ACHIEVEMENTS

Molecular characterization of marine cyanobacteria:
India is one of the major marine biodiversity “hotspots” of the world with a vast coastal area of @7700 km. The diverse organisms of “hotspots” should be conserved and catalogued for prosterity. Cyanobacteria, the unique group of oxygenic photoautotrophic prokaryotes possess extensive morphological diversity ranging from single celled, colonial to differentiated multicellular forms with branching patterns. Due to their wide diversity and adaptability, they could survive in varied marine ecological niches such as estuaries, coral reefs, mangroves, salt pans, backwaters, rocky shores and sandy shores. The cyanobacterial strains of various Indian coastal areas are catalogued and conserved for various biotechnological purposes in our repository. Traditionally, cyanobacteria are classified on the basis of morphological approach but certain morphological criteria used for classification have only ecotypic value which leads to misinterpretation of identification. Hence the identification has also to be carried out at the genetic level using molecular approach which offers the possibility to estimate the biodiversity upto species level. Precise identification of cyanobacterial strains in our germplasm and to optimize the accuracy of taxonomic identification is most vital. Since NFMC has a rich marine microalgal collection of @630 strains, we are concerned in the development of foolproof identification method for cyanobacterial taxa using 3 molecular markers such as 16S rRNA, 16-23S ITS region and cpc regions along with the traditional morphological method. Gene details sequenced so far were accessible in Gene sequencing portal of NFMC website (http://www.nfmc.res.in).
Comparative analysis of cyanobacterial superoxide dismutases, a canonical form
Superoxide dismutases (SOD) are ubiquitous metalloenzymes that catalyze the disproportion of superoxide to peroxide and molecular oxygen through alternate oxidation and reduction of their metal ions. In general, SODs are classified into four forms by their catalytic metals namely; FeSOD, MnSOD, Cu/ZnSOD and NiSOD. In addition, a cambialistic form that uses Fe/Mn in its active site also exists. Cyanobacteria, the oxygen evolving photosynthetic prokaryotes, produce reactive oxygen species that can damage cellular components leading to cell death. Thus, the co-evolution of an antioxidant system was necessary for the survival of photosynthetic organisms with SOD as the initial enzyme evolved to alleviate the toxic effect. Cyanobacteria represent the first oxygenic photoautotrophs and their SOD sequences available in the databases lack clear annotation.
Our in silico analysis on the sequence conservation and structural analysis of Fe (Thermosynechococcus elongatus BP1) and MnSOD (Anabaena sp. PCC7120) reveal the sharing of N and C terminal domains. At the C terminal domain, the metal binding motif in cyanoprokaryotes is DVWEHAYY while it is D-X-[WF]-E-H-[STA]-[FY]-[FY] in other pro- and eukaryotes. The cyanobacterial FeSOD differs from MnSOD at least in three ways viz.
  1. FeSOD has a metal specific signature F184X3A188Q189.......T280......F/Y303 while, in Mn it is R184X3G188G189......G280......W303,
  2. aspartate ligand forms a hydrogen bond from the active site with the outer sphere residue of W243 in Fe where as it is Q262 in MnSOD; and
  3. two unique lysine residues at positions 201 and 255 with a photosynthetic role, found only in FeSOD.
Further, most of the cyanobacterial Mn metalloforms have a specific transmembrane hydrophobic pocket that distinguishes FeSOD from Mn isoform. Cyanobacterial Cu/ZnSOD has a copper domain and two different signatures G-F-H-[ILV]-H-x-[NGT]-[GPDA]-[SQK]-C and G-[GA]-G-G-[AEG]-R-[FIL]-[AG]- C-G, while Ni isoform has an nickel containing SOD domain containing a Ni-hook HCDGPCVYDPA.
Our study or the first time have unraveled the ambiguity among cyanobacterial SOD isoforms. NiSOD is the only SOD found in lower forms; whereas, Fe and Mn occupy the higher orders of cyanobacteria. Cyanobacteria harbor either Ni alone or a combination of Fe and Ni or Fe and Mn as their catalytic active metal while Cu/Zn is rare and cambialistic form is absent. SOD is important, which produces OH radical and H2O2, which helps in decolourization of effluent. Thereby SOD is involved in killing pathogens in sewage.

The active site residues of Fe Superoxide dismutase of Thermosynechococcus elonagtus. (PDB: 1gv3).
The active site residues of Mn Superoxide dismutase of Anabaena sp. ( PDB: 1my6)

Publication:
1. Priya B, Premanandh J, Dhanalakshmi, Seethalakshmi, D, Prabaharan D, and Uma L. (2007) Comparative analysis of cyanobacterial SOD a canonical form , BMC Genomics Vol :8; 458 (4.03))
2. Balakrishnan Priya, Reddi K Sivaprasanth, Vincent Divya Jensi, Lakshmanan Uma, Gopalakrishnan Subramanian, Dharmar Prabaharan, (2010). Characterization of manganese superoxide dismutase from a marine cyanobacterium. Leptolyngbya valderiana BDU20041. Saline Systems 6:6 (3.09).
Siderophore mediated uranium sequestration
Increasing contamination of the environment by uranium on account of its mining and disposal of tailings is a worldwide problem. Microbial interactions with metals form an important part of the natural biogeochemical processes and have important consequences for human society. It is therefore, vital to advance our understanding of the metal-microbe interactions that may include physical and chemical adsorption, ion exchange co ordination, complexation, chelation and micro-precipitation in order to develop suitable bioremediation strategies for metal contaminated sites. Among microbes, cyanobacteria represent a morphologically diverse group of oxygenic, gram-negative photosynthetic prokaryotes, which are widely distributed in freshwater, marine and terrestrial environments. These organisms respond and adapt to most stress conditions and are often abundant in metal contaminated environments. Siderophores, constitute a major class of naturally occurring chelators that includes hydroxamate, catecholate, and carboxylic acid functional groups secreted by microorganisms in various habitats, which bind to iron and mediate its transport to the cell. These metal chaperones are majorly specific for iron. Inspite of its specification for iron complexation, marine cyanobacterium S.elongatus BDU130911 was evaluated for siderophore production and its specificity to complex with uranium through in vitro and in silico analysis. Wet lab studies corroborate the siderophore production in marine cyanobacterium S.elongatus BDU130911 and were identified as hydroxamate type. Also hydroxamate siderophore complexation with uranium was estimated to be 50% by Chrome Azurol S modified assay. In order to substantiate wet lab analysis, in silico docking was performed between Desferroxamine (a standard hydroxamate type) with Fe (III) and UO22+. Docking studies validates the hydroxamate siderophore to bind effectively with Fe (III) and UO22+and their binding affinity constant remains > 2Aº. This finding was the first report in marine cyanobacteria to elucidate uranium siderophore complexation through in vitro and in silico analysis.
Desferroxamine - Fe(III)

Desferroxamine - UO22+

Publication:
Rashmi, V., ShylajaNaciyar M., Rajalakshmi,R., D'Souza, S.F., Prabaharan,D., Uma, L., 2013. Siderophore mediated uranium sequestration by marine cyanobacterium Synechococcus elongatus BDU 130911. Bioresour. Technol. 130: 204-210.
Marine cyanobacterial carbon sequestration
Rapidly growing concern on global warming is attributed to the elevated CO2 in the atmosphere and has instigated the necessity to find an efficient way to mitigate carbon dioxide. Currently, CO2 can be sequestered by chemical, biological and geological methods. Biological carbon sequestration has gained attention as it results in the production of biomass and energy. Cyanobacteria the oxy - phototrophs possess much higher growth rate and ability to fix CO2 while capturing the solar energy with much greater efficiency over the terrestrial plants. Having evolved at CO2 rich atmospheres, these organisms are plausible candidates for carbon sequestration. Eighteen organisms representing three different orders have been screened from the National Facility for Marine Cyanobacteria for their carbon dioxide tolerance. The selected plausible strain was grown with continuous flow of carbon dioxide at concentration near to flue gas. The major carbon fixing enzymes of C3 and C4 cycles are also being studied for their role in carbon fixation at higher concentration of CO2. In silico analysis of CO2 fixing enzymes Phosphoenol pyruvate carboxylase and Ribulose 1,5 bis phosphate carboxylase/oxygenase untangles the ambiguity among cyanobacteria which can be up-regulated for high CO2 fixing capabilities. Our current research throws light upon calcifying potentials of the cyanobacteria at continuous carbon dioxide in order to convert CO2 to insoluable form as CaCO3.

Experimental setup for growing cyanobacteria at its substrate continuous CO2


docking pose of PEPCase with phosphoenolpyruvate
Glutathione S-Trasnferases
Cyanobacteria will serve as promising bioremediator to clean up the environment. It evolved with catabolic potential that eliminates numerous natural and synthetic compounds that are considered infallible. Glutathione S- Transferase (GST) is a multifunctional phase II detoxification enzyme that act as a first line of defense against chemically induced toxicity in all pro and eukaryotes. The current research performs a genome wide hunt to portray the presence of GST enzymes, types and its evolution and it also comprehend the binding affinity and expression toward xenobiotics compounds. Cyanobacteria, a primordial organism to harbor GSTs, give an ample understanding on immense potential of cyanobacterial glutathione S-transferases towards pollution abatement.
(i) GST - Endosulfan docking pose

(ii) GST - Chi

(iii)GST - Omega
Marine green alga as a potential candidate for biodiesel production
Globally, biodiesel is a firm expanding industry that is facing a growing dilemma of exploring feedstock. There are many options in this area, but unlike solar, nuclear, and fossil fuels, biodiesel have the capability of providing a fuel source ideally suited to replace fossil fuel and fulfil the existing demand. Microalgae are tiny sun light driven cell factories having been projected as one of the most promising feedstock for biodiesel production since they accumulate oil and exhibits faster growth compared to other energy crops without competing for arable land. Our research on microalgal biodiesel production was initiated with ten marine green algae from different geographical regions of south east coast in Tamil Nadu. Chlorella sp. BDU G91771 possessed approximately 23% lipid content. Biodiesel production is feasible only if hyper lipid producing promising strain is used. With this context, high lipid Chlorella sp. BDUG 91771 by external stimuli has been carried out. Out door mass cultivation to the tune of 5KL in an economic medium is also tried. Additionally, overexpressing genes responsible for enhancing lipid content in microalgae is being carried out using bioinformatics approach.
Marine cyanobacteria - the plausible fourth generation bioenergy feedstock
Green chemistry proposes to synthesize environmentally benign compounds with the utilization of renewable feedstocks and develops design for energy efficiency. Cyanobacteria the versatile oxygenic photosynthetic microbes were found in oil bubbles in quartz dated 1000 million years ago and are the potent fourth generation biodiesel feedstock over the conventional plant feed crops. The rich cyanobacterial diversity of National Facility for Marine Cyanobacteria representing 19 genera and 41 species has widened the arena of bioenergy research. A vast number of 250 cyanobacterial strains have been screened for their lipid production and has identified about 12 strains with a high lipid yield of 15 % and above. The fatty acid profile of these strains revealed the incidence of middle to long chain fatty acids, the prerequisite for biodiesel. Our current research focusses on the fatty acid metabolic pathway in cyanobacteria and emphases in the comprehensive analysis of key regulatory enzymes involved in the up regulation of fatty acid biosynthesis - towards the production of better quality biodiesel.
Temperature response study of psychrophilic and mesophilic cyanobacteria
Cyanobacterial diazotrophs play a major role in biogeochemical cycling of carbon and nitrogen in tundra ecosystem. The microbial and biological processes in tundra ecosystem are slower when compared to other ecosystems due to low soil temperature. Cyanobacteria have an inherent ability to fix atmospheric nitrogen and carbon in varied thermal extremes including Antarctic lake ice where the temperature is always below 0º C and in hot spring mats where the temperature is about 55º C. Thus, the cyanobacteria comprises of psychrophilic, psychrotolerant, mesophilic and thermophilic strains showing wide thermal adaptability. The survey of microalgal flora in the polar region Ny-Alesund, (76º53’E), Svalbard, Spitsbergen during Arctic summer IV expedition (2011-12) resulted in psychrophilic germplasm comprising about 65 psychrophilic cyanobacteria and 40 psychrophilic green algae maintained at 4 ± 2ºC and 15 ± 2ºC. As we have virtuous prosperity of both the psychrophilic and mesophilic strains, research to explore the physiological response and nitrogen metabolising abilities of psychrophilic and mesophilic strains at different temperatures regimes to understand their essential role in C and N cycle in tundra ecosystem is on.

Home         NFMC       Bioinformatics centre       Marine Biotechnology     Contact us