Cyanobacterium Anabaena Torulosa Research Articles

Inorganic polyphosphate accumulation protects a marine, filamentous cyanobacterium, Anabaena torulosa against uranium toxicity

The intricate dynamics of inorganic polyphosphate (polyP) in response to phosphorus (P) limitation and metal exposure typical of contaminated aquatic environments is poorly understood. Cyanobacteria are important primary producers in aquatic environments that are exposed to P stringency as well as metal contamination. There is a growing concern regarding migration of uranium, generated as a result of anthropogenic activities, into the aquatic environments owing to high mobility and solubility of stable aqueous complexes of uranyl ions. The polyP metabolism in cyanobacteria in context of uranium (U) exposure under P limitation has hardly been explored. In this study, we analyzed the polyP dynamics in a marine, filamentous cyanobacterium Anabaena torulosa under combination of variable phosphate concentrations (overplus and deficient) and uranyl exposure conditions typical of marine environments. Polyphosphate accumulation (polyP+) or deficient (polyP−) conditions were physiologically synthesized in the A. torulosa cultures and were ascertained by (a) toulidine blue staining followed by their visualization using bright field microscopy and (b) scanning electron microscopy in combination with energy dispersive X-ray spectroscopy (SEM/EDX). On exposure to 100 μM of uranyl carbonate at pH 7.8, it was observed that the growth of polyP+ cells under phosphate limitation was hardly affected and these cells exhibited larger amounts of uranium binding as compared to polyP− cells of A. torulosa. In contrast, the polyP− cells displayed extensive lysis when exposed to similar U exposure. Our findings suggest that polyP accumulation played an important role in conferring uranium tolerance in the marine cyanobacterium, A. torulosa. The polyP-mediated uranium tolerance and binding could serve as a suitable strategy for remediation of uranium contamination in aquatic environments.

Priming maize seeds with cyanobacteria enhances seed vigour and plant growth in elite maize inbreds.

Biofilm formation of a nitrogen-fixing cyanobacterium Anabaena torulosa with a beneficial fungus Trichoderma viride (An-Tr) was examined under laboratory conditions. A gradual enhancement in growth over A. torulosa alone was recorded in the biofilm, with 15-20% higher values in nitrogen fixation, IAA and exopolysaccharide production illustrating the synergism among the partners in the biofilm. To investigate the role of such biofilms in priming seed attributes, mesocosm studies using primed seeds of two maize inbred lines (V6, V7) were undertaken. Beneficial effects of biofilm (An-Tr) were recorded, as compared to uninoculated treatment and cyanobacterial consortium (Anabaena-Nostoc; BF 1-4) at both stages (7 and 21 DAS, days after sowing) with a significant increase of more than 20% in seedling attributes, along with 5-15% increment in seed enzyme activities. More than three- to fivefold higher values in nitrogen fixation and C-N mobilizing enzyme activities, and significant increases in leaf chlorophyll, proteins and PEP carboxylase activity were observed with V7-An-Tr biofilm. Cyanobacterial inoculation brought about distinct changes in the soil phospholipid fatty acid profiles (PLFA); particularly, significant changes in those representing eukaryotes and anaerobic bacteria. Principal component analyses illustrated the significant role of dehydrogenase activity and microbial biomass carbon and distinct elicited effects on soil microbial communities, as evidenced by the PLFA. This investigation highlighted the promise of cyanobacteria as valuable priming options to improve mobilization of nutrients at seed stage, modulating the abundance and activities of various soil microbial communities, thereby, enhanced plant growth and vigour of maize plants.

Unusual Versatility of the Filamentous, Diazotrophic Cyanobacterium Anabaena torulosa Revealed for Its Survival during Prolonged Uranium Exposure.

Reports on interactions between cyanobacteria and uranyl carbonate are rare. Here, we present an interesting succession of the metabolic responses employed by a marine, filamentous, diazotrophic cyanobacterium, Anabaena torulosa for its survival following prolonged exposure to uranyl carbonate extending up to 384 h at pH 7.8 under phosphate-limited conditions. The cells sequestered uranium (U) within polyphosphates on initial exposure to 100 μM uranyl carbonate for 24 to 28 h. Further incubation until 120 h resulted in (i) significant degradation of cellular polyphosphates causing extensive chlorosis and cell lysis, (ii) akinete differentiation followed by (iii) extracellular uranyl precipitation. X-ray diffraction (XRD) analysis, fluorescence spectroscopy, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy established the identity of the bioprecipitated uranium as a U(VI) autunite-type mineral, which settled at the bottom of the vessel. Surprisingly, A. torulosa cells resurfaced as small green flakes typical of actively growing colonies on top of the test solutions within 192 to 240 h of U exposure. A consolidated investigation using kinetics, microscopy, and physiological and biochemical analyses suggested a role of inducible alkaline phosphatase activity of cell aggregates/akinetes in facilitating the germination of akinetes leading to substantial regeneration of A. torulosa by 384 h of uranyl incubation. The biomineralized uranium appeared to be stable following cell regeneration. Altogether, our results reveal novel insights into the survival mechanism adopted by A. torulosa to resist sustained uranium toxicity under phosphate-limited oxic conditions.IMPORTANCE Long-term effects of uranyl exposure in cyanobacteria under oxic phosphate-limited conditions have been inadequately explored. We conducted a comprehensive examination of the metabolic responses displayed by a marine cyanobacterium, Anabaena torulosa, to cope with prolonged exposure to uranyl carbonate at pH 7.8 under phosphate limitation. Our results highlight distinct adaptive mechanisms harbored by this cyanobacterium that enabled its natural regeneration following extensive cell lysis and uranium biomineralization under sustained uranium exposure. Such complex interactions between environmental microbes such as Anabaena torulosa and uranium over a broader time range advance our understanding on the impact of microbial processes on uranium biogeochemistry.

Deciphering the biochemical spectrum of novel cyanobacterium-based biofilms for use as inoculants

An investigation was done using cyanobacterium Anabaena torulosa as a matrix for developing novel biofilms, with anti-grazer traits against microfauna/pathogenic fungi, through co-inoculation of agriculturally important bacteria and fungi. The biofilms generated were evaluated after 4, 6 and 9 weeks of incubation for the activity of hydrolytic enzymes and fungicidal activity against phytopathogenic fungi. The activity of β-1,3-glucanase, in general, showed a gradual increasing trend up to 9 weeks, while endoglucanase activity was highest after 6 weeks of incubation, and a 40–50% reduction in chitosanase activity was recorded by the end of 9 weeks of incubation. Observations revealed that the fungus–cyanobacterium biofilms, especially Anabaena–Aspergillus awamori, exhibited the highest activity of β-1,3-glucanase and ranked second in terms of chitosanase activity. Fungicidal activity was recorded up to 9 weeks in most of the biofilms, and the highest values were recorded in cyanobacterium–Bacillus and cyanobacterium–fungus biofilms. Such biofilms were also tested against selected nematodes in microcosm experiments, which revealed no significant deleterious effects. The biocontrol activity of such biofilmed preparations against phytopathogenic fungi, but not towards selected nematodes, illustrates their promise in agriculture as potential inoculants that can effectively establish in soil.

Performance of a Cyanobacteria Whole Cell-Based Fluorescence Biosensor for Heavy Metal and Pesticide Detection

Whole cell biosensors always face the challenge of low stability of biological components and short storage life. This paper reports the effects of poly(2-hydroxyethyl methacrylate) (pHEMA) immobilization on a whole cell fluorescence biosensor for the detection of heavy metals (Cu, Pb, Cd), and pesticides (dichlorophenoxyacetic acid (2,4-D), and chlorpyrifos). The biosensor was produced by entrapping the cyanobacterium Anabaena torulosa on a cellulose membrane, followed by applying a layer of pHEMA, and attaching it to a well. The well was then fixed to an optical probe which was connected to a fluorescence spectrophotometer and an electronic reader. The optimization of the biosensor using several factors such as amount of HEMA and drying temperature were undertaken. The detection limits of biosensor without pHEMA for Cu, Cd, Pb, 2,4-D and chlorpyrifos were 1.195, 0.027, 0.0100, 0.025 and 0.025 μg/L respectively. The presence of pHEMA increased the limits of detection to 1.410, 0.250, 0.500, 0.235 and 0.117 μg/L respectively. pHEMA is known to enhance the reproducibility of the biosensor with average relative standard deviation (RSD) of ±1.76% for all the pollutants tested, 48% better than the biosensor without pHEMA (RSD = ±3.73%). In storability test with Cu 5 μg/L, the biosensor with pHEMA performed 11.5% better than the test without pHEMA on day-10 and 5.2% better on day-25. pHEMA is therefore a good candidate to be used in whole cell biosensors as it increases reproducibility and enhances biosensor storability.

Evaluating the influence of novel cyanobacterial biofilmed biofertilizers on soil fertility and plant nutrition in wheat

Microbial inoculants suffer from the major problem of poor survival in rhizospheric soil and their plant growth-promoting abilities are dependent upon their competence in this niche. An interesting observation regarding the biofilms, which urged us to evaluate them with crop, was the increased nitrogen-fixing potential or ARA (Acetylene Reduction Activity), even after 10 and 24 weeks of incubation under laboratory conditions. Our present investigation was therefore aimed toward evaluating novel biofilmed preparations developed using cyanobacterium Anabaena torulosa as a matrix for agriculturally useful bacteria (Azotobacter, Mesorhizobium, Serratia and Pseudomonas) in wheat crop. The performance of such phototroph–heterotroph biofilmed preparations was evaluated using individual cyanobacterium, available bacterial inoculants and dual cultures of the partners. Comparison of biofilms was undertaken using RP (Rock phosphate) for phosphate solubilizers –Serratia, Pseudomonas and their combinations only. Nitrogen-fixers –Azotobacter, Mesorhizobium and their combinations, including biofilmed preparations were compared using SSP (single super phosphate) as phosphate source. An enhancement in ARA in the range of 40–50% was recorded in the treatments inoculated with biofilms even after 14 weeks of inoculation, as compared to 4 weeks old samples. The performance of Anabaena–Serratia biofilm and dual culture inoculant, in the presence of RP was most promising, as they exhibited highest ARA even at harvest stage. Anabaena–Pseudomonas biofilm showed highest P uptake, illustrating the interrelationships of nitrogen fixation with increased P uptake by plant. The observations highlighted the synergism among the partners, which emphasizes the need for evaluation at field level for their promise as a green technology for agriculture.

Toxicological impact of anilofos on some physiological processes of a rice field cyanobacterium Anabaena torulosa

This study deals with the toxicological impact of the herbicide anilofos on photosynthesis, respiration, nitrogen assimilation, and antioxidant system in a diazotrophic rice field cyanobacterium Anabaena torulosa. Treatment of anilofos (1.25, 2.5, and 5 mg L−1) affected growth, photosynthetic pigments, photosynthesis, and respiration of the cyanobacterium. Although all the photosynthetic pigments were affected, a maximum effect of the herbicide was observed on phycocyanin (51% reduction) followed by the carotenoids. The effect of the herbicide on photosynthetic pigments resulted in 57% decrease in photosynthetic O2 evolution. Studies on the photochemical activity demonstrated that both photosystems (PS I and PS II) were affected by the herbicide. Decrease in the photosynthesis rate resulted in decreased nitrogen assimilation, as revealed by reduced nitrate (20%) and ammonium (26%) uptake and decreased activities of nitrogenase (63% decrease) and glutamine synthetase (22% decrease). This ultimately resulted in the reduced growth of the organism. Activities of superoxide dismutase, catalase, and peroxidase in the presence of anilofos increased by 1.8–3.5 times over control cultures. Proline content increased by 1.6 times, while the content of ascorbate decreased slightly. These results indicate that the organism was able to tolerate the herbicide stress by activating oxidative stress defense mechanism.

Interaction between carbon and nitrogen metabolism during akinete development in the cyanobacterium Anabaena torulosa

Nutrient enrichment with a nitrogen (as nitrate) or carbon (as fructose) source to unaerated diazo and photoautorophic cultures of the cyanobacterium Anabaena torulosa induced early development of akinetes with high frequency. When cultures under any mode of nutrition were aerated, akinetes were not differentiated. Unaerated cultures with nitrate nitrogen or fructose exhibited higher respiratory rates and nitrogen assimilation compared to aerated cultures. This was evidenced by increased respiratory O2 uptake and high activities of pyruvate kinase, malate dehydrogenase (NAD+), nitrogenase and nitrate reductase signifying that akinete forming unaerated cultures exhibited high carbon dissimilation and nitrogen assimilation resulting in high nitrogenous build up in the cells. Aerated, non-akinete cultures, on the other hand, were associated with low respiratory O2 uptake, low pyruvate kinase and malate dehydrogenase (NAD+) activities, suggesting that carbon dissimilation was not favoured either in presence of nitrate or fructose. Moreover, higher activity of NADP+ linked malate dehydrogenase and lower nitrate reductase activity in aerated cultures led to a high carbon and low nitrogen content of the cells resulting in high cellular C:N ratio. The results suggest that interaction between carbon and nitrogen metabolism regulates akinete development in A. torulosa.

Total Structure and Inhibition of Tumor Cell Proliferation of Laxaphycins

From a mixed assemblage of Lyngbya majuscula rich marine cyanobacteria, we isolated a series of cell growth inhibitory cyclic peptides. The structures of the two major components, laxaphycins A (1) and B (2), and of two minor peptides, laxaphycins B2 (3) and B3 (4), were determined by spectroscopic methods and degradative analysis. Absolute configurations of natural and nonproteinogenic amino acids were determined by a combination of hydrolysis, synthesis of noncommercial residues, chemical derivatization, and HPLC analysis. The organism producing the laxaphycins was identified as the cyanobacterium Anabaena torulosa. The antiproliferative activity of laxaphycins was investigated on a panel of solid and lymphoblastic cancer cells. Our results demonstrate that in contrast to laxaphycin A, laxaphycin B inhibits the proliferation of sensitive and resistant human cancer cell lines and that this activity is strongly increased in the presence of laxaphycin A. This effect appears to be due to an unusual biological synergism.

Nutrient stress causes akinete differentiation in cyanobacteriumAnabaena torulosa with concomitant increase in nitrogen reserve substances

Addition of nitrogen source (nitrate), carbon sources (acetate, citrate and fructose), depletion of nutrients (phosphate-free nitrate medium), dilution of medium (2, 4 and 8 times diluted nitrate medium) under unaerated conditions induced akinete differentiation in Anabaena torulosa. Aerated cultures under the same conditions did not differentiate akinetes. The amounts of reserve metabolites--glycogen and cyanophycin (multi-L-arginyl-poly-L-aspartic acid) granule polypeptide (CGP)--were determined in unaerated and aerated cultures, and at different stages of growth and akinete differentiation. The addition of nitrate, acetate, citrate and fructose under unaerated conditions resulted in the accumulation of glycogen and CGP in higher amounts after 4 d (akinete initiation); the CGP content further changed at mature free akinetes phase. Higher accumulation of reserve products was also observed under nutrient deficiency (phosphate-depleted or diluted media) after 4 d of cultivation. Under aerated conditions reserve product accumulation was considerably lower. Thus a low accumulation of reserve products in aerated cultures showed that aeration probably somehow relieves the organism from a nutritional stress.

A novel potassium deficiency-induced stimulon in Anabaena torulosa.

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase of A. torulosa and the glycine betaine-binding protein of E. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria

Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.

Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.

Phosphorus deficiency, nitrogen assimilation and akinete differentiation in the cyanobacteriumAnabaena torulosa

Anabaena torulosa is unable to fix N2 and to differentiate akinetes in a P-deficient nitrate-free medium. In a P-deficient medium with nitrate, the NO3 − assimilation and period of akinete differentiation are of the same order of magnitude as in a P-containing nitrate medium. It is suggested that regulation of akinete differentiation in P-deficient organism proceeds through the regulation of the N-assimilating system. At the time of akinete differentiation, cellular P is excreted into the medium which leads to a decrease of cellular P.

Cloning of salinity stress-induced genes from the salt-tolerant nitrogen-fixing cyanobacterium Anabaena torulosa

A subtractive hybridization procedure was used to clone genes of the cyanobacterium Anabaena torulosa expressed in response to salt stress. The method uses total RNA from salt-treated cells, labeled in vitro, as the probe. Hybridization to restriction digests of total DNA was used for interspecies comparison; the procedure was also successful for isolation of cosmids by colony hybridization, semiquantitative dot blots, and cosmid characterization by Southern blotting. Analysis of eleven independent cosmids containing genes whose transcription is abundantly induced by salt suggests that a substantial portion of the A. torulosa genome, probably in excess of 100 kilobases, responds to salt.

Induction of the Formation of Akinetes and Microcycle Akinetes in the Cyanobacterium Anabaena torulosa

Summary Induction of akinete formation in Anabaena torulosa ensued with high frequency in nitrogen/carbon sources or their combinations in 10 d old cultures while nitrogen free medium supported low frequency of akinetes by 20th d. The akinetes in nitrogen/carbon sources have undergone germination too readily and the hormogones accumulated granular contents resulting in a second generation of small circular akinetes. This has been designated as microcycle akinete formation. The increase of light intensity and volume of medium with nitrate or nitrite induced the formation of akinetes and microcycle akinetes without any change in the period of akinete formation. The addition of 2,4-dinitrophenol DNP), streptomycin and chloroamphenicol in the two nitrogen sources reduced the frequency of akinetes, germination and microcycle akinetes. Inhibition of nitrate utilization by 3-(3,4-dichlorophenyl)-N-N′dimethyl urea (DCMU) completely suppressed growth, akinete and microcycle akinete formation. In control cultures, nitrate uptake reached a steady state level with akinete and microcycle akinete differentiation. The contents of glycogen and cyanophycin granule polypeptide (CGP) decreased from akinete stage to microcycle akinete formation as well as a decline in the activity of glutamine synthetase (GS). The significance of these features has been discussed.

Enhancement of Cyanobacterial Salt Tolerance by Combined Nitrogen

Presence of certain nitrogenous compounds in the growth medium significantly enhanced the salt tolerance of the fresh-water cyanobacterium Anabaena sp. strain L-31 as well as the brackish water cyanobacterium Anabaena torulosa. Among these, nitrate, ammonium, and glutamine were most effective followed by glutamate and aspartate. These nitrogenous compounds also inhibited Na(+) influx in both Anabaena spp. with the same order of effectiveness as that observed for protection against salt stress. The inhibition of Na(+) influx on addition of the nitrogenous substances was rapid; nitrate and ammonium inhibited Na(+) influx competitively. Proline and glycine did not affect Na(+) influx and also had no influence on the salt tolerance of either Anabaena sp. The observed protection was not consequent to a stimulatory effect of combined nitrogen on growth per se. Uptake of NO(3) (-) and NH(4) (+) increased during salt stress but was not correlated with growth. Intracellular levels of NO(3) (-) and NH(4) (+) were found to be inadequate to constitute a major component of the internal osmoticum. The results suggest that inhibition of Na(+) influx by combined nitrogen is a major mechanism for protection of cyanobacteria against salt stress.

Effect of Sodium on Nitrogen Fixation in Anabaena torulosa and Plectonema boryanum

The cyanobacterium Anabaena torulosa required sodium and molybdenum for nitrogenase activity and diazotrophic growth. Addition of sodium or molybdenum to cultures deficient in either element restored nitrogenase activity. Heterocyst differentiation was unaffected by sodium but molybdenum deficiency enhanced differentiation. The non-heterocystous cyanobacterium Plectonema boryanum 594 also required sodium for nitrogenase activity but synthesized presumptive nitrogenase component I and II proteins during sodium deficiency. The results show that in cyanobacteria nitrogenase is synthesized even in the absence of sodium but functions only in its presence.

Impairment of photosynthesis by sodium deficiency and its relationship to nitrogen fixation in the cyanobacterium Anabaena torulosa

Impairment of photosynthesis by sodium deficiency and its relationship to nitrogen fixation in the cyanobacterium Anabaena torulosa

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.