Physicochemical characterization of C-phycocyanin from Plectonema sp. and elucidation of its bioactive potential through in silico approach.

C-phycocyanin (C-PC), the integral blue-green algae (BGA) constituent has been substantially delineated for its biological attributes. Numerous reports have illustrated differential extraction and purification techniques for C-PC, however, there exists paucity in a broadly accepted process of its isolation. In the present study, we reported a highly selective C-PC purification and characterization method from nontoxic, filamentous and non-heterocystous cyanobacterium Plectonema sp. C-PC was extracted by freeze-thawing, desalted and purified using ion-exchange chromatography. The purity of C-PC along with its concentration was found to be 4.12 and 245 µg/ml respectively.  Comparative characterization of standard and purified C-PC was performed using diverse spectroscopic techniques namely Ultra Violet-visible, fluorescence spectroscopy and Fourier transform infrared (FT-IR). Sharp peaks at 620 nm and 350 nm with UV-visible and FT-IR spectroscopy respectively, confirmed amide I bands at around 1638 cm-1 (C=O stretching) whereas circular dichroism (CD) spectra exhibited α-helix content of secondary structure of standard 80.59% and 84.59% of column purified C-PC. SDS-PAGE exhibited two bands of α and ß subunits 17 and 19 kDa respectively. HPLC evaluation of purified C-PC also indicated a close resemblance of retention peak time (1.465 min, 1.234 min, 1.097 min and 0.905 min) with standard C-PC having retention peak timing of 1.448 min, 1.233 min and 0.925 min. As a cautious approach, the purified C-PC was further lyophilized to extend its shelf life as compared to its liquid isoform. To evaluate the bioactive potential of the purified C-PC in silico approach was attempted. The molecular docking technique was carried out of C-PC as a ligand-protein with free radicals and α-amylase, α-glucosidase, glycogen synthase kinase-3 and glycogen phosphorylase enzymes as receptors to predict the free radical scavenging (antioxidant) and to target antidiabetic property of C-PC. In both receptors free radicals and enzymes, ligand C-PC plays an important role in establishing interactions within the cavity of active sites. These results established the antioxidant potential of C-PC and also give a clue towards its antidiabetic potential warranting further research.

Untargeted metabolomics of the alkaliphilic cyanobacterium Plectonema terebrans elucidated novel stress-responsive metabolic modulations.

Alkaliphilic cyanobacteria are suitable candidates to study the effect of alkaline wastewater cultivation on molecular metabolic responses. In the present study, the impact of wastewater, alkalinity, and alkaline wastewater cultivation was studied on the biomass production, biochemical composition, and the alkalinity responsive molecular mechanism through metabolomics. The results suggested a 1.29 to 1.44-fold higher biomass production along with improved lipid, carbohydrate, and pigment production under alkaline wastewater cultivation. The metabolomics analysis showed 1.2-fold and 5.54-fold increase in the indole-acetic acid and phytoene biosynthesis which contributed to overall enhanced cell differentiation and photo-protectiveness. Furthermore, lower levels of Ribulose-1,5-bisphosphate (RuBP), and higher levels of 2-phosphoglycerate and 3-phosphoglycerate suggested the efficient fixation of CO2 into biomass, and storage compounds including polysaccharides, lipids, and sterols. Interestingly, except L-histidine and L-phenylalanine, all the metabolites related to protein biosynthesis were downregulated in response to wastewater and alkaline wastewater cultivation. The cells protected themselves from alkalinity and nutrient stress by improving the biosynthesis of sterols, non-toxic antioxidants, and osmo-protectants. Alkaline wastewater cultivation regulated the activation of carbon concentration mechanism (CCM), glycolysis, fatty-acid biosynthesis, and shikimate pathway. The data revealed the importance of alkaline wastewater cultivation for improved CO2 fixation, wastewater treatment, and producing valuable bioproducts including phytoene, Lyso PC 18:0, and sterols. These metabolic pathways could be future targets of metabolic engineering for improving biomass and metabolite production. SIGNIFICANCE: Alkalinity is an imperative factor, responsible for the contamination control and biochemical regulation in cyanobactera, especially during the wastewater cultivation. Currently, understanding of alkaline wastewater responsive molecular mechanism is lacking and most of the studies are focused on transcriptomics of model organisms for this purpose. In this study, untargeted metabolomics was employed to analyze the impact of wastewater and alkaline wastewater on the growth, CO2 assimilation, nutrient uptake, and associated metabolic modulations of the alkaliphilic cyanobacterium Plectonema terebrans BERC10. Results unveiled that alkaline wastewater cultivation regulated the activation of carbon concentration mechanism (CCM), glycolysis, fatty-acid biosynthesis, and shikimate pathway. It indicated the feasibility of alkaline wastewater as promising low-cost media for cyanobacterium cultivation. The identified stress-responsive pathways could be future genetic targets for strain improvement.

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria.

Leptolyngbya boryana (Plectonema boryanum) is a diazotrophic cyanobacterium lacking heterocysts. How nitrogen fixation is regulated in filamentous nonheterocystous cyanobacteria remains unclear. Here we describe a large 50-kb nitrogen fixation (nif) gene cluster in L. boryana containing 50 genes. This gene cluster contains 14 nif genes (nifBSUHDKVZT and nifPENXW), two genes encoding transcriptional regulators showing high similarity to ChlR (chlorophyll regulator) and PatB, three genes encoding ferredoxin, three genes encoding cytochrome oxidase subunits, and 28 genes encoding nif-related proteins and proteins with putative or unknown functions. Eleven mutants lacking one gene or a subset of genes were isolated. Five of them did not grow under diazotrophic conditions, including two mutants lacking the transcriptional regulators. Although the chlR homolog-lacking mutant showed a normal level of nitrogenase activity, various intermediates of chlorophyll biosynthesis were accumulated under micro-oxic conditions. The phenotype suggested that ChlR activates the expression of the genes responsible for anaerobic chlorophyll biosynthesis to support energy supply for nitrogen fixation. In another mutant lacking the patB homolog, no transcripts of any nif genes were detected under nitrogen fixation conditions, which was consistent with no activity. Constitutive expression of patB in a shuttle vector resulted in low but significant nitrogenase activity even under nitrate-replete conditions, suggesting that the PatB homolog is the master regulator of nitrogen fixation. We propose to rename the patB homolog as cnfR, after cyanobacterial nitrogen fixation regulator.

Hydrogen generation through indirect biophotolysis in batch cultures of the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum.

The nitrogen-fixing nonheterocystous cyanobacterium Plectonema boryanum was used as a model organism to study hydrogen generation by indirect biophotolysis in nitrogen-limited batch cultures that were continuously illuminated and sparged with argon/CO(2) to maintain anaerobiosis. The highest hydrogen-production rate (i.e., 0.18 mL/mg day or 7.3 micromol/mg day) was observed in cultures with an initial medium nitrate concentration of 1 mM at a light intensity of 100 micromol/m(2) s. The addition of photosystem II (PSII) inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not reduce hydrogen-production rates relative to unchallenged controls for 50 to 150 h, and intracellular glycogen concentrations decreased significantly during the hydrogen generation period. The insensitivity of the hydrogen-production process to DCMU is indicative of the fact that hydrogen was not derived from water splitting at PSII (i.e., direct biophotolysis) but rather from electrons provided by intracellular glycogen reserves (i.e., indirect biophotolysis). It was shown that hydrogen generation could be sustained for long time periods by subjecting the cultures to alternating cycles of aerobic, nitrogen-limited growth and anaerobic hydrogen production.

Oxygen sensitivity of a nitrogenase-like protochlorophyllide reductase from the cyanobacterium Leptolyngbya boryana.

Dark-operative protochlorophyllide (Pchlide) oxido-reductase (DPOR) is a nitrogenase-like enzyme that catalyzes Pchlide reduction, the penultimate step of chlorophyll a biosynthesis. DPOR is distributed widely among oxygenic phototrophs such as cyanobacteria, green algae and gymnosperms. To determine how DPOR operates in oxygenic photosynthetic cells, we constructed two shuttle vectors for overexpression of Strep-tagged L-protein (ChlL) and Strep-tagged NB-protein (ChlN-ChlB) in Leptolyngbya boryana (formerly Plectonema boryanum) and introduced them into mutants lacking chlL and chlB. Both transformants restored the ability to produce chlorophyll in the dark. The DPOR activity was reconstituted by L-protein and NB-protein purified from the transformants under anaerobic conditions. L-protein activity disappeared within 5 min of exposure to air while NB-protein activity persisted for >30 min in an aerobic condition, indicating that the L-protein of DPOR components is the primary target of oxygen in cyanobacterial cells. These results suggested that the DPOR from an oxygenic photosynthetic organism did not acquire oxygen tolerance during evolution; but that the cyanobacterial cell developed a mechanism to protect DPOR from oxygen.

Chromatin fiber structure and plectonemic model of chromosome condensation in Drosophila cells.

Reversible permeable cells have been used to isolate chromatin structures during the process of chromosome condensation. Analysis of individual structures slipping out from nuclei after reversal of permeabilization revealed that chromosomes of Drosophila cells consist of small units called rodlets. The fluorescent images of chromatin fibers were subjected to computer analysis allowing the computer-aided visualization of chromatin fibers. The zig-zag array of fibers consisting of 12-15 nucleosomes with a length of 270-330 nm (average 300 nm) showed decondensed extended strings, condensed loops, and coiled condensed loops. Theoretical considerations leading to the plectonemic model of chromatin condensation are based on experimental data, and give an explanation how the 30 chromatin fibers are formed and further condensed to the 300 nm chromatin loops in Drosophila cells.

Dissolution of dead corals by euendolithic microorganisms across the northern Great Barrier Reef (Australia).

Spatial and temporal variabilities in species composition, abundance, distribution, and bioeroding activity of euendolithic microorganisms were investigated in experimental blocks of the massive coral Porites along an inshore-offshore transect across the northern Great Barrier Reef (Australia) over a 3-year period. Inshore reefs showed turbid and eutrophic waters, whereas the offshore reefs were characterized by oligotrophic waters. The euendolithic microorganisms and their ecological characteristics were studied using techniques of microscopy, petrographic sections, and image analysis. Results showed that euendolithic communities found in blocks of coral were mature. These communities were dominated by the chlorophyte Ostreobium quekettii, the cyanobacterium Plectonema terebrans, and fungi. O. quekettii was found to be the principal agent of microbioerosion, responsible for 70-90% of carbonate removal. In the offshore reefs, this oligophotic chlorophyte showed extensive systems of filaments that penetrated deep inside coral skeletons (up to 4.1 mm) eroding as much as 1 kg CaCO3 eroded m(-2) year(-1). The percentage of colonization by euendolithic filaments at the surface of blocks did not vary significantly among sites, while their depths of penetration, especially that of O. quekettii (0.6-4.1 mm), increased significantly and gradually with the distance from the shore. Rates of microbioerosion (0.1-1.4 kg m(-2) after 1 year and 0.2-1.3 kg m(-2) after 3 years of exposure) showed a pattern similar to the one found for the depth of penetration of O. quekettii filaments. Accordingly, oligotrophic reefs had the highest rates ofmicrobioerosion ofup to 1.3 kg m(-2) year(-1), whereas the development of euendolithic communities in inshore reefs appeared to be limited by turbidity, high sedimentation rates, and low grazing pressure (rates < 0.5 kg m(-2) after 3 years). Those results suggest that boring microorganisms, including O. quekettii, have a significant impact on the overall calcium carbonate budget of coral reef ecosystems, which varies according to environmental conditions.

Synthesis of palladium nanoparticles by reaction of filamentous cyanobacterial biomass with a palladium(II) chloride complex.

The interaction of cyanobacterial biomass (Plectonema boryanum UTEX 485) with aqueous palladium(II) chloride (PdCl2 degrees ) has been investigated at 25-100 degrees C for up to 28 days. We report that the release of organic materials from the cyanobacteria promoted the precipitation of Pd(0) as crystalline spherical and elongate nanoparticles (< or =30 nm), both in solution and as dispersed and encrusted nanoparticles on cyanobacterial cells. In contrast, under abiotic conditions at 100 degrees C, palladium hydride (PdHx) was the principal palladium phase precipitated, with only minor amounts of palladium metal.

Solenoids and plectonemes in stretched and twisted elastomeric filaments.

We study the behavior of a naturally straight highly extensible elastic filament subjected to large extensional and twisting strains. We find that two different phases can coexist for a range of parameter values: the plectoneme and the solenoid. A simple theory based on a neo-Hookean model for the material of the filament and accounting for the slender geometry suffices to explain these observations, and leads to a phase diagram that is consistent with observations. Extension and relaxation experiments on these phases show the presence of large hysteresis loops and sawtooth-like force-displacement curves which are different for the plectoneme and the solenoid.

Common freshwater cyanobacteria grow in 100% CO2.

Cyanobacteria and similar organisms produced most of the oxygen found in Earth's atmosphere, which implies that early photosynthetic organisms would have lived in an atmosphere that was rich in CO2 and poor in O2. We investigated the tolerance of several cyanobacteria to very high (>20 kPa) concentrations of atmospheric CO2. Cultures of Synechococcus PCC7942, Synechocystis PCC7942, Plectonema boryanum, and Anabaena sp. were grown in liquid culture sparged with CO2-enriched air. All four strains grew when transferred from ambient CO2 to 20 kPa partial pressure of CO2 (pCO2), but none of them tolerated direct transfer to 40 kPa pCO2. Synechococcus and Anabaena survived 101 kPa (100%) pCO2 when pressure was gradually increased by 15 kPa per day, and Plectonema actively grew under these conditions. All four strains grew in an anoxic atmosphere of 5 kPa pCO2 in N2. Strains that were sensitive to high CO2 were also sensitive to low initial pH (pH 5-6). However, low pH in itself was not sufficient to prevent growth. Although mechanisms of damage and survival are still under investigation, we have shown that modern cyanobacteria can survive under Earth's primordial conditions and that cyanobacteria-like organisms could have flourished under conditions on early Mars, which probably had an atmosphere similar to early Earth's.

Macromolecule metabolism and photosynthetic functions in blue-green algae treated with virginiamycin, an inhibitor of protein synthesis.

The M component of virginiamycin inhibited growth of Plectonema boryanum under both photoautotrophic and heterotrophic conditions. Though the S component of this antibiotic had no apparent activity per se, it enhanced the inhibitory action of its partner. Cells incubated with suitable concentrations of either M or M + S stopped growing and lysed. Loss of the colony-forming capacity occurred quickly in the presence of M + S and slowly in the presence of M alone. Virginiamycin M inhibited protein synthesis in autotrophically and heterotrophically growing Plectonema. This effect was very rapid and could be reversed by removing the antibiotic. The S component did not block the incorporation of amino acids into proteins, but prevented the reversibility of the inhibitory effect of M. Virginiamycin M or S did not affect the photosynthetic oxygen development (Hill's reaction) in Plectonema. Moreover, carbon dioxide photoassimilation and formation of chlorophyll were inhibited only after an appreciable lag. Deoxyribonucleic acid synthesis was blocked virtually without delay by virginiamycin M. Since virginiamycin inhibited protein synthesis in a similar fashion in the unicellular Anacystis nidulans, as well as in the filamentous P. boryanum, the mechanism of action of this antibiotic is probably the same in all blue-green algae.