Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

Impact of different abiotic stresses on growth, photosynthetic electron transport chain, nutrient uptake and enzyme activities of Cu-acclimated Anabaena doliolum

This study provides a comparative account of the effects of cadmium, temperature, ultraviolet-B and sodium chloride on the growth, photosynthesis, nutrient uptake and enzyme activities of untreated control and copper-acclimated Anabaena doliolum. Reduction in all the studied parameters, except carotenoids, was maximum for sodium chloride followed by ultraviolet-B, temperature and cadmium treatments, the reduction being greater in control than acclimated A. doliolum. Among the various parameters, photosystem II was most sensitive for all the stresses in both control and acclimated A. doliolum. Likewise, O2 evolution was more susceptible to various stressors than 14C uptake. Ammonium uptake and glutamine synthetase (GS, EC 6.3.1.2) were the least affected parameters. As compared to control, acclimated Anabaena exhibited higher ATP content under normal conditions. These results attest our hypotheses that acclimated Anabaena was physiologically more robust than control and that salinity was more injurious to the test organism than other abiotic stresses investigated.     

Influence of chromium on some physiological variables ofAnabaena doliolum: interaction with metabolic inhibitors

The impact of 2,4-dinitrophenol and chlorophenyl dimethylurea on ATP content carbon fixation, O₂ evolution, nitrogenase activity and Cr uptake ofAnabaena doliolum has been studied. 2,4-Dinitrophenol has been found to be more toxic than chlorophenyldimethylurea for all these processes. However, when Cr toxicity to above variables was assessed in their presence the interaction was less than additive. An initial (10–15 min) concentration-dependent rapid Cr uptake, followed by a slow one, indicates a biphasic uptake. A significant inhibition of Cr uptake in the presence of both these metabolic inhibitors suggests the involvement of metabolic processes in Cr uptake.     

Involvement of a cadmium-induced low molecular weight protein in regulating cadmium toxicity in the diazotrophic cyanobacterium Anabaena doliolum

This study demonstrated the production of a cadmium-induced low molecular weight (3.5 kDa), buthionine sulfoximine (BSO) sensitive protein in Anabaena doliolum. Production of this protein was accompanied by a decrease in the glutathione level of the cell. Cadmium was found to be differentially toxic to carbon fixation, O₂ evolution, ATP content, nitrate reductase, nitrogenase, alkaline phosphatase and ATPase of control (untreated), BSO, cadmium and (cadmium + BSO) pre-treated A. doliolum. Toxicity was maximum in BSO-grown cells followed be control (untreated), cadmium + BSO and least in cadmium-grown A. doliolum. Cadmium and (cadmium + BSO)-grown cells registered an increased lipid production, reduced metal uptake and low K⁺, Na⁺ loss. In spite of equal cadmium uptake rates, a significant difference in toxicity between cadmium-grown and (cadmium + BSO)-grown cultures was, however, noticed. Better performance of physiological and biochemical variables of cadmium-grown A. doliolum and its tolerance to cadmium could be due to the synthesis of low molecular weight cadmium binding protein (presumably phytochelatin) as well as an increased production of lipid.     

Mycosporine-like amino acids (MAAs) profile of a rice-field cyanobacterium Anabaena doliolum as influenced by PAR and UVR

The mycosporine-like amino acid (MAA) profile of a rice-field cyanobacterium, Anabaena doliolum, was studied under PAR and PAR + UVR conditions. The high-performance liquid chromatographic analysis of water-soluble compounds reveals the biosynthesis of three MAAs, mycosporine-glycine (lambda (max) = 310 nm), porphyra-334 (lambda (max) = 334 nm) and shinorine (lambda (max) = 334 nm), with retention times of 4.1, 3.5 and 2.3 min, respectively. This is the first report for the occurrence of mycosporine-glycine and porphyra-334 in addition to shinorine in Anabaena strains studied so far. The results indicate that mycosporine-glycine (monosubstituted) acts as a precursor for the biosynthesis of the bisubstituted MAAs shinorine and porphyra-334. Mycosporine-glycine was under constitutive control while porphyra-334 and shinorine were induced by UV-B radiation, indicating the involvement of UV-regulated enzymes in the biotransformation of MAAs. It seems that A. doliolum is able to protect its cell machinery from UVR by synthesizing a complex set of MAAs and thus is able to survive successfully during the summer in its natural brightly lit habitats.     

Enhancement of nitrogen fixation inAnabaena flos-aquae (Cyanobacteria) via low-dose ultrasonic treatment

Low-dose (50 W), short-duration (5 min) ultrasonic treatments were delivered to log phase batch cultures of the cyanobacteriumAnabaena flos-aquae grown in nitrogen-free medium. Near the mid-point of nine-day experimental periods, ultrasonically-treated cultures exhibited higher growth rates, enhanced chlorophylla content, and greater acetylene reduction capacity when compared to cultures grown with reciprocal shaking alone. Heterocyst frequency was markedly higher in ultrasonically-treated cultures over the duration of five-day experimental periods.Author for correspondence     

Salinity and Copper-Induced Oxidative Damage and Changes in the Antioxidative Defence Systems of Anabaena doliolum

Summary This study provides first-hand information on the salinity and copper-induced oxidative damage and its protection in Anabaena doliolum by the antioxidant defence system. Oxidative damage measured in terms of lipid peroxidation, electrolyte leakage and H₂O₂ production was induced by different concentrations of NaCl and Cu²⁺. A greater electrolyte leakage by NaCl than Cu²⁺ supported the hypothesis of salinity being more injurious than copper. To explore the survival strategies of A. doliolum under NaCl and Cu stress, enzymatic antioxidant activities e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) and nonenzymatic antioxidant contents such as glutathione reduced (GSH), ascorbate, α-tocopherol, and carotenoid were measured. A general induction in SOD and APX activities as well as ascorbate and α-tocopherol contents was found under NaCl and Cu²⁺ stress. In contrast to this, an appreciable decline in GR activity, GSH pool and carotenoid content under Cu²⁺ and an increase under NaCl stress were observed. CAT activity was completely inhibited at high doses of NaCl but stimulated following Cu²⁺ treatment. The above results suggest the involvement of APX and CAT in the scavenging of H₂O₂ under Cu²⁺ stress. In contrast to this, only APX was involved in H₂O₂ scavenging under salt stress. Our postulate of Cu²⁺-mediated antagonism of salt stress can be explained by a conceivable reversion of Na⁺-induced disturbance of cellular homeostasis by redox active Cu²⁺.     

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability inAnabaena sp. strain PCC 7120

When deprived of combined nitrogen, aerobically-grown filaments ofAnabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus inAnabaena.     

Symbiotic algal nitrogenase activity and heterocyst frequency in sevenAzolla species after phosphorus fertilization

Seven species ofAzolla (A. caroliniana, A. microphylla, A. nilotica, A. filiculoides, A. mexicana, A. rubra, A. pinnata the last from both Malaysia and India) grown in pots of flooded soil were subjected to three different treatments with respect to P: none, single application, split application. The experiments were carried out under greenhouse conditions. Heterocyst frequency inAnabaena azollae and acetylene reducing activity (ARA) were studied in successiveAzolla leaves. Both variables increased from the first leaf (shoot apex) to the last one (before branch) in all species in the presence or absence of P. However, heterocyst frequency, ARA andAzolla biomass were all less in the treatment lacking P. Heterocyst frequency inA. azollae, ARA and biomass ofAzolla were higher when P was applied in split doses than in the other treatments.Azolla plants exhibited more ARA than the isolated leaves.     

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N₂-grown [peptide I] and NO₃-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N₂-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.     

Influence of culture density,pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris

The potential of alginate-immobilized Anabaena doliolum and Chlorella vulgaris was assessed for removal of nutrients (NO _inf3 ^sup- and NH _inf4 ^sup+ ) and metals (Cr₂O _inf7 ^sup2- and Ni²⁺) at different biomass concentrations (0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt l^-1) and pH values (4 to 10). Though uptake of all these substances was higher in concentrated algal beads (0.25, 0.49 and 1.22 g dry wt l^-1), their rate of uptake was significantly (P<0.001) lower than that of low (0.05 g dry wt l^-1) cell density beads. For A. doliolum, there was no significant difference in uptake rates for beads having densities of 0.05 and 0.1 g dry wt l^-1. Chlorella vulgaris, however, showed maximum efficiency at 0.1 g dry wt l^-1. Uptake of both the nutrients and the metals was maximal at pH 7 followed by pH 8, 6, 9, 10, 5 and 4. Of the different substances (organic acids and divalent cations) used, humic acid was most efficient in decreasing metal uptake. Mg²⁺ was, however, more efficient than Ca²⁺ in decreasing Ni²⁺ uptake. Immobilized algae with a cell density of 0.1 g dry wt l^-1 were the most efficient for nutrient and metal removal at pH 6 to 8.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

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.     

Action of 2,4-dichlorophenoxyacetic acid and rifampicin on heterocyst differentiation in the blue-green alga,Nostoc linckia

2,4-Dichlorophenoxyacetic acid, a commonly used herbicide, increased the growth of the filamentous blue-green alga,Nostoc linckia at doses upto 100 μg /ml. The herbicidetreated N₂-cultures showed enhanced heterocyst frequency and N₂-growth. Thus, the herbicide stimulated algal growth at the expense of molecular nitrogen under aerobic growth conditions. Rifampicin caused chain formation of heterocysts. This was effectively counteracted by 2,4-dichlorophenoxyacetic acid, suggesting a biological interaction between them at the level of the heterocyst spacing control mechanism.     

Factors modulating differentiation of spores: Characterization of mutants defective in sporulation in the cyanobacterium Anabaena doliolum (AdS strain)

Summary Mutants of Anabaena doliolum (AdS strain) altered with respect to the time of initiation and degree of sporulation were isolated following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine and hydroxylamine. The non-sporulating mutant showed a high phycocyanin (Pc): chlorophyll a (chl a) ratio (ca. 7.2) as compared to sporulating strains (Pc:chl a, 4.7–5.3). Also this strain seemed to have higher RNA pools per unit of genomic material as reflected in a higher RNA:DNA ratio. The data suggest that degradaton of phycocyanin and controlled RNA synthesis are prerequisites for sporulation. Mutants exhibiting non-sporulation and delayed initiation of sporulation accumulated more nitrogen through nitrogen fixation, probably indicating nitrogenase function over an extended vegetative phase.     

Inhibition of nitrogen fixation by nitrite inAnabaena variabilis

Addition of nitrite to rapidly growing, nitrogen-fixing filaments ofAnabaena variabilis caused an immediate drop in nitrogenase activity. This was followed by a transient induction of nitrite reductase, recovery of nitrogen fixation and cyanobacterial growth. The experiments with isolated heterocysts and a partially purified nitrogenase preparation from heterocysts showed that nitrite primarily exerted its inhibitory effect by inactivating nitrogenase irreversibly, rather than interfering with photosynthetic energy conservation.Abbreviations ATCC American type culture collection - Chl chlorophyll - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - Tes 2-{[2 hydroxy-1,1-bis(hydroxymethyl)ethyl] amino} ethane sulfonic acid     

Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 mol·m^-2·s^-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2–3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluenceresponse relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.Abbreviations and symbols BL blue light - V _m membrane potential - V ₈ surface potential     

Removal and assessment of toxicity of Cu and Fe toAnabaena doliolum andChlorella vulgaris using free and immobilized cells

Alginate-immobilized and free cells ofAnabaena doliolum andChlorella vulgaris were compared for their use in the removal and toxicity bioassays of Cu and Fe. A decrease in toxicity with regard to growth and uptake of NO ₃ ^– and NH ₄ ⁺ was noticed following immobilization of both the organisms. In contrast, immobilized cells had higher uptake rates of Cu and Fe suggesting that immobilization offers protection against metal toxicity. Compared with free cells, the immobilized cells showed greater efficiency for metal removal, even over three repeated cycles, though with a gradual decrease in efficiency in the second and third cycles. This reduction in removal efficiency was, however, more pronounced for Fe withA. doliolum and for Cu withC. vulgaris. The ease in harvesting and potential for repeated use makes the immobilized cells good tools for scavenging heavy metals from metal-contaminated environments.