Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

Growth and regulation of heterocyst and nitrogenase by fixed nitrogen sources were studied comparatively in parent and glutamine auxotrophic mutant of Anabaena cycadeae. The parent strain grew well on N2, NH+4 or glutamine while the mutant strain grew on glutamine but not on N2 or NH+4. The total lack of active glutamine synthetase in the mutant strain thus appears to be the reason for its observed lack of growth in N2 or NH+4, which explains why it is a glutamine auxotroph and at the same time shows glutamine synthetase to be the sole primary ammonia assimilating enzyme. NH+4 repression of heterocyst and nitrogenase in the mutant and the parental strains and their derepression by L-methionine-DL-sulfoximine suggest that NH+4 per se and not glutamine synthetase mediated pathway of ammonia assimilation is the initial repressor signal of heterocyst and nitrogenase in A. cycadeae.     

Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica.

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaema cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective election donors to nitro genase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum.     

Glyoxylate decreases the oxygen sensitivity of nitrogenase activity and photosynthesis in the cyanobacterium Anabaena cylindrica

Raising the pO₂ reduced nitrogenase activity (C₂H₂ reduction) of Anabaena cylindrica for both glyoxylate-treated (5 mM) and untreated cells. The stimulation caused by glyoxylate, however, increased with increases of pO₂ from 2 to 99 kPa. As the pO₂ increased the net CO₂ fixation was lowered (Warburg effect) while the CO₂ compensation point increased. Glyoxylate partly relieved this sensitivity of net photosynthesis to oxygen and reduced the compensation point considerably. The cells used were preincubated in the dark to exhaust photosynthetic pools. A more pronounced reduction in sensitivity of nitrogenase to oxygen for glyoxylate-treated cells was evident when a preincubation in air with reduced pCO₂ (13 l l^-1) was used. This was, however, not evident until after a 10-h incubation in air. Before this point 2 kPa O₂ sustained the highest nitrogenase activity. Addition of 0.5 and 5 mM of HCO ₃ ^- to Anabaena cultures preincubated at low CO₂ levels (29 l l^-1) abolished the stimulatory effect of glyoxylate on the nitrogenase. Thus, the results sustain the suggestion that glyoxylate may act as an inhibitor of photorespiratory activities in cyanobacteria and can be used as a means of increasing their nitrogen and CO₂ fixation capacities.Abbreviation RuBP ribulose 1,5-bisphosphate     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

The effects of acetaldehyde on nitrogenase, hydrogenase and photosynthesis in the cyanobacterium Anabaena cylindrica.

Acetaldehyde was shown to be an irreversible inhibitor of nitrogenase, hydrogenase, CO2 fixation and growth in the cyanobacterium Anabaena cylindrica, but had no effect on photosynthetic electron flow as measured by Methyl Viologen-dependent O2 uptake. The concentration-dependence of the inhibition of nitrogenase and hydrogenase activities was determined, and it was shown that acetaldehyde inhibition poses problems for anaerobic experiments in which the activities of these enzymes are measured in the presence of the frequently used glucose/glucose oxidase/catalase/ethanol O2 trap. It is suggested that acetaldehyde may find use as an inhibitor in experiments designed to separate electron flow through the photosystems from consequent fixation of CO2 and N2.     

GTP-binding proteins in a cyanobacterium Anabaena cylindrica

GTP-binding proteins were detected in a crude extract containing membrane components of Anabaena cylindrica. The crude extract was treated with 1% Lubrol PX and was fractionated by gel filtration. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented in the presence of 0.1 mM GTP and in the presence of 0.1 mM ATP. Six fractions of these GTP-binding proteins, tentatively designated GA1 to GA6, were ADP-ribosylated by pertussis toxin. GA3, GA4 and GA5 had Km values of 10, 60 and 7 nM, respectively. The molecular weights of some of these GTP-binding proteins were reduced after being labelled with [35S]GTP gamma S.     

Evidence for the occurrence of the alternative, vanadium-containing nitrogenase in the cyanobacterium Anabaena variabilis

Abstract Anabaena variabilis can be grown with dependence on either molybdenum (Mo) or vanadium (V) in the medium with essentially the same growth rates. Vanadium cultures reduce C₂H₂ to C₂H₄ and partly (to 2–3%) to C₂H₆. These C₂H₄ and C₂H₆ formations can be shown to be strictly light dependent, proving that the gases are formed by the cyanobacterium. C₂H₄ and C₂H₆ productions are accompanied by a H₂ formation which is much higher than in Mo cultures. Maximal C₂H₂-formation rates are 2/3 lower in V-grown cells compared to Mo control cultures. This is the first demonstration of a light-dependent ethane formation and of the occurrence of the alternative nitrogenase in any phototroph.     

Uninduced ammonia release by the nitrogen-fixing cyanobacterium Anabaena

Abstract Certain strains of the nitrogen-fixing cyanobacterium Anabaena were found to release varying quantities of ammonia without any induction, both in the presence and absence of combined nitrogen (nitrate) in the medium, during the different phases of their growth. In general, growth and ammonia release were comparable in both media, although there were strain differences. 3 patterns of ammonia release were observed in different strains during the growth period. They were: (1) release pattern parallel to the growth curve; (2) a continuous increase in release; and (3) release showing a bimodal curve.     

A note on the effects of associated micro-organisms on the growth and nitrogenase activity of the cyanobacterium Anabaena cylindrica

Pseudomonas spp., Micrococcus spp. and Fusarium oxysporum promote growth and nitrogenase activity in Anabaena cylindrica. Respiratory CO₂ production by the microbial associate appears to be the main benefit to the cyanobacterium. Some stimulation of nitrogenase, however, appears to be due to other causes in associations with Pseudomonas and Fusarium.     

Characteristics of the nitrogenase system of the blue-green alga Anabaena cylindrica

Summary The requirements for activity of blue-green algal nitrogenase have been studied. The optimal concentration ranges for ATP and Na₂S₂O₄ are 2-3 mM and 4-10 mM respectively. A magnesium requirement has been confirmed but the enzyme is not specific for Mg²⁺, Co²⁺ and Mn²⁺ will also support activity but Ca²⁺, Cu²⁺ and Zn²⁺ will not. The partially purified enzyme is soluble and specific activities of 50–100 nmoles C₂H₄/mg protein/min have been obtained. The biochemical characteristics of the enzyme, as determined in studies using enzyme inhibitors, are similar to those of bacterial and legume nitrogenases in that the enzyme is a metallo-protein containing iron and reduced thiol groups and the redox capacity of the enzyme involves a possible valency change in the iron. The transfer of electrons from H₂ via a bacterial hydrogenase has been shown to be mediated, at least in part, by ferredoxin. The role of ferredoxin and the interrelationships between photosynthesis, reductant pool and hydrogen metabolism are discussed in the light of recent results obtained by ourselves and other workers.     

The activation of glutamine synthetase from the cyanobacterium Anabaena cylindrica by thioredoxin

Abstract The present communication defines the conditions under which thioredoxin activates glutamine synthetase from Anabaena cylindrica . Effects are obtained at pH values around neutrality, and the activation is affected by Mg²⁺ in the assays. The thioredoxin systems from A. cylindrica and spinach are functionally interchangeable in the activation of glutamine synthetase. The enzyme is efficiently activated by thioredoxin_m and also by thioredoxin_f, but at much higher concentrations. Thioredoxin_m has previously been shown to activate NADPH-dependent malate dehydrogenase and isocitrate dehydrogenase from cyanobacteria. It is speculated that thioredoxin_m plays a role in the differentiation of vegetative cells to heterocysts.     

Occurrence and identification of UDP-N-acetylmuramyl-pentapeptide from the cyanobacterium Anabaena cylindrica

UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) is well known to be a key intermediate of bacterial peptidoglycan biosynthesis. We first detected the occurrence of UDP-MurNAc-pentapeptide in the cyanobacterium Anabaena cylindrica (NIES-19), and identified the structure of this pentapeptide by two-dimensional 1H-1H and 1H-13C NMR correlation experiments and mass spectra.     

Modifiers of heterocyst repression and spacing and formation of heterocysts without nitrogenase in the cyanobacterium Anabaena variabilis.

Twelve amino acid analogs and related compounds were screened for their ability to induce heterocysts in ammonia-repressed, undifferential filaments of Anabaena variabilis. As has been previously described, 1-methionine-dl-sulfoximine induces both heterocysts and nitrogenase. In contrast, dl-7-azatryptophan and beta-2-thienyl-dl-alanine were found to induce heterocysts but not nitrogenase activity (measured as acetylene reduction) even under microaerobic conditions. When the initial ammonium concentration was reduced, dl-7-azatryptophan-treated cultures sequentially produced heterocysts and then nitrogenase activity, but nitrogenase was detected only when a parallel culture without analog also became capable of acetylene reduction. Neither of the two latter analogs affected gamma-glutamyl transferase activity in crude extracts. All three analogs significantly reduced the mean interheterocyst distance in nitrogen-fixing cultures.     

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH₄Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO₂ fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO₂ (3%) lowered the release, if not given as a longer pretreatment (as CO₂ or HCO ₃ ^- ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N₂-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.Abbreviations MSX L-methionine-Dl-sulfoximine - INH isonicotinic acid hydrazide - RuDP ribulose 1,5-diphosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GS glutamine synthetase - GOGAT glutamate synthase - DTT Dl-dithiothreitol     

Transport and accumulation of nickel ions in the cyanobacterium Anabaena cylindrica

The uptake of nickel ions by the cyanobacterium Anabaena cylindrica was studied. Nickel transport was dependent on the membrane potential of the cells and the rate of uptake was decreased in the dark or by the addition of inhibitors, including uncouplers and electron transport inhibitors, which decreased or abolished the membrane potential of cells. The transport process obeyed hyperbolic kinetics, with a high affinity (apparent Km = 17 +/- 11 (SEM) nM) and low turnover number (maximum velocity = 22.3 +/- 5.4 (SEM) pmol h-1 mg dry wt-1 of cells or flux rate of 3.1 nmol h-1 m-2 of plasma membrane surface area). The process was also apparently specific for Ni2+, the rate being unaffected by the presence of a range of other metal ions in large excess. Equilibrium experiments showed that, over a range of nickel ion concentrations, the cells concentrated Ni2+ by a factor of 2700 +/- 240 (SEM)-fold, corresponding to a chemical diffusion potential for Ni2+ of 101 mV. It was concluded that the cells transport nickel ions by a carrier-facilitated transport process with the concentration factor for the ions being determined by the cell membrane potential according to the Nernst equation.     

Regulation of urea uptake by ammonia in the cyanobacterium Anabaena doliolum

Abstract The urea uptake system was studied with regard to its repression and derepression in the cyanobacterium, Anabaena doliolum . The uptake of urea was energy-dependent and was repressed in ammonia grown cells. Repression of the urea uptake by ammonium did not require ammonium assimilation or de novo protein synthesis, suggesting that ammonium itself was the repressor signal. The derepression of the urea uptake system, however, required de novo protein synthesis and glutamine synthetase activity.     

Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.