642 ORF (642-bp, located 191-bp downstream of alr4641) and alr4641 are not co-transcribed, signifying that alr4641 forms a monocistronic operon. The promoter sequence found immediately upstream of this start site showed the presence of FurA (a transcriptional repressor) binding site (Figure 2) and the purified FurA protein from Anabaena bound the DNA fragment containing this sequence (Additional file 1). Gonzalez et al. have shown production of the Alr4641 protein to be reduced in the FurA-overexpressing Anabaena PCC7120. All these results imply that FurA regulates transcription of alr4641 in Anabaena .
In response to different abiotic stresses, the 2-Cys-prx gene transcript was enhanced in Synechococcus PCC7942 but not Synechocystis PCC6803 suggesting that transcriptional induction of 2-Cys-prx differs among cyanobacteria . In Anabaena, along with methyl viologen and H2O2, gamma radiation (a physical agent that causes oxidative stress) could also enhance production of the alr4641 transcript or the Alr4641 protein (Figure 1) . Interestingly, salinity as well as osmotic stress also increased production of the Alr4641 protein (Figure 1), indicating that Alr4641 may be a multiple stress induced protein. This is not unusual as several environmental stresses (salinity, drought, heavy metals, heat shock etc.) generate ROS , which in turn may ultimately enhance production of the Alr4641 protein.
Employing pull down assay and SPR analysis, NTRC was shown to physically interact with Alr4641 (Figures 5E, 7F) suggesting that NTRC is likely to be the physiological reductant of Alr4641 in Anabaena. In vivo, on treatment with H2O2, the recombinant AnC56S+ strain (that over-expresses Alr4641C56S) showed higher ROS levels compared to the An4641+ strain (that over-expresses the wild-type Alr4641) (Figure 7G). This underscores the importance of peroxidatic cysteine of Alr4641 for detoxification of H2O2 in vivo in Anabaena.
In general, the purified 2-Cys-Prx isolated from different organisms co-exists in various forms i.e. dimeric, decameric and high molecular wt. complexes . In contrast, in its native form, Alr4641 as well as its cysteine mutants appeared as decamers in vitro, (Figure 3) or in vivo (Figure 7E). Moreover, irrespective of the redox state (i.e. whether oxidized or reduced), Alr4641 appeared as decamer (Figure 3) indicating that the disulfide bonds are not involved in oligomerization.
Excess H2O2 over-oxidizes the peroxidatic cysteine, rendering Alr4641 incapable of peroxidase activity . Interestingly, high dose of γ-radiation could also over-oxidize the 2-Cys-Prx (Figure 6B) preventing the formation of disulfide bond. Post irradiation, on SDS PAGE, the monomeric form of Alr4641 disappeared and only the dimeric form could be seen after 24 h, indicating the reversion of the over-oxidised Alr4641protein to its normal form (Figure 6C). It is suggested that the recovery of the over-oxidized Alr4641 is facilitated by the sulfiredoxin (Srx) protein, which reverses the over-oxidation in vivo .
In Anabaena, depletion of combined nitrogen from medium leads to formation of heterocysts, the specialized cells that fix nitrogen . It should be noted that in spite of lowered O2 content, ROS are generated by photosystem I (PSI) and respiratory electron transport in heterocysts . Also, when the ratio of nitrogenase reductase to O2 is greater than 4, nitrogenase reductase reduces oxygen to H2O2 without being inactivated by oxygen . Thus the H2O2 produced in heterocysts has to be removed promptly. Prxs are known to be differentially distributed among the vegetative cells and heterocysts  and among the four PrxQ-like proteins (Alr3183, Alr2503, Alr2375 and Alr2556) from Anabaena, only Alr2375 was detected in heterocysts . In our study, promoter activity (P alr4641 -GFP fluorescence) as well the Alr4641 protein was observed in heterocysts as well as vegetative cells (Figure 2), indicating that it may play a role in detoxifying peroxides in vegetative cells and heterocysts.
Hydrogen peroxide (H2O2) is a commonly occurring reactive oxygen species (ROS) in biological systems and cyanobacteria have been shown to be generally more sensitive to H2O2 than other phototrophs . As Anabaena PCC7120 does not show good catalase activity [8,27], it is suggested that Prxs may be the principal components that detoxify H2O2 in this organism. H2O2 damages photosynthetic apparatus and severely affects Fv/Fm in several cyanobacteria . In the cyanobacterium Microcystis aeruginosa, treatment with H2O2 enhanced reactive oxygen species (ROS) accumulation, which caused destruction of pigment synthesis and led to cell death . Treatment of the wild-type Anabaena PCC7120 with H2O2 caused (a) enhanced levels of ROS (b) decrease in photosynthetic activities and (c) loss in viability (Figure 8). However, all the above-mentioned deleterious effects were alleviated in An4641+ strain, indicating that Alr4641 can protect Anabaena from oxidative stress.
The present study has identified Alr4641 as an abiotic stress induced protein that plays an important role in protecting Anabaena from oxidative stress. The Alr4641 protein was found to be unique from the other reported 2-Cys-Prxs i.e. the redox state and not its oligomerization status dictated the functional switch between the peroxidase or chaperone activity of this protein. Key attributes of Alr4641 like dual function, inherent transcriptional/translational induction under different stresses, localization in both vegetative cells and heterocysts, ability to use various reducing agents for detoxifying peroxides, reflect the versatile role played by the protein in Anabaena. The recombinant Anabaena strain over expressing Alr4641 exhibited higher tolerance to oxidative stress, thus establishing its potential to serve as stress-tolerant biofertilizers in paddy fields.
Organism and growth conditions
Anabaena PCC7120 cultures were grown in BG-11 liquid medium, pH 7.0 with combined nitrogen (17 mM NaNO3) under continuous illumination (30 μE m−2 s−1), with shaking (100 rpm) or without shaking (as still culture) at 27°C ± 2°C. Growth was assessed by monitoring the content of chlorophyll a mL−1 of culture volume . E. coli cells were grown in Luria-Bertani (LB) medium in the presence of appropriate antibiotics at 37°C with shaking at 150 rpm. The antibiotics used were 10 μg neomycin mL−1 (Nm10) in BG-11 liquid media and 25 μg neomycin mL−1 (Nm25) in BG-11 agar plates for recombinant Anabaena PCC 7120; and 34 μg chloramphenicol mL−1 (Cm34), 50 μg kanamycin mL−1 (Kan50) or 100 μg carbenicillin mL−1 (Cb100) for E. coli. The E. coli and Anabaena strains and plasmids used in the study are indicated in Additional file 5.
Cloning of alr4641, alr4641C56S alr4641C178S, furA and ntrc into pET16b
The alr4641 ORF was PCR amplified using gene-specific primers, alr4641fwd and alr4641rev, from Anabaena PCC7120 chromosomal DNA (Additional file 6). The PCR product (612-bp) obtained was digested with NdeI and BamHI and cloned into similarly digested pET16b (Additional file 5) to obtain plasmid pET4641. The pET4641 insert was sequenced to confirm the nucleotide sequence integrity of the cloned gene. A point mutation, leading to substitution of the Cys codon at positions 56 or 178 to Ser codon was introduced into the alr4641 ORF by PCR directed site-specific mutagenesis using overlapping PCR as described earlier . The PCR products (alr4641C56S and alr4641C178) containing the desired mutation were cloned into pET16b vector between the NdeI and BamHI restriction enzyme sites for over-expression in E. coli and named pET4641C56S and pET4641C178S respectively. The furA (all1691) or the ntrc (all0737) ORF was amplified with specific primers (described in Additional file 6) employing Anabaena PCC7120 genomic DNA as template. Restriction enzyme sites for NcoI and BamHI were incorporated in the forward and the reverse primers respectively. The reverse primer also had six His codons (shown in bold) followed by a stop codon. PCR product was purified, digested, and ligated to the NcoI-BamHI digested pET16b vector to give rise to pETFurA and pETNTRC respectively. All the resultant clones were confirmed by DNA sequencing.
Over-production and purification of recombinant proteins
Over-production of the His-tagged Alr4641, Alr4641C56S, Alr4641C178S, FurA and NTRC proteins in E. coli BL21pLysS and their subsequent purification was performed by affinity chromatography using Ni-NTA matrix as described earlier . The purified Alr4641 protein was also used to immunize rabbits for generating specific antiserum. The primary and booster immunizations and collection of the antiserum were performed at a commercial facility (Merck, India).
Size exclusion chromatography (SEC)
HPLC (AKTApure, USA) was performed using Superdex 200 10/300 GL column equilibrated at a flow rate of 0.5 ml min−1 at 25°C in Tris-buffer (20 mM, pH 7.2) containing NaCl (50 mM).
The chaperone activity of purified proteins was measured by using 1 μM malate dehydrogenase (MDH) as substrate in 50 mM HEPES-NaOH (pH 8.0) buffer at 55°C with various Prx concentrations (4:1, 2:1, 1:1, 1:2 MDH:Prx molar ratio). Turbidity (A360) due to substrate aggregation at 55°C was monitored in a spectrophotometer (JASCO, Japan) equipped with a thermostatic cell holder. When desired, the purified Alr4641/Alr4641C56S/Alr4641C178S proteins were individually treated with DTT (5 mM) or H2O2 (10 mM) for 10 min, passed through a desalting column and employed for the chaperone assay as described above. Assays were performed at least 4 times and representative curves are shown in the figure.
Peroxidase activity assay
For DTT dependent peroxidase assay, reaction mixture (1 ml) containing HEPES-NaOH (50 mM, pH 7.0) and desired concentrations of Prx proteins were pre-incubated with DTT (3 mM) for 10 min at 37°C, followed by the addition of H2O2 (200 μM). The reaction was stopped after 10 min by addition of TCA (10%, v/v). Subsequently, 0.2 volume of ferrous ammonium sulfate (10 mM) and 0.1 volume of potassium thiocyanate (2.5 M) were added. Absorbance of the red colored complex was spectrophotometrically measured at 480 nm. The amount of residual H2O2 remaining in the reaction was calculated from a standard calibration curve prepared by using known concentrations of H2O2. Trx-dependent peroxidase reactions were performed in a 50 μl reaction mixture containing HEPES-NaOH (50 mM, pH 7.0), E. coli thioredoxin A (TrxA, 5 μM), E. coli thioredoxin reductase (TR, 0.5 μM), NADPH (0.25 mM), Prx Protein or its mutant variants (0.05-1 μg), and H2O2 (200 μM). For GSH-dependent peroxidase activity, the typical reaction mixture contained HEPES/NaOH (50 mM, pH 7.0), NADPH (0.25 mM), glutathione reductase (GR, 0.2 μM), Prx protein (1 μg) and reduced glutathione (GSH, 5 mM). The reaction was started by addition of 100 μM peroxide substrate. The residual amount of peroxide was determined by ferrithiocyanate system as mentioned above. NTRC-dependent peroxidase activity of Alr4641 with different peroxide substrates was determined as described by Pascual et al. (2011) .
Protein electrophoresis, Western blotting and immunodetection
Purified Alr4641, Alr4641C56S and Alr4641C178S were resolved electrophoretically by 12% SDS-PAGE with or without DTT (10 mM). These three proteins were also resolved by native PAGE and stained with CBB. Total cellular proteins from Anabaena cultures were extracted using Laemmli’s buffer  and electrophoretically separated by 12% SDS-PAGE. The gel was electroblotted on to a nitrocellulose membrane as described earlier . In case of native PAGE, for efficient transfer, the gel was immersed in 1X SDS-PAGE running buffer, incubated at 70°C for 30 min and electroblotted on to the nitrocellulose membrane. Immunodetection was carried out with the Alr4641 antiserum.
Pull down assay
In the pull-down experiment, His-tagged NTRC (500 μg) was allowed to bind to Ni-NTA agarose slurry (100 μl) in assay buffer (50 mM Tris, 200 mM NaCl, 5 mM immidazole) for 2 h at 4°C, followed by washing with same buffer to remove the unbound NTRC. Cytosolic extract of An4641+ (800 μg protein) was incubated with NTRC-bound Ni-NTA agarose overnight at 4°C with constant rocking. In the control experiment, only Ni-NTA agarose was incubated with cytosolic fraction of An4641+. In both the cases, agarose was centrifuged at 5000 g for 5 min at 4°C, washed thrice with the assay buffer, boiled with cracking buffer and resolved on 12.5% SDS-PAGE. The gel was transferred on to a nitrocellulose membrane and probed with anti-Alr4641 antibody.
For co-immuno-precipitation, the His-tagged NTRC (50 μg) was allowed to incubate with His-tagged Alr4641 (50 μg) in co-immunoprecipitation buffer (50 mM Tris-Cl pH 7.5, 15 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 and protease inhibitor cocktail obtained from Sigma) at 4°C in duplicate. To one vial, Alr4641 antiserum was added and the components were allowed to interact for 6 h at 4°C with constant shaking. No antibody was added to the other vial (negative control). After that, a slurry of protein-G agarose beads (50 μl) was added to both the vials and these were kept shaking overnight at 4°C. Next day, beads were precipitated by centrifuging at 1200 g for 5 min at 4°C and washed thrice with the co-immuno-precipitation buffer. Subsequently, sample buffer was added to beads and the proteins extracted by boiling. The extracted proteins were separated on SDS-polyacrylamide (12.5%) gels, and visualized by staining.
Surface plasmon resonance (SPR) analysis
Autolab Esprit SPR system was used for surface plasmon resonance analysis with bare gold sensor chip. At 20°C, about 250 response units of Alr4641 was loaded onto the bare gold chip employing the EDC-NHS chemistry (Autolab ESPIRIT User manual SPR) followed by extensive washing with buffer H (10 mM HEPES, 100 mM NaCl, pH 7.5). Different concentrations (4.33, 8.66, 10.39 and 12.99 μM) of the NTRC protein were injected onto the Alr4641-bound sensor chip at 33.3 μL min−1 flow rate in independent experiments. The NTRC was allowed to interact with the immobilized Alr4641 for 300 s before washing off with buffer H. The data were processed and equilibrium constant (KD) was calculated using Autolab kinetic evaluation software (V5.4) provided with the instrument.
Gel retardation assays (GRA)
Primers 4641Prom_GRAFwd and 4641Prom_GRARev were annealed to form a 39-bp dsDNA (i.e. alr4641 promoter), which was used for the gel shift assays with the purified FurA protein. The end labeling of DNA fragments with digoxygenin (DIG) and the subsequent GRA, in the presence of the non-specific competitor poly (dI-dC), was performed as described by the manufacturer (Roche).
Metal catalyzed oxidation (MCO) assay for antioxidant activity
The plasmid DNA (pBluescript, 1 μg) was subjected to MCO by incubating FeCl3 (20 μM) and DTT (5 mM) at room temperature for 30 min. Purified proteins (2–20 μg) were added to the reaction mixture and further incubated for 4 h. Subsequently, DNA integrity was assessed by electrophoresis of reaction samples on agarose (0.8%, TBE, pH 7) gels.
Oxidation and reduction of the Alr4641 protein
The purified Alr4641 protein was oxidized with H2O2 (10 mM) for 30 min or reduced by addition of DTT (5 mM). The samples were analyzed for chaperone activity as described earlier or resolved on Native PAGE and visualized by CBB staining.
Northern blotting-hybridization and dot blot analysis
Isolation of Anabaena PCC7120 total RNA and subsequent Dot blot or Northern blotting-hybridization analysis with the alr4641 DIG-labeled DNA probe was performed as described earlier .
Rapid amplification of cDNA ends (RACE)
The total RNA isolated from the wild-type Anabaena PCC7120 cells stressed with H2O2, for 1 h was treated with DNase I and re-purified using commercial spin columns (Nucleospin RNA clean-up XS, Macherey Nagel). The reverse primer EXTERN-4641RACE-Rev (Additional file 6) was employed for cDNA synthesis. Tailing of cDNA using dATP and terminal transferase, the subsequent PCR with oligo dT-anchor primer and an internal gene-specific primer (INTER-4641RACE-Rev) was performed exactly as described (5’/3’ RACE kit, 2nd Generation, Roche).
Construction of GFP promoter
A 600-bp DNA fragment (upstream of the alr4641 gene) that contained the alr4641 promoter was amplified with suitable primers and cloned just upstream of gfp (reporter gene) in pAM1956 employing the restriction enzymes KpnI and SacI (construct named as pAM4641prom). This construct was conjugated into Anabaena PCC7120 and exconjugants (An4641 prom) were selected on BG-11/N+ plates containing neomycin (25 μg ml−1) and subjected to microscopic analysis.
Anabaena PCC7120 was grown aerobically in nitrogen-free BG-11 liquid medium. Heterocysts were isolated from whole filaments using modified protocol as described by Cha et al., 2007 . Anabaena culture was harvested and subjected to several freeze-thaw cycles in Tris-buffer (20 mM Tris, 1 mM EDTA, pH 7.4). After centrifugation for 5 min at 1000 g, the sedimented cells were suspended in the same buffer, glass beads (600 μM) were added and the suspension was vortexed for 2 min. Subsequently, the suspension was centrifuged at 150 g for 5 min to obtain heterocysts in the supernatant. Enrichment of greenish yellow heterocysts was achieved by repeatedly washing (7–8 times) and centrifuging (150 g, 5 min) the heterocyst pellet.
Construction of pAM4641 plasmid and over-expression of Alr4641 protein in Anabaena PCC7120
The alr4641 DNA fragment (~0.66-kb) from pET4641 was subcloned, downstream of the strong P psbA1 promoter, into the pFPN vector  employing the restriction enzymes NdeI and BamHI (plasmid called pFPN4641). Subsequently, the alr4641 gene along with the P psbA1 promoter was transferred as a SalI–XmaI fragment from pFPN4641 to appropriately digested E. coli/Anabaena shuttle vector pAM1956  to obtain pAM4641. Using a conjugal E. coli donor [HB101 (pRL623 + pRL443)], pAM4641 was conjugated into Anabaena PCC7120 as described earlier . Exconjugants were selected on BG-11/N+ plates containing neomycin (25 μg ml−1) and repeatedly subcultured. The transformed Anabaena strain thus obtained (designated An4641+) was maintained on BG-11/N+ plates containing neomycin.
The wild-type Anabaena PCC7120 and the recombinant An4641+ cells were subjected to 1 mM H2O2 treatment for 1 day. Culture aliquot (200 μl) of the above-mentioned Anabaena cells (5–6 μg ml−1 chlorophyll a) was incubated in white fluorescent light (24 W m−2) for 5 min and followed by addition of NaH14CO3 (20 mM, specific activity 0.5 mCi mmol−1). The reaction was stopped after 10 min by addition of 400 μl of 6 N acetic acid to the reaction mixture. The acid stable product was counted in a liquid scintillation counter with 0.4% BBOT [2, 5-bis (5-tert-butylbenzoxazole-2-yl) thiophen] dissolved in a solution containing toluene and absolute ethanol (v/v, 65:35). The experiment was performed twice with three replicate samples each time.
The content of the reactive oxygen species (ROS) in Anabaena strains treated with H2O2 for 1 day and in respective controls cells was measured with Dichlorodihydrofluorescein diacetate (DCHFDA) . Briefly, DCHFDA (10 μM final concentration) was added to cells suspended in BG-11 medium (3 μg chlorophyll a ml−1). Cells were incubated for 20 min in dark at 25°C. Fluorescence emission (λ ex = 490 nm and λ em = 520 nm) of the control or H2O2 (1 mM)-treated cells was measured immediately afterwards. Experiments were repeated thrice and average values are reported.
Determination of oxidative stress tolerance of An4641+ strain
Three-day-old Anabaena cultures of the wild-type Anabaena PCC7120 (WT) as well as An4641+ (in triplicates) were inoculated in a fresh growth medium at a chlorophyll a density of 3 μg ml−1 and subjected to H2O2 (1 mM) stress in tubes (without shaking) under illumination for 2 days. Growth was monitored in liquid cultures by determination of chlorophyll a content .
Amino acid sequence was analysed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or SMART (http://smart.embl-heidelberg.de/) algorithms [57,58]. Promoter, upstream of the transcriptional start site, was identified by a promoter search program (www.softberry.com). DNA-binding consensus sequence (GATAATGATAATCA TTATC) of the E. coli FurA protein was used to identify corresponding sequences from DNA upstream of the alr4641 ORF using the LALIGN program (www.ch.embnet.org).
We would like to thank Dr. S. K. Apte for constant encouragement and support during the course of this work. Expertise provided by Dr. (Mrs.) J. K. Sainis (for PAM Fluorimetry) and Dr. Subhash Bihani (for CD spectropolarimetry) is highly appreciated. Dr. Rachna Agarwal is gratefully acknowledged for helpful discussion during the preparation of the manuscript.
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