Cyanobacteria, the oxygen-evolving photosynthetic prokaryote originated ~3.5 billion years ago, occupy a credential position between pro- and eukaryotes 1 . The resultant tandem operation of two photosystems is now known as oxygenic or plant-type photosynthesis 1 . This marked the turning point in the evolution of earth, opening up the era for aerobes. For the survival of cyanobacteria with oxygenic photosynthesis, the selection pressure led to the evolution of SODs 2 3 4 5 6 7 . Cyanobacteria are well documented for its ability to maintain the antioxidant levels by releasing H₂O₂ into the environment 2 3 4 5 . The prime armory for the release of H₂O₂ is superoxide dismutases. The first implication on the protective role of cyanobacterial SOD in photo-oxidative damage was shown in Anacystis nidulans 6 .

SODs are generally classified according to their metal species which acts as redox-active center to catalyze the dismutation reaction O₂ ^- + 2H⁺ → O₂ + H₂O₂. Cu/ZnSOD type consists of Cu(II) and Zn(II) at the active site, MnSOD has Mn(III), the third FeSOD possess Fe(III), and a fourth NiSOD contains Ni(II/III). Generally SODs are localization specific. MnSOD is found in the cytosol and thylakoid membrane, whereas Fe and NiSOD in the cytosol and Cu/Zn is periplasmic in location but cyanobacteria have multiple forms of each type encoded by more than one gene 7 . The existence of multiple SODs may result from the fact that the cells of cyanobacteria are divided into compartments by internal membranes 8 . Since O₂ ^- ions are negatively charged and cannot cross the phospholipids bilayer readily, they are efficiently trapped within the compartment where they are generated. This may have been selected for the evolution of multiple SODs in compartmentalized cells 9 .

Different levels of expressions in terms of SOD isoenzymes resulted in response to oxidative stress caused by various abiotic factors such as, pesticides 10 , lignin and its model dye 11 and nutrient limitation 12 particularly of Fe/MnSOD. In addition, our hitherto reports on marine cyanobacterium, Leptolyngbya valderiana BDU 20041 on exposure to synthetic dye has shown that the organism decolourized Acid black1, C.I. 20470 (100 mG L^-1) to 85.6% in 12 days 13 14 . The active moiety involved in decolourization was adjudged to be AOS, mainly H₂O₂ (40 μM of H₂O₂ mG^-1 Chl h^-1) which is excreted by the organism into the milieu 14 . The SOD activity was found to be induced by two-fold in the presence of dye suggesting a significant role in the production of H₂O₂ 14 .

In the present study, we proposed to analyze the role of SOD in L. valderiana BDU 20041 during oxidative stress at the molecular level in order to more precisely identify its isoform. Hence, an attempt was made to PCR amplify, sequence and identify the metalloform of the SOD from the marine cyanobacterium, L. valderiana BDU20041 of Indian subcontinent.

ROS are repeatedly reported to be the inevitable by-products of biological redox reaction and normal metabolism in humans, animals, plants and algae 15 . Cyanobacteria are oxygenic photosynthetic organisms that are prone to the oxidative stress due to the facts that they contain an array of photosynthetic pigments and that they both produce oxygen during photosynthesis and consume oxygen during respiration. It has been estimated that 1% of O₂ consumed by cells is diverted to produce ROS in various subcellular loci 16 .

The increased production of ROS and the resultant oxidative stress are considered to be the initial event and act as an alert signal for the organisms under several environmental stresses, such as light, temperature and UV. Inevitably, ROS also react with biologically important molecules such as lipids, proteins and DNA, inducing oxidative damage in membranes and the photosynthetic apparatus, which probably results in the death of cells 15 . Here we focus on synthetic dye induced oxidative stress and oxidative damage in cyanobacteria.

In the present study, the dye dosed stress response of L. valderiana BDU20041 was tested in nitrogen free ASN III medium for a stipulated period of 12 days. The response to stress was determined by the organism's decolourizing efficiency which varied among the duration tested (Figure 1). This shows that decolourization was only due to the metabolic activity of the organisms and not an abiotic factor. The results compliance with the decolourization studies by cyanobacteria 17 18 .

Earlier reports deciphers that oxidative stress induces formation of ROS by multiple pathways 15 19 . Firstly, the photosynthetic pigments such as chlorophylls and phycobilins in cyanobacteria that act as photosensitizers (PS) under stress 15 . It is noteworthy that the energy of excited chlorophylls or phycobilins is utilized efficiently for photosynthesis under normal growth conditions. However, the inhibition of photosynthesis or the electron transport chain under stress may elevate the photosensitization process as well as the formation of ROS in this way.

Secondly, the essential role of the photosynthetic electron transport chain in the life of cyanobacteria promotes the possibility of subjection to oxidative stress. The probable electron transfer from the electron transport chain, especially in photosystem I (PSI), to molecular oxygen, the way to quench extensive excitation energy, is an alternative source of ROS. Photoreduction of molecular oxygen by the primary electron acceptor in the PSI complex is thought to be the main source of superoxide in illuminated cells 19 .

Hence in the present study, experiments were carried out to evaluate the above phenomena as a response of L. valderiana BDU20041 to oxidative stress caused by C.I. Acid Black 1.

First, dye treated L. valderiana BDU20041 showed increase in chlorophyll a proportional to the rate of decolourization (Figure 2) which suggests that dye hinders the availability of light for the photosynthetic machinery. In addition, there was a slight variation in the phycobilins of dye untreated and treated L. valderiana BDU20041 at the end of 12 days (Figure 3). These changes in chlorophyll and phycobilins during decolourization corroborate with the first phenomena mentioned above on the organism's potential to strive against oxidative stress caused by C.I. Acid Black 1.

The second notable feature is the total ROS produced in stress response which is represented as 2', 7'- Dichorofluorescein (DCF) with relative fluorescence intensity at 520 nm. Generally, ROS levels increase in response to abiotic stress 20 . Decreased levels of ROS initially in the 3^rd h with L. valderiana BDU20041 dosed with dye may be due to its involvement in degradation whereas, within 24 h, the organism increases the rate of ROS production and showed sustenance (Figure 4).

The initial decrease in total ROS in dye dosed culture could be attributed to two reasons (i) role of ROS in dye decolourization 21 22 23 (ii) the free radical generated passes through the Asada-Halliwell pathway initiated by SOD that converts the highly reactive oxy-radicals through an array of reaction to less toxic forms 19 24 . Further, the sustenance of ROS clearly depicts the stress response in L. valderiana BDU20041 as cyanobacteria maintain their antioxidants level by release of reactive oxygen species into the milieu 5 24 . Studies with eukaryotic and bacterial systems showed that, low levels of AOS are indispensable to act in cellular signaling and in the control of gene expression 25 . Because of the dual functions of ROS, a tight control of their concentrations may be anticipated, which requires a delicate balance of systems involved in their generation and destruction 26 .

Hence, the third significant trait studied was the response of SOD enzyme to oxidative stress (in this case, C.I. Acid Black 1). SOD activity was found to increase in the 3^rd h and on further exposure to dye, the activity increased two-fold and got sustained thereafter (Figure 5). This obviously proves that SOD plays a significant role in alleviating oxidative stress in L. valderiana BDU20041.

As the initial oxy-radical product to be formed under any oxidative stress is the superoxide radical (O₂ ^-.) which upon further reaction within the cell can generate more ROS such as hydroxyl radicals and singlet oxygen. Superoxide dismutases are metalloenzymes that dismutase these superoxide radical to hydrogen peroxides. Their activity increases on exposure to oxidative stress 16 . Hence, they can be depicted as the central dogma of antioxidative system. Hence, in our study attempts was made to isolate and characterize the SOD gene involved in abating oxidative stress caused by C.I. Acid Black 1.

The SOD gene from the DNA isolated of marine cyanobacterium, L. valderiana BDU20041 was amplified using the below mentioned primers. Electrophoresis of the amplified products of gradient PCR (61°C) showed a band of 550 bp and none in negative control (Figure 6). The nucleotide sequence of the partial SOD gene of 432 bp was submitted to GenBank database (AY974247) and their deduced aminoacid was 144 residues (AAX84682) (Figure 7). The computational analysis on the sequenced partial SOD gene comprised of an N-terminal and a C-terminal region from 1 to 63 and 70 to 143 residues respectively with a theoretical molecular weight of 15.5 KDa.

When the obtained sequence was aligned within cyanobacterial SOD, MnSOD of Thermosynechococcus elongates BP1 (BAC07589) and Leptolyngbya boryana (P50058) had maximum homology of 47%. The least homology of 43% was shared with Leptolyngbya boryana (P50056). The homology difference could be attributable to partial SOD gene, in particular C-terminal region where most of the aminoacids are highly conserved residues along with motif region (DVWEHAYY).

Further, the isolated sequence on analysis (Figure 8) showed the presence of first 3 residues of SOD motif region DVWEHAYY (D₂₈₁-W₂₂₃) along with conserved residues-glycine (G₅₃), histidine (H₅₅), phenylalanine (F₆₁), serine (S₁₀₅), tryptophan (W₁₀₈), leucine (L₁₀₉), arginine (N₁₂₅) and glutamine (Q₁₂₆). These highly conserved residues were found to be precise for cyanobacterial MnSOD as described by Priya et al 7 .

All living systems have only one of each type of SOD in the various cellular compartments indicating that they have far more complex antioxidant defenses than other organisms. As per the findings of Parker and Blake 27 , Jackson and Copper 28 and Priya et al 7 our analysis of gene sequence of L. valderiana BDU20041 shows that the residues histidine (H₄, H₅₈) and aspartate (D₁₄₁) plays a role in active sites (Figure 8, 9). In addition, the presence of important metal specific residues viz., glycine (G₅₄), isoleucine (I₈₁), glycine (G₁₀₆), tryptophan (W₁₀₇) and proline (P₁₂₉) concludes that the metalloform isolated from L. valderiana BDU20041 is that of Mn (Figure 8, 9).

The elucidated structure possesses 6 helices and 3 sheets (Figure 10). Homology modeling with MnSOD of L. valderiana BDU 20041 showed a similarity of 63.76% with Bacillus anthracis (1XUQ) and 60.98% with Deinococcus radiodurans (1Y67) (Figure 11). The RMSD value of superposed structure shows that the alpha carbons are at 0.88A and backbone carbon is at 0.85. This further substantiates that the isolated SOD gene is MnSOD with its co-coordinating residues at position His₄, His₅₈ and Asp₁₄₁ (Figure 9).

The radial neighbor-joining (NJ) analysis of all cyanobacterial SODs from public database (NCBI/DDBJ/EMBL) showed four distinct metalloforms viz., Mn, Fe, Cu/Zn and NiSODs. The SOD gene from L. valderiana BDU 20041 is found to be grouped with MnSODs (Figure 12).

The identified MnSOD indicates that it is one of the most probable means through which marine cyanobacterium, L. valderiana BDU20041 alleviates oxidative stress caused by abiotic factors (C.I. Acid Black 1).

The authors declare that they have no competing interests.

BP and KRS carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. VDJ helped in carrying out the structural comparison. GS helped in fine tuning of the manuscript. LU and DP conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.