Nitrogenase activity in extracts of heterocystous and non-heterocystous blue-green algae

Summary Cell-free extracts capable of acetylene reduction and cyanide reduction have been prepared from heterocystous (Anabaena cylindrica) and non-heterocystous (Plectonema boryanum 594) blue-green algae. Extracts from Anabaena were obtained from cultures grown in blulk under aerobic conditions, while the Plectonema cultures were grown in bulk on nitrate-nitrogen, then washed free from nitrate and sparged with A/CO₂ for 40 h after which time maximum nitrogenase activity was detected. The nitrogenases of both algae are similar and resemble in many respects nitrogenases from bacteria and legumes. Activity is located primarily in a 40,000xgx15 min supernatant fraction and the rate of C₂H₂ reduction observed is about 10 per cent of whole cell activity. ATP and a source of reducing power (Na₂S₂O₄) are required for efficient functioning of the enzyme. ATP-dependent hydrogen evolution occurs, the extracts are cold labile and highly sensitive to oxygen and the oxygen inhibition is irreversible.     

Nitrogen fixation (acetylene reduction) associated with communities of heterocystous and non-heterocystous blue-green algae in mangrove forests of Sinai

Summary High rates of nitrogen fixation (acetylene reduction) are associated with communities of heterocystous and non-heterocystous blue-green algae, which are widespread and abundant in the coastal mangrove forests of the Sinai Peninsula.Heterocystous forms, particularly representatives of the Rivulariaceae, grow in aerobic environments, where nitrogenase activity may be limited by the availability of nutrients such as Fe and PO₄–P. Desiccated communities of Scytonema sp. reduce acetylene within ten minutes of wetting by tidal sea water. Communities dominated by the non-heterocystous Hydrocoleus sp., Hyella balani, Lyngbya aestuarii, Phormidium sp. and Schizothrix sp., occur in close contact with anaerobic sediments and reduce acetylene in the dark as well as in the light.Nitrogen fixation in all these communities is light dependant and may be supplemented by an alternative source of reductant in the dark. The indications are that nitrogen fixation by these communities of blue-green algae, makes a significant contribution to the overall nitrogen input of the mangrove ecosystem.     

Acetylene reduction by nitrogen-fixing blue-green algae

Summary Known nitrogen-fixing species of blue-green algae are capable of reducing acetylene to ethylene, but acetylene is not reduced by Anacystis nidulans, which does not fix nitrogen. Cycad root nodules which contain blue-green algae as endophytes reduce acetylene. Acetylene reduction is inhibited by carbon monoxide. Nitrate or ammonium-nitrogen has no immediate effect on algae reducing acetylene, but algae grown on nitrate-nitrogen gradually lose their capacity to reduce acetylene. Nitrate-nitrogen also inhibits heterocyst formation in these algae and there is a fairly direct correlation between the abundance of heterocysts in a particular sample and its capacity to reduce acetylene. Aphanizomenon flosaquae reduces acetylene and fixes nitrogen in unialgal culture and there is strong presumptive evidence that these reductions are carried out by the alga rather than by associated bacteria. The molar ratios of ethylene: ammonia produced vary within the range 1.4–1.8.     

Mesosomes in blue-green algae

Summary Mesosome-like, unit-membrane structures are clearly defined in the blue-green algae, Spirulina and three strains of Synechococcus, after osmium or potassium permanganate fixation and observation with the electron microscope. The membranous structures are distinct from the photosynthetic membranes and, in the case of Spirulina, are frequently observed in cells and can occur in large volume within the cell.     

Contiguous organization of nitrogenase genes in a heterocystous cyanobacterium

The organization of the three structural nitrogen fixation (nif) genes that encode nitrogenase (nif K and nif D) and nitrogenase reductase (nif H) have been examined in a number of cyanobacteria. Hybridization of Anabaena 7120 nif gene probes to restriction endonuclease-digested genomic DNA has shown (a) that cyanobacteria incapable of N₂ fixation have no regions of DNA with significant homology to the three nif probes, (b) that Pseudanabaena sp., a nonheterocystous cyanobacterium, has a contiguous nif KDH gene cluster, and (c) that in contrast with other heterocystous cyanobacteria, Fischerella sp. has a contiguous nif KDH gene cluster.     

Hydrocarbons in green and blue-green algae

Liquid column chromatography and thin-layer chromatography were used to determine the total content of hydrocarbons and gas chromatography was used to evaluate composition of hydrocarbons in green algae (Chlorella kessleri, C. vulgaris, Chlorella sp.,Scenedesmus acutus, S. acuminatus, S. obliquus) and the blue-green alga (Spirulina platensis) cultivated under autotrophic or heterotrophic conditions. InC. kessleri cultivated under heterotrophic conditions the content of hydrocarbons was found to be about 10^-2 % (per dry mass), whereas under autotrophic conditions it was about 10^-3 % (per dry mass). The highest content of hydrocarbons was detected in species of the genusScenedesmus cultivated autotrophically (10^-1 %). Heptadecane and hexacosane were found as major alkanes, 1-heptadecene was detected among alkenes.     

Immunostimulating activity of the lipopolysaccharides of blue-green algae]

The whole cells of blue-gree algae and lipopolysaccharides isolated from these cells were shown to stimulate the production of macro-(mainly) and microglobulin antibodies in rabbits. The macro- and microphage indices in rabbits increased significantly after the injection of LPS isolated from blue-green algae 24--48 hours before infecting the animals with a virulent Y. pseudotuberculosis strain. Besides, the inhibiting action of this strain on the migration of phagocytes to the site of infection was abolished immediately after the injection. The use of the indirect hemagglutination test allowed to prove the absence of close antigenic interrelations between blue-green algae and the following organisms: Spirulina platensis, Microcystis aeruginosa, Phormidium africanum and P. uncinatum.     

Morphology-habitat associations in blue-green algae

Morphological and habitat features have been recorded on punchcards for three genera in the Oscillatoriaceae and two in the Nostocaceae. As a result it is concluded that generic divisions in the Oscillatoriaceae are arbitrary and artificial, but that in the Nostocaceae, Cylindrospermum represents a grouping of distinct forms to a greater degree than does the amorphous genus Anabaena.

Some morphological comparisons between the two groups are attempted and detectable associations between morphological and habitat features are suggested : together these may contribute to an eventual separation of taxa on a more logical basis than at present.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

A re-examination of the problem of photochemical nitrogen reduction by blue-green algae

Summary Although it was possible in the light in the absence of carbon dioxide to obtain a ratio of nitrogen fixed to oxygen evolved in nitrogen-starved cells of A. cylindrica near to 1:1.5, that quoted by other workers, ratios varying between 1:0.9 and 1:3.0 were also obtained. The amount of oxygen evolved under the same conditions by normal cells in the presence of pyruvate was increased considerably. Since the addition of pyruvate also resulted in increased carbon dioxide output in the dark with the same algal material, oxygen output in the light was attributed to the production of factors necessary for carbon assimilation.Addition of pyruvate to nitrogen-starved and normal cells in the light resulted in similar rates of oxygen evolution after an initially higher rate in the starved cells. The ratio of overall nitrogen fixed to oxygen evolved, was 1:6.6 for the starved cells and 1:6.4 for the normal cells, showing that the presence of an added substrate increased oxygen output relative to nitrogen uptake.¹⁴CO₂ was recovered from sodium pyruvate-1-¹⁴C in flasks incubated in the dark, showing that, at least in the dark, pyruvate was decarboxylated.The interpretation of these results is that endogenous and exogenous substrates available to cells of A. cylindrica become decarboxylated and that, in the light, carbon dioxide produced may be assimilated photochemically with accompanying oxygen evolution. This interpretation has been discussed in relation to reports of photochemical nitrogen reduction in blue-green algae.