Peter Rowell

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.

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.

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of 14CH3NH3+uptake. Initial accumulation (up to 60 s) was independent of CH3NH3+metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH3NH3+was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH3NH3+metabolism, but did not affect the CH3NH3+transport system.NH4+, when added after the addition of 14CH3NH3+, caused the efflux of free CH3NH3+; when added before 14CH3NH3+, NH4+inhibited its uptake indicating that both NH4+and CH3NH3+share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH3NH3+accumulation indicating that the transport system was Δψ-dependent. At pH 7 and at an external CH3NH3+concentration of 30 μmol dm-3, A. variabilis showed a 40-fold intracellular accumulation of CH3NH3+(internal concentration 1.4 mmol dm-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a Δψ-dependent NH4+transport system suggesting that the reduced inhibitory effect of NH4+on nitrogenase cannot be attributed to the absence of an NH4+transport system but is probably related to the reduced GS activity of the cyanobiont.

Alanine dehydrogenase of the N2-fixing blue-green alga, Anabaena cylindrica.

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The Kmapp for NAD+ is 14 μM and 60 μM at low and high NAD+ concentrations, respectively. The Kmapp for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The Kmapp for NH4+varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N2-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N2-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.

Thioredoxin as a Modulator of Glucose-6-phosphate Dehydrogenase in a N2-Fixing Cyanobacterium

Summary: Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme involved in fixed carbon dissimilation in photosynthetic micro-organisms; in heterocystous cyanobacteria it may also be implicated in the supply of reductant to nitrogenase. In crude cell-free extracts of the N2-fixing cyanobacterium Anabaena variabilis G6PDH activity was reversibly deactivated by the thiol agent dithiothreitol in the presence of a low molecular weight protein (12000 mol. wt). Glucose 6-phosphate reversed deactivation when added at high concentration, or prevented deactivation if added with the thiol. NADP+, which, like glucose 6-phosphate, is a G6PDH substrate, also deactivated the enzyme; deactivation was reversed or prevented by adding glucose 6-phosphate or glutamine. Purified thioredoxin from Anabaena cylindrica, at very low concentrations (2 nm), deactivated purified G6PDH in a manner identical to that observed when crude extracts were used in the presence of dithiothreitol. Glutathione did not affect the enzyme.

Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N2-fixing cultures were treated with 10 mM NH4+or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH4+. When the enzyme was deactivated by simultaneously adding NH4+and placing the culture in darkness, reactivation occurred on reillumination and removal of NH4+. The removal of NH4+in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH4+-treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH4+. The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.

Electron donation to ferredoxin in heterocysts of the N2-fixing alga Anabaena cylindrica

Glucose-6-phosphate dehydrogenase activity is soluble in Anabaena cylindrica and is 6-7 times higher in heterocysts than in vegetative cells of N2 or NH4+-grown cultures. Vegetative cell activity is inhibited by light in vitro, but in heterocyst extracts activity is equally high in the light and dark. This inhibition occurs at equal rates in the presence and absence of thylakoid fragments. NADPH inhibits glucose-6-phosphate dehydrogenase activity competitively with NADP+ and non-competitively with glucose-6-phosphate. ATP inhibition also occurs and is competitive with NADP+ and non-competitive with glucose-6-phosphate. Dithiothreitol inhibits activity but ribulose 1,5-diphosphate, ADP, phosphoenol pyruvate, glutamine and glutamate do not affect activity. Ferredoxin-NADP+ oxidoreductase activity was measured as cytochrome c reduction and as 2,6-dichlorophenol indophenol reduction. Ferredoxin-NADP+ oxidoreductase activity remains in solution on centrifugation of extracts at 100000 g x 60 min and in such preparations activity is equally high in vegetative cell and in heterocyst material in the light and dark. In 1000 g x 30 min and in 35000 g x 30 min supernatant fractions cytochrome c reduction is immediately inhibited by light in preparations of vegetative cells, but activity in heterocyst preparations is not inhibited by light. Inhibition by light can be reversed by placing the preparations in the dark. The addition of fractions containing vegetative cell thylakoids inhibits cytochrome c reduction by the 100000 g x 60 min supernatant enzyme from vegetative cells and from heterocysts but the addition of fractions containing heterocyst thylakoids has no such effects. Of various compounds tested, including potential inhibitors of ferredoxin-NADP+ oxidoreductase, only NADP+ inhibits activity. This inhibition is competitive with NADPH. Antibody to Scenedesmus ferredoxin inhibits cytochrome c reduction. The presence of glucose-6-phosphate dehydrogenase and ferredoxin-NADP+ oxidoreductase in heterocysts provides a route of electron transfer from glucose to ferredoxin which is light-independent and which may be regulated by the intracellular levels of oxidized and reduced pyridine nucleotides.

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

SummaryEthylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N2 and liberated NH4+ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH4+ was obtained in air-lift reactors at rates of up to 50 μmol NH4+ mg chl a−1 h−1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed 15N2 and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to 15N2, showed that highest initial 15N2-labelling was into NH4+. After 12 min little further increase in the NH4+label occurred while that in the amide group of glutamine and in glutamate continued to increase. The 15N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of 15N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total 15N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which 14N2 was added to cephalodia previously exposed to 15N2, showed that the NH4+pool rapidly became depleted of 15N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The 15N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the 15N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.

Role of extracellular polysaccharide in the colonization of wheat (Triticum vulgare L.) roots by N2-fixing cyanobacteria

The characteristics of the mucilaginous sheaths of the cyanobacteria Nostoc 2S9B and Anabaena C5 and their role in the formation of associations with the roots of wheat plants grown in liquid culture have been assessed. Light and scanning electron microscopy revealed that the filaments of Nostoc 2S9B that formed a tight association with the root surface were contained in a firm mucilaginous shell. In contrast, filaments of Anabaena C5 formed a loose association and were easily detached from the mucilage that had a sheet-like appearance and tended to disintegrate as the culture aged. Similarly, there was a tight attachment of the isolated polysaccharide from Nostoc 2S9B to the root surface and a loose attachment of the Anabaena C5 polysaccharide. When the crude polysaccharide from Nostoc 2S9B was freed from proteins by phenol or pronase treatment, its ability to adhere to the root surface was lost or considerably reduced, suggesting that a protein component contributes to the tight attachment of Nostoc 2S9B. The crude polysaccharide preparation from Nostoc 2S9B contained 2.8% (w/w) protein while that from Anabaena C5 was only 0.6% (w/w) protein. The purified exopolysaccharide from Nostoc 2S9B contained three neutral sugars and glucuronic acid, whereas fucose and a uronic acid were the main components of that from Anabaena C5. Washing the roots or treating them with different sugars did not alter the ability of Nostoc 2S9B to colonize the root surface, indicating that cyanobacterial attachment may not be specific.