N2-fixing Cyanobacterium Anabaena Research Articles

Isolation, biosynthesis and antimicrobial activity of gold nanoparticles produced with extracts of Anabaena spiroides.

Multidrug-resistant (MDR) pathogenic bacteria have become dangerous in bringing sporadic outbreaks in public health and nosocomial spreads from the addition of antibacterials/antibiotics continually. Obviously, the pharmacy world is in search of antibacterials that would be invincible by the evolved bacteria. Green synthesis of gold-nanoparticles (AuNps) was focused on the use of aqueous chloroauric acid (HAuCl4) and cell-free aqueous extract of the N2-fixing cyanobacterium (blue-green alga) Anabaena spiroides collected from a brackish-water, Bay of Bengal at Puri, Odisha; green-synthesized AuNps could be used as antibacterials against MDR bacteria. The synthesized AuNps were subjected to the following characterizations, UV-Vis spectrophotometry, SEM-EDX, XRD and ART-FTIR analysis. An absorption peak at 538nm by UV-Vis spectrophotometry and the FTIR analysis confirmed the presence of AuNps. A. spiroides-AuNps were monitored for antibacterial activities against MDR pathogenic bacterial strains isolated from clinical samples, namely, Klebsiella oxytoca, MRSA and Streptococcus pyogenes, in vitro; the individual antibiograms of those bacteria were known. The recorded MIC dose values were 25, 20 and 30mg A. spiroides-AuNps (As-AuNps) against K. oxytoca, MRSA and S. pyogenes, in vitro, respectively. Thus, As-AuNps bear promises as possible antibacterials, in future.

Maximize Growth and Productivity of Rice by Using N2-Fixing Anabaena oryzae and Spirulina platensis Extract

This study highlights the role of both inoculation with N2-Fixing cyanobacterium Anabaena oryzae and spraying with cyanobacterium Spirulina platensis extract and their combinations on growth and productivity of the two rice varieties (Sakha108 and Giza178) under different doses of nitrogen fertilizers. The experiment was laid out in a split - plot design with four replications; the two rice varieties were located in the main plots and fertilizer treatments were placed in the sub-plots. Chlorophyll content of flag leaf, plant height, number of panicle/m2 and panicle length, number of filled grain/panicle, one thousand grain weight, straw and grain yields (t/ha) were measured. Bioactive compounds and amino acid production of Spirulina were determined by GC–MC and amino acid analyzer, respectively. The results show Spirulina extract contains eighteen amino acid and several bioactive molecules and application of mixture Anabaena oryzae and Spirulina platensis extract with 75% recommended dose of nitrogen or recommended dose of N fertilizer gave nearly the highest value of growth characters, grain yield and yield components compared with other treatments in both seasons. This study describes the role of Anabaena oryzae and spraying with Spirulina platensis extract through plant-microbe interactions and the effect of this reaction on the growth and productivity of rice crop.

Identification of two genes required for heptadecane production in a N2-fixing cyanobacterium Anabaena sp. strain PCC 7120

Cyanobacteria photosynthetically produce long-chain hydrocarbons, which are considered as infrastructure-compatible biofuels. However, native cyanobacteria do not produce these hydrocarbons at sufficient rates or yields to warrant commercial deployment. This research sought to identify specific genes required for photosynthetic production of alkanes to enable future metabolic engineering for commercially viable production of alkanes. The two putative genes (alr5283 and alr5284) required for long-chain hydrocarbon production in Anabaena sp. PCC 7120 were knocked out through a double crossover approach. The knockout mutant abolished the production of heptadecane (C17H36). The mutant is able to be complemented by a plasmid bearing the two genes along with their native promoters only. The complemented mutant restored photosynthetic production of heptadecane. This combined genetic and metabolite (alkanes) profiling approach may be broadly applicable to characterization of knockout mutants, using N2-fixing cyanobacteria as a cellular factory driven by solar energy to produce a wide range of commodity chemicals and drop-in-fuels from atmospheric gases (CO2 and N2 gas) and mineralized water.

Effect of Metal Cofactors of Key Enzymes on Biohydrogen Production by Nitrogen Fixing Cyanobacterium Anabaena siamensis TISIR 8012

In N2-fixing cyanobacteria, three enzymes are involved in the H2 metabolism. Nitrogenase catalyzes the N2 fixation which produces H2 as a by-product. The produced H2 is taken up to protons and electrons by an activity of uptake hydrogenase. Reversible enzyme catalyzes both reactions of the H2 evolution and the H2 uptake. These enzymes are all metalloenzyme. The cyanobacterial nitrogenase normally requires molybdenum and iron as cofactors; however nitrogenase of few cyanobacterial species is dependent on vanadium. The cyanobacterial uptake and reversible hydrogenase requires nickel and iron as cofactors. This research aimed to investigate the effect of these metal cofactors on H2 production and hydrogenase activity by N2-fixing cyanobacterium Anabaena siamensis TISTR 8012 isolated from rice paddle field in Thailand. The result showed that A. siamensis cells incubated in N-deprived BG11 medium (BG110) gave clearly higher H2 production rate and hydrogenase activity than those in normal BG11 medium. Under nitrogen deprivation, an increase of iron, nickel, and molybdenum concentrations obviously enhanced H2 production rate. But only higher iron concentrations increased hydrogenase activity, indicating that the iron metal assisted in the function of reversible hydrogenase activity. In addition, vanadium seemed not to be a metal cofactor of key enzymes involving in H2 production in A. siamensis. The optimal concentrations of iron, nickel and molybdenum ions for H2 production rate by A. siamensis were 60 μM, 4 μM and 4 μM, respectively. The highest H2 production rate of 0.057 µmolH2 mg chl a-1 h-1 was observed in cells incubated in BG110 medium supplemented with 4 μM nickel ion.

Increasing Hydrogen Production Efficiency of N2-Fixing Cyanobacterium Anabaena siamensis TISTR 8012 by Cell Immobilization

H2 produced by cyanobacteria is one of the interesting alternative energy carriers for the future. The filamentous N2-fixing cyanobacterium Anabaena siamensis TISTR 8012 is a potential microorganism for H2 production. It can produce H2 via both photosynthesis and nitrogen fixation processes. This study aimed to increase the efficiency of H2 production by immobilization of A. siamensis cells. The result showed that H2 production rate by A. siamensis grown in BG110 medium was higher than that in BG11 and Allen-Arnon media due to an increase of the heterocyst cells. The sulfur deprivation during adaptation period for 24 hours increased its H2 production rate. The optimal conditions for H2 production by immobilized cells were immobilization with alginate and using 150 gel beads in 20 mL glass vial. In immobilized cells, an addition of 0.5% fructose resulted in a 2-fold increase of H2 production rate. Finally, the reducing agents β–mercaptoethanol and methylviologen enhanced H2 production rate with 3.092 and 2.426 µmolH2 mg chl a-1 h-1, respectively, in A. siamensis immobilized cells whereas NADH, dithiothreitol and sodium dithionite were not capable of increasing H2 production rate.

Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120

Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N(2)) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N(2)-fixing cyanobacteria. In this report, five putative AspAT genes (alr1039, all2340, alr2765, all4327 and alr4853) were identified in the N(2)-fixing heterocystous cyanobacterium Anabaena sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H(6)) were overexpressed in Escherichia coli and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity in vitro using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed in vitro. Alr4853-H(6) showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and alr4853 was further identified to be a Fox gene (requiring fixed N(2) for growth in the presence of oxygen). Four out of five P(aspAT)-gfp transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that alr4853 expression was increased by 2.3-fold after 24 h of N(2) deprivation. Taken together, these findings add to our understanding of the role of AspATs in N(2)-fixing within heterocystous cyanobacteria.

Enhanced Resistance to UV-B Radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by Repeated Exposure

In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N2-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats.

Improving conversion efficiency of solar energy to electricity in cyanobacterial PEMFC by high levels of photo-H2 production

In this study, we described an efficient electrical power generating system containing cyanobacterial photo-H2 production and custom-built proton exchange membrane fuel cell (PEMFC). The filamentous N2-fixing cyanobacterium Anabaena cylindrica was used as the photo-H2 producer. A photosynthesis inhibitor-diuron (DCMU) was used for the enhancement of photo-H2 production in the culture under argon gas. For the first time, a total of 1.0 μM DCMU was found to be the most effective treatment, as this produced 3.6 fold higher levels of H2 in microalgae. By measuring polarization curve, the gas mixture collected from the culture was proven to be an effective fuel for electrical generation through a custom-built PEMFC. When the PEMFC was directly combined with the culture tube, the cells generated as much as 843 mV during a 5-day incubation due to the efficient conversion of solar energy to H2 by A. cylindrica. Light energy conversion efficiency (LCE) for both solar energy to H2 and solar energy to electricity were also determined. The LCE for the cyanobacterial conversion of solar energy to H2 reached a peak at four days with a maximal value of 2.05% and an average value of 1.70% ± 0.17. The corresponding LCE for the conversion of solar energy to electricity in this system was 1.13% at peak and 0.94% ± 0.09 on average.

Nucleotide sequencing and transcriptional analysis of uptake hydrogenase genes in the filamentous N2-fixing cyanobacterium Anabaena siamensis

The hupSL encoding uptake hydrogenase of the N2-fixing cyanobacterium Anabaena siamensis was cloned and sequenced. Anabaena siamensis hupS and hupL contained 963 and 1,596 bp, respectively. The predicted gene products for hupS and hupL consisted of 320 amino acids with a molecular mass of 35,436 Da and 531 amino acids with a molecular mass of 60,704 Da, respectively. Their deduced amino acid sequences showed higher than 90% and 88% similarity for HupS and HupL respectively, compared to those of other cyanobacteria including non N2-fixing cyanobacteria. The intergenic 195-bp sequence between hupS and hupL showed no homology to others among cyanobacteria but it still contained the repetitive sequence, which could form a hairpin structure commonly found in other N2-fixing cyanobacteria. RT-PCR analysis showed higher transcription level of hupS and hupL in Anabaena siamensis grown in BG110 under anaerobic conditions. Nickel exogenously added to the culture was also found to increase hupL transcript.

HstK, a cyanobacterial protein with both a serine/threonine kinase domain and a histidine kinase domain: implication for the mechanism of signal transduction

Two distinct families of protein kinases are involved in signal transduction: Ser, Thr and Tyr kinases, which are predominantly found among eukaryotes, and His kinases, as part of bacterial two-component signalling systems. Genetic studies in Arabidopsis and Saccharomyces have demonstrated that bacterial-type two-component systems may act upstream of Ser/Thr kinases in the same signalling pathway, but how this coupling is accomplished remains unclear. In the present study, we report the characterization of a protein kinase, HstK, from the N2-fixing cyanobacterium Anabaena sp. PCC 7120, that possesses both a Ser/Thr kinase domain and a His kinase domain. Proteins with a structural architecture similar to that of HstK can be found in the eukaryote, Schizosaccharomyces pombe, and the bacterium, Rhodococcus sp. M5. HstK was present in cells grown with NH4+ or N2 as the nitrogen source, but was absent in cells grown with NO3−. The hstK gene was inactivated and the mutant phenotype was characterized. The catalytic domain of the Ser/Thr kinase of HstK functionally replaced that of Hog1p, a well-characterized protein kinase required for the response to high osmolarity in the S. cerevisiae heterologous system. The unusual multidomain structure of HstK suggests that a two-component system could be directly coupled to Ser/Thr kinases in the same signal transduction pathway.

Interactive effects of UV-B and heavy metals (Cu and Pb) on nitrogen and phosphorus metabolism of a N2-fixing cyanobacterium Anabaena doliolum

This study demonstrated a general decrease in uptake of NH4+, urea and PO43−, activities of nitrogenase, glutamine synthetase, urease, alkaline phosphatase and ATPase following ultraviolet-B (UV-B) treatment and metal exposure, separately. As compared with an individual stressor the effect of their combination was more detrimental to the above processes. In contrast, a significant increase in NO3− uptake and nitrate reductase activity, following exposure of Anabaena doliolum to UV-B was observed. Kinetic study of all the processes demonstrated that the UV-B-induced structural change(s) in the enzymes/carriers could be responsible for uptake and assimilation of these nutrients. However, the metals seem to compete for the binding sites of the above enzymes/carriers. A synergistic interaction of UV-B and metals (Cu/Pb) seems to be due to an increased metal uptake by an altered membrane permeability, as a result of peroxidation of membrane lipids in UV-B exposed cells.

Short- and long-term effects of ammonium on photodependent nitrogen fixation in wetland rice fields of Spain

Short- and long-term experiments were conducted in the rice fields of Valencia, Spain, to determine the ecological significance of ammonium on nitrogen fixation. A significant inhibition of nitrogenase activity by ammonium, at concentrations higher than 0.5mM, was observed after 8h of incubation in short-term experiments done with a bloom of the N2-fixing cyanobacterium Anabaena sp. In a second set of short-term experiments for in situ assays of nitrogenase activity in the field, a significant correlation between nitrogenase activity and the number of N2-fixing cyanobacteria in soil was found. No significant inhibition of nitrogenase activity by ammonium at concentrations up to 2mM was observed in these assays after 24h of incubation. This lack of inhibition was probably due to the rapid decrease in ammonium content in the flood water. Only 5% of the ammonium initially added remained in the water 24h later. In the long-term experiments, nitrogenase activity was assayed in plots fertilized with 0, 70 and 140kgNha–1, over the cultivation cycle, for 5 years. A partial inhibition of nitrogenase activity by deep-placed N fertilizers was observed. Differences were only significant in 2 years. Mean results from 5 years only showed significant differences between plots fertilized with 0 and 140kgNha–1. The partial inhibition of nitrogenase activity by ammonium increased over the cultivation cycle. Inhibition was only significant in September, at the end of the cultivation cycle.

Lipid content and fatty acid composition in N2-fixing cyanobacterium Anabaena doliolum as affected by molybdenum deficiency

Anabaena doliolum grown under molybdenum deficiency produced less biomass (on a dry wt basis) and the cells had lower protein content but higher carbohydrate content than Mo-grown material. Molybdenum deficiency led to a slight decrease in chlorophyll a, a 1.5-fold increase in carotenoids and a 1.4-fold increase in total lipid but there was no difference in the lipid profiles of Mo-enriched and Mo-deficient cells. Molybdenum deficiency caused increases in the cell contents of digalactosyl diacylglycerol and phosphatidylglycerol and decreases in monogalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol lipids. The concentration of unsaturated C18 fatty acids was lower in the Mo-deficient cells.

Heterocyst envelope thickness, heterocyst frequency and nitrogenase activity in Anabaena flos-aquae: influence of exogenous oxygen tension

SUMMARY: The heterocyst envelope of the N2-fixing cyanobacterium Anabaena flos-aquae thickened as exogenous O2 partial pressure (pO2) was increased from 5 to 40 kPa. The majority of the thickening occurred in the glycolipid layer area of the envelope. Such thickening appears to be an O2-induced mechanism for providing a greater O2 diffusion barrier against O2 inhibition of nitrogenase. Nitrogenase activity decreased at pO2 levels above ambient (20 kPa), indicating that a thicker envelope is not completely effective as a barrier to O2 diffusion. However, when cultures grown at 10 kPa and 40 kPa pO2 were transferred to ambient pO2, the 40 kPa pO2 cells showed higher nitrogenase activity 24 h after transfer compared to those grown at 10 kPa or ambient pO2, indicating O2-protection of nitrogenase by thicker heterocyst envelopes. Heterocyst frequency was lowest at 20 kPa O2.

Response ofAnabaena doliolum to bimetallic combinations of Cu, Ni and Fe with special reference to sequential addition

This study reports physiological features of a N2-fixing cyanobacteriumAnabaena doliolum in response to metal mixtures. Exposure of the cyanobacterium to Cu, Ni and Fe individually, as well as in combinations (Cu + Ni, Cu + Fe, Ni + Fe), showed marked differences in growth inhibition, nutrient uptake (NH4+ and NO3−), photosynthesis, ATP content, nitrate reductase, glutamine synthetase and urease activities. The response to metal combinations was also dependent upon the order in which the metals were added. The Cu-Ni combination resulted in synergistic interaction, in contrast to the antagonism of Cu-Fe and Ni-Fe. Pre-addition of Fe protected the cyanobacterium against Cu and Ni toxicity. Statistically significant (P < 0.005) inhibition of all the processes following metal supplementation was observed. This study suggests that carbon fixation is the most suitable variable for assessing heavy metal toxicity.

Localization of ribulose 1,5-bisphosphate carboxylase/oxygenase in the N2-fixing cyanobacterium Anabaena cylindrica

Localization of ribulose 1,5-bisphosphate carboxylase/oxygenase in the N2-fixing cyanobacterium Anabaena cylindrica

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica.

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.

Thioredoxin as a Modulator of Glucose-6-phosphate Dehydrogenase in a N2-Fixing Cyanobacterium

Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme involved in fixed carbon dissimilation in photosynthetic micro-organisms; in heterocystous cyanobacteria it may also be implicated in the supply of reductant to nitrogenase. In crude cell-free extracts of the N2-fixing cyanobacterium Anabaena variabilis G6PDH activity was reversibly deactivated by the thiol agent dithiothreitol in the presence of a low molecular weight protein (12000mol. wt). Glucose 6-phosphate reversed deactivation when added at high concentration, or prevented deactivation if added with the thiol. NADP+, which, like glucose 6-phosphate, is a G6PDH substrate, also deactivated the enzyme; deactivation was reversed or prevented by adding glucose 6-phosphate or glutamine. Purified thioredoxin from Anabaena cylindrica, at very low concentrations (2 nm), deactivated purified G6PDH in a manner identical to that observed when crude extracts were used in the presence of dithiothreitol. Glutathione did not affect the enzyme.

Structural features of calcium alginate entrapped cyanobacteria modified for ammonia production

The photosynthetic N2-fixing cyanobacterium Anabaena ATCC 27893 was immobilized by entrapment in beads of calcium alginate gel and used in a three-phase fluidised bed reactor for NH 4 + production from air, light and water. NH 4 + release from the beads was induced by adding L-methionine-DL-sulphoximine (MSX) which inhibits glutamine synthetase, the primary NH 4 + assimilating enzyme. The morphological features of the beads and entrapped cyanobacterium have been examined by light and scanning electron microscopy before, during and after the period of NH 4 + production. The cyanobacterium appeared morphologically normal throughout the 792 h experimental period with its filamentous form and heterocyst content being sustained throught. Our results indicate that Anabaena ATCC 27893 is well able to withstand prolonged entrapment and varying experimental conditions while entrapped.

Sustained ammonia production by immobilized filaments of the nitrogen-fixing cyanobacterium Anabaena 27893

Whole filaments of the N2-fixing cyanobacterium Anabaena ATCC 27893 have been immobilized by entrapment in calcium alginate gel beads. In a continuous flow fluidized bed reactor sustained photosynthesis, N2-fixation, and ammonia production have been achieved over a 130 hour period, the longest tested.