W. D. P. Stewart

Acetylene reduction by nitrogen-fixing blue-green algae

SummaryKnown 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.

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N2-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH3 being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N2-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH3 pool increases. MSO alleviates the inhibitory effect of exogenous NH4+ on nitrogenase synthesis and heterocyst production. The results suggest that in N2-fixing cultures of A. cylindrica the primary NH3 assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH4+ but is GS, GOGAT, or a product of their reactions.

Carbohydrate Accumulation and Osmotic Stress in Cyanobacteria

SUMMARY: In a comprehensive survey of the carbohydrate accumulation profiles of more than 70 strains of cyanobacteria three organic osmotica (glucosylglycerol, sucrose and trehalose) have been identified in both freshwater and marine isolates under conditions of osmotic stress. While the trend was towards glucosylglycerol accumulation in marine strains and sucrose accumulation in freshwater forms, there were no absolute differences between cyanobacteria isolated from each habitat. There was also no clear link between genus and the type of carbohydrate accumulated.

Nitrogenase activity in the blue-green alga Plectonema boryanum strain 594

SummaryThe non-heterocystous filamentous blue-green alga, Plectonema boryanum strain 594 reduces acetylene to ethylene, incorporates 15N2 into cell protoplasm, and grows readily in medium free of combined nitrogen, when incubated in a gas phase without added oxygen. Cells grown in the presence of 50 mg/l of ammonium-nitrogen do not reduce acetylene, and a concentration of 0.015 atm. CO in the gas phase inhibits nitrogenase activity completely but inhibits 14CO2 incorporation by only 28%. Nitrogenase activity is inhibited after 2 h treatment with 3×10-5 M DCMU and is inhibited completely in air.

Effects of Aerobic and Anaerobic Conditions on Growth and Metabolism of Blue-Green Algae

The blue-green algae Anabaena flos-aquae and Nostoc muscorum may reduce acetylene to ethylene most actively at pO2 levels below 0.2 atm. High pO2 levels inhibit acetylene reduction, nitrogen fixation, respiration and 14CO2 fixation in A. flos-aquae. The effect is not solely via an inhibition of nitrogenase activity because inhibition of 14CO2 fixation by a species of Phormidium which does not fix nitrogen, and by A. flos-aquae grown on combined nitrogen also occurs. The inhibition of acetylene reduction in Anabaena is reversible in short-term experiments, the rate of recovery being rather similar irrespective of the pO2level to which the alga was subjected initially. 3(3,4-dichlorophenyl)-1-1-dimethyl urea (DCMU) at a concentration of 3 x 10-5 M, which completely inhibits oxygen evolution and acetylene reduction but not respiration by aerobically grown Anabaena cultures also inhibits acetylene reduction in Na2S-grown cultures. Salicylaldoxime at a concentration of 10-4 M which partially inhibits electron flow from photosystem II inhibits acetylene reduction to a greater extent under aerobic conditions than in the presence of Na2S. The presence of Na2S also results in the removal of free oxygen from the medium in the pH range at which Anabaena normally grows. The data suggest that under our conditions (1) the photolysis of water, or photosystem II is essential for the growth of A. flos-aquae in the presence of H2S ; (2) H 2S may provide electrons when reducing power from the photolysis of water is reduced but not inhibited completely; (3) H2S prevents an accumulation of oxygen in the medium during photosynthesis. These physiological findings from the laboratory may help to explain why blue-green algae live not only in aerobic environments but also in habitats where reducing conditions may prevail.

Osmotic adjustment and organic solute accumulation in unicellular cyanobacteria from freshwater and marine habitats

The intracellular concentrations of low-molecular weight carbohydrates and quaternary ammonium compounds present in 26 axenic isolates of unicellular cyanobacteria have been studied over a range of external salinity from freshwater up to 300% seawater (100%=35‰ S). In all cases, a single carbohydrate, either sucrose or glucosylglycerol, was identified as the principal organic osmoticum, showing major variation in response to the external salt concentration; quaternary ammonium compounds were present in osmotically insignificant amounts. Glucosylglycerol was accumulated as primary osmoticum by nine of the isolates from saline habitats and by five of the freshwater isolates; trace amounts of sucrose were also prsent. The remaining twelve freshwater strains accumulated sucrose as sole osmoticum. Glucosylglycerol-accumulating strains grew over the widest salinity range (up to 200 to 250% seawater), whether isolated from saline or non-saline habitats. Sucrose-accumulating strains were more stenohaline, growing only in up to 50 to 100% seawater and showing no sustained growth in hypersaline media (>100% seawater). The data suggest that (1) glycosylglycerol accumulation is not unique to marine cyanobacteria, and (2) the upper salinity limit for growth may be linked to organic solute accumulation, rather than habitat, with glucosylglycerol-accumulating isolates having a greater potential for growth in salt-stressed conditions than sucrose accumulators.

Purification and some Properties of Glutamine Synthetase from the Nitrogen-fixing Cyanobacteria Anabaena cylindrica and a Nostoc sp.

Summary: Glutamine synthetase has been purified to homogeneity from two N2-fixing cyanobacteria, Anabaena cylindrica and a species of Nostoc (the phycobiont of Peltigera canina). The activities of the A. cylindrica enzyme in the biosynthetic and transferase assays were, respectively, 9.4 and 32 μmol product formed min−1 (mg protein)−1; the corresponding values for the Nostoc sp. enzyme were 6.5 and 20. Stabilization of the enzyme required Mg2+, glutamate, EDTA and a thiol reagent to be present during purification. The molecular weight of the A. cylindrica enzyme was 591000 as estimated by sedimentation analysis, 660000 by gel filtration and 565000 by polyacrylamide gel electrophoresis; the Nostoc sp. enzyme gave values of 630000 by gel filtration and 575000 by electrophoresis. The molecular weights of the sub-units of each enzyme were approximately 49000 to 50 000. Electron microscopy revealed that each molecule was composed of 12 sub-units arranged in two superimposed hexagonal rings. The maximum diameter of the rings was 13.6 nm and the distance between the centres of adjacent sub-units was 4.9 nm. When dialysed in the absence of stabilizing ligands the A. cylindrica enzyme lost activity and the protein band characteristic of the native enzyme was replaced by three bands with approximate molecular weights of 510000, 310000 and 130000. These sub-species re-associated and activity was restored by adding 2-mercaptoethanol and substrates. A similar reversible deactivation has been observed with glutamine synthetase from photosynthetic eukaryotes and yeast but no similar data have been reported for a N2-fixing prokaryote.

Pathways of glycollate metabolism in the blue-green alga Anabaena cylindrica

Summary1.Exogenous glycollate was assimilated by the blue-green alga Anabaena cylindrica.2.About 50% of the C-1 carbon of 14C-1-glycollate (i.e.25% of the total carbon) was released as 14CO2 in the dark and also in the light in the presence of DCMU. Most of the 14CO2 released in the light in the absence of DCMU was refixed.3.Assimilation was almost completely inhibited by α-hydroxy-2-pyridinemethane sulphonic acid, an inhibitor of enzymic glycollate oxidation. Cell extracts catalyzed the oxidation of glycollate to glyoxylate at rates sufficient to account for the in vivo assimilation.4.Isonicotinylhydrazide, an inhibitor of the conversion of glycine to serine in higher plant/green algae glycollate metabolism, did not significantly affect glycollate metabolism in A. cylindrica. Short-term labelling experiments with 14C-1-glycollate in the light and dark did not show a significant metabolism of 14C via glycine and serine. However, the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate to glycerate were demonstrated in cell extracts, although the activity of the enzyme catalyzing the metabolism of serine to hydroxypyruvate was not sufficient to account for the in vivo rate of glycollate assimilation.5.Cell extracts catalyzed the enzymic condensative decarboxylation of glyoxylate to tartronic semialdehyde and also the enzymic reduction of tartronic semialdehyde to glycerate. The activities in extracts were sufficient to account for the total in vivo photoassimilation of glycollate. The specific activity of malate synthase was insufficient to account for the total photometabolism of glycollate but exceeded the in vivo rate in the dark.6.On the basis of the inhibitor and kinetic experiments and in terms of the enzymes detected, it appears that in the light glycollate is metabolized mainly via glyoxylate → tatronic semialdehyde → glycerate → 3-phosphoglycerate → (glycolytic pathway) → pyruvate → alanine plus tricarboxylic acid cycle and related compounds. The bulk of the CO2 released in the light is probably refixed via the Calvin cycle. In the dark, the glyoxylate, produced from exogenous glycollate, appears to be metabolized mainly by malate synthase directly to malate.

Akinetes of the Cyanobacterium Nostoc PCC 7524: Macromolecular Composition, Structure and Control of Differentiation

Summary: Synchronized akinete differentiation occurred following the transition from exponential to non-exponential (linear) growth, the major trigger being energy limitation. Young akinetes first accumulated cyanophycin, then developed a multilayered extracellular envelope and a thickened wall. The dry weight, chlorophyll a, glycogen and carbon contents of mature akinetes were greater than those of vegetative cells, while their contents of DNA, RNA, protein, phycocyanin and nitrogen were similar to those of vegetative cells. Akinetes were resistant to desiccation and low temperatures, but not to temperatures above the maximum for vegetative cell growth. In N2-grown cultures heterocyst differentiation ceased at the end of exponential growth, while cell division continued, and akinetes first appeared in a regular pattern at a fixed distance (9 cells) from the nearest heterocyst. Exogenous NH4 + inhibited the differentiation of heterocysts and, in their absence, akinetes developed in irregular positions. The regular spatial pattern imposed on akinete differentiation by heterocysts was, like the heterocyst spatial pattern itself, independent of N2 fixation. Similar changes in both patterns induced by 7-azatryptophan suggested that they share a common mechanism of control.

Osmotic adjustment in cyanobacteria from hypersaline environments

The intracellular concentrations of the monovalent inorganic cations K+ and Na+, low molecular weight carbohydrates and quaternary ammonium compounds have been determined for 4 strains of cyanobacteria (Aphanothece halophytica, Coccochloris elabens, Dactylococcopsis salina and Synechocystis DUN52) originally isolated from hypersaline habitats (i.e. habitats with a salinity greater than that of seawater) over a range of external salt concentration (from 50% to 400% seawater). Intracellular cation levels (Na+ and K+) were determined to be within the range 80–320 mmol · dm-3 (cell volume), showing only minor changes in response to salinity. Intracellular carbohydrates were found to comprise a negligible component of the intracellular osmotic potential [at 2–19 mmol · dm-3 (cell volume)], throughout the salinity range. Quaternary ammonium compounds, however, were recorded in osmotically significant quantities [up to 1,640 mmol · dm-3 (cell volume)] in these strains, showing major variation in response to salinity. Thus Synechocystis DUN 52 showed an increase in quaternary ammonium compounds in the oder of 1,200 mmol · dm-3 between 50% and 400% seawater medium, accounting for a significant proportion of the change in external osmotic potential.Examination of intact cells and cell extracts using 13C and 1H nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of the quaternary ammonium compound glycine betaine as the major osmoticum in the 4 strains; no other compounds were detected during NMR assays. These results suggest a common mechanism of osmotic adjustment, involving quaternary ammonium compounds, in cyanobacteria from hypersaline environments.