Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis

The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the first time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5°C. The uptake hydrogenase gene hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.     

Common evolutionary origin of planktonic and benthic nitrogen-fixing oscillatoriacean cyanobacteria from tropical oceans

The filamentous cyanobacteria belonging to the genus Hydrocoleum (Blennothrix) are among the most common mat-forming cyanobacteria in tropical oceans. We present here the evidence that these benthic cyanobacteria are morphologically and phylogenetically very close to the planktonic species of Trichodesmium. Genetic relationship was established independently with regard to sequences of the 16S rRNA gene, nifH gene, and phycocyanin and phycoerythrin intergenic spacers. The species of both genera formed a common distinct branch in phylogenetically reconstructed cyanobacterial trees, suggesting that the main constituents of cyanobacterial benthos and plankton have an early common origin and both represent major contributors to nitrogen budget of tropical oceans today as in the distant geological past.     

Studies on mineral phosphate solubilization by cyanobacteria Westiellopsis and Anabaena

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in pH of the media and thin layer chromatography Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas Chromatographic-Mass Spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.     

Alkali-labile precursors of dimethyl sulfide in marine benthic cyanobacteria

By the method of cold alkali hydrolysis, 29 marine benthic cyanobacteria were screened for production of alkali-labile precursors of dimethyl sulfide (DMS) including dimethylsulfoniopropionate (DMSP), a compound of significant importance in marine environments. Concentrations of DMS precursors ranged from undetectable to 0.8 mmol (g Chl a)^–1. The data correspond to some previous investigations concerning DMSP content of marine cyanobacteria and suggest that marine benthic cyanobacteria are only minor producers of DMSP. Received: 3 July 1997 / Accepted: 21 October 1997     

Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent     

Restriction mapping of genomic DNA from five cyanophages infecting the heterocystous cyanobacteria Nostoc and Anabaena

The positions of the cleavage sites of several restriction endonucleases were mapped in DNA isolated from the cyanophages A-1L, A-4L, AN-10, AN-13 and AN-23, which were replicated in Anabaena PCC 7120. Similarities within the maps show AN-13 and AN-23 to be closely related strains. A-1L and AN-10 also appear to be related, but show more divergence. The maps of A-1L and AN-10 are circular, apparently as the result of circular permutation of sequences. The maps of the other cyanophages are linear.     

Molecular differentiation of the heterocystous cyanobacteria,Nostoc and Anabaena,based on complete NifD sequences

The segregation of Nostoc and Anabaena into separate genera has been debated for some time. The nitrogen fixation gene nifD was completely sequenced from representatives of these genera and analyzed phylogenetically, by using the representatives of other genera of the heterocystous cyanobacteria as outgroups. We were clearly able to differentiate between Nostoc and Anabaena in all analyses used. Our data suggest that Nostoc and Anabaena should remain as separate genera. Received: 16 November 2001 / Accepted: 14 December 2001     

Interaction of boron and calcium in the cyanobacteria Anabaena and Synechococcus

Although some studies have reported an interaction between boron (B) and calcium (Ca²⁺) in higher plants, there is little evidence for a similar relationship in cyanobacteria. The present study was designed to determine the effect of a supplement of boron to Ca²⁺-deficient cultures of Anabaena PCC 7119 and Synechococcus PCC 7942. Grown under Ca²⁺ deprivation, Anabaena had a slow growth rate and a low photosynthetic pigment content that was related to an inhibition of photosynthesis. Ca²⁺-deficient cells showed a lack of cohesiveness of the heterocyst envelope layers, which was consistent with a rapid decline in nitrogenase activity. A supplement of B led to partial recovery from the effects caused by lack of Ca²⁺. Similarly, low Ca²⁺ had inhibitory effects on growth and metabolism of Synechococcus cultures. In this case, the effect of a B supplement depended on the concentration of Ca²⁺ in the growth medium. When Ca²⁺ was present at normal concentration. B was not required, at least no more than trace amounts. However, when the Ca²⁺ concentration decreased, B was required at increasing levels. An effect of boron on uptake and/or on the binding of Ca²⁺ in cyanobacteria is proposed.     

Glycollate oxidation by thylakoids of the cyanobacteria Anabaena cylindrica,Nostoc muscorum and Chlorogloea fritschii

The intracellular distribution of glycollate dehydrogenase EC 1.2.1.17 has been investigated in extracts of the cyanobacteria (blue-green algae) Anabaena cylindrica, Nostoc muscorum and Chlorogloea fritschii. Most of the enzyme activity was associated with a chlorophyll-containing cell-free pellet, which also exhibited Photosystem I and II activities. Sucrose density gradient centrifugation of this washed pellet resulted in the formation of a green band within which maximal chlorophyll concentration and enzymic glycollate oxidation coincided. Antiserum raised to this fraction obtained from A. cylindrica inhibited glycollate dehydrogenase and Photosystem II activity. The data indicate that most of the cyanobacterial glycollate dehydrogenase is associated with the thylakoids and thus provide evidence for the dual role of these membranes in photosynthetic and respiratory processes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide     

Environmental conditions in high mountain lakes containing toxic benthic cyanobacteria

In glacial lakes on an alpine pasture in Switzerland, benthic cyanobacteria produced microcystin, a cyclic hepatotoxic heptapeptide. The cyanobacteria formed dense mats on sediments and submerged stones. The mats consisted mainly of Oscillatoria limosa, Phormidium konstantinosum (= Oscillatoria tenuis) and Tychonema granulatum (= Oscillatoria granulata). In order to characterize the ecological conditions of these cyanobacteria, nutrient concentrations were determined, and an automatic data acquisition station was installed in one of the lakes. It continuously recorded air temperature, global irradiance, precipitation, atmospheric pressure, wind speed and direction; as well as temperature, pH, oxygen content and conductivity of the lake water. The nutrient situation in the lakes was mainly influenced by the erosion of the gneissic catchment by glacial meltwater and by precipitation. In the glacial lakes, the concentrations of calcium, iron, magnesium, sodium and sulphate increased throughout the summer season. Conductivity values of 4–110 μS cm^-1 represent generally low nutrient concentrations. Nevertheless, iron concentrations of up to 20 μM occurred. Biomass, expressed as protein concentration, as well as the microcystin content of the cyanobacterial mats varied within one season and between different years (1994 and 1995). In one cyanobacterial mat community, biomass and microcystin concentrations were highest at the same time, in an other one the microcystin content was maximal three weeks after the highest biomass concentration was reached. Our observations suggest that biomass and toxin production in the mats were strongly influenced by mechanical stress, temporary desiccation and high irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of the light-limited growth of the nitrogen-fixing cyanobacteria Anabaena and Aphanizomenon

The effect of simultaneous N₂ fixation and light limitation on the growth of two strains of Anabaena sp. Bory de St. Vincent and Aphanizomenon flos-aquae (L.) Ralfs was investigated using continuous cultures. Under severely light-limited conditions, Aphanizomenon showed a broader absorption spectrum (due to the presence of phycoerythrin), a higher maximum efficiency of photosynthesis, a higher steady-state N₂ fixation activity and a higher growth affinity for light than did Anabaena . On the other hand, under light saturation, Anabaena showed a higher maximum rate of O₂ production and a higher maximum specific growth rate than Aphanizomenon . These monoculture results characterize Anabaena and Aphanizomenon , in relative terms, as a 'sun' and a 'shade' species respectively, and are in accordance with field observations. The difference between the two species in their acclimatory response is discussed in terms of a species-specific alteration of the PSI:PSII stoichiometry. Besides the species-specific modulation of the accessory pigments, such an acclimation would provide a biochemical basis for the observed physiological differences. The monoculture results were used to differentiate the niches of the two species and suggested that Aphanizomenon would competitively displace Anabaena under N₂-fixing, light-limited conditions. However, when both species were grown together, Anabaena became dominant and seemed to be the superior competitor for light. In order to explain this finding, the possible effects of release of allelopathic compounds, or dynamic aspects of light supply, are discussed.