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 3rd 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 [21–23] (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 3rd 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).