Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

Some properties of an NADH-benzyl viologen reductase from azotobacter vinelandii

1. NADH-benzyl viologen reductase, solubilized by acetone extraction, was purified about 10-fold from small particles of Azotobacter vinelandii.

2. The purified enzyme preparation was free from hydrogenase activity. Either NADH or NADPH served as an electron donor for the reduction of benzyl viologen. This reaction is reversible.

3. The essential thiol groups of the enzyme are protected since they do not react with N-ethylmaleimide and p-chloromercuribenzoate inhibits only after it has been preincubated with the enzyme.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Exopolysaccharide production by free and immobilized microbial cultures

The batch production of different exopolysaccharides (alginate, xanthan, pullulan, dextran) by free and immobilized microbial cultures was investigated. First, conventional free-cell cultures were performed to obtain control fermentation parameters and macromolecular characteristics of exopolysaccharides. Then microbial cultures were immobilized in composite agar layer/microporous membrane structures and tested for polysaccharide production. The immobilized-cell system proved unsuitable for xanthan and pullulan production. Owing to the fouling of the microporous membrane by the polysaccharide, dextran production by immobilized Leuconostoc mesenteroides also was inefficient. More promising results have been obtained with immobilized Azotobacter vinelandii cultures. The amount of alginate produced by immobilized A. vinelandii represented about 60% of that recovered from a free-cell culture, whereas the polysaccharide yield reached 35% instead of 9% for the free counterpart. These results are compared to the macromolecular characteristics of exopolysaccharides.     

Biosynthesis of alginate. 3. Tritium incorporation with polymannuronic acid 5-epimerase from Azotobacter vinelandii

Incubation of polymannuronic acid in tritiated water with the polymannuronic acid C-5-epimerase isolated from Azotobacter vinelandii led to incorporation of tritium into the glycuronan. Hydrolysis of the labelled glycuronan and separation of the uronic acids demonstrated that 92% of the activity was present in the guluronic acid produced by the epimerase. There was also a small, but significant, incorporation into the mannuronic acid. The results are discussed in relation to the present knowledge of the enzymic epimerisations, and it is suggested that the first step in the reaction is an abstraction of H-5.     

The nitrogen fixation system of photosynthetic bacteria II. Chromatium nitrogenase activity linked to photochemically generated assimilatory power

The nitrogenase activity (measured by N₂ or acetylene reduction) of cell-free extracts of the photosynthetic bacterium Chromatium was coupled to photochemically generated ATP and reductant. The ATP was formed through cyclic photophosphorylation by bacterial chromatophores. The reductant (reduced ferredoxin) was generated by a heated preparation (incapable of O₂ and ATP production) of spinach chloroplasts. The nitrogenase activity of Chromatium extracts was supported by reduced Chromatium or Clostridium pasteurianum ferredoxin but not by that of spinach chloroplasts.     

氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响

共生根瘤的固氮效率受外界氮素的严格调控。除固氮酶活性外,豆血红蛋白(Lb)浓度亦是反应固氮能力的重要指标。为明确氮水平对生物固氮作用的影响,以大豆(Glycine max)为材料,在低氮(0.53 mmol·L–1)条件下接种根瘤菌,30天后再进行高氮(5.3、10、20、30和40 mmol·L–1)处理7天,分析L...     

EPR studies by Fe isotopic substitution on the nature of an unknown electron acceptor in Azotobacter vinelandii phosphorylating particles

1. EPR ⁵⁷Fe isotopic substitution studies provide unequivocal evidence that the g = 2.011 signal found in oxidized Azotobacter vinelandii phosphorylating particles is due to an iron-containing structure. The broadening constant determined as a result of this electron—nuclear hyperfine interaction was 15.7 G.

2. A similar signal found in a number of iron—sulfur containing proteins was found by quantitative EPR estimations to exist in a variable but substantial concentration when compared to the intensity of the reduced g = 1.9 type EPR resonance.

3. Reaction of the phosphorylating particles with excess potassium ferricyanide resulted in an alteration of the initial g = 2.011 iron signal resulting in the detection by microwave power studies of at least two different iron species which exhibited major g-values at 1.992 and 2.027.     

Catalytic reduction of acetylene in the presence of molybdenum and iron clusters, including FeMo cofactor of nitrogenase

Acetylene was reduced by zinc amalgam in the presence of three synthetic polynuclear complexes: {[Mg₂Mo₈O₂₂(OMe)₆(MeOH)₄]⁻²·[Mg(MeOH)₆]²⁺}6MeOH (I), (Bu₄N)₂[Fe₄S₄(SPh)₄] (II), [Me₄N][VFe₃S₄Cl₃(DMF)₃]·2DMF (III) and the iron-molybdenum cofactor of nitrogenase Azotobacter vinelandii MoFe₇(S²⁻)₉·homocitrate, FeMo-co (IV). Thiophenol was found to greatly facilitate the reaction in the presence of complexes I, II, IV. The reaction is catalytic and for I and IV proceeds at the amalgam surface. Thiophenol seems to increase the adsorption of the complexes, serving as an electron bridge to transfer electrons to the catalyst. In the case of II a homogeneous reduction of the substrate occurs presumably after the cluster reduction at the surface and with III the catalytic reduction proceeds only under the action of sodium amalgam; no thiophenol cocatalytic action is observed. Relevance to N₂ enzymatic reduction is discussed.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Isolation and characterization of a metronidazole-resistant (Mtn-R) mutant strain of Nostoc muscorum affected in ammonium regulation of heterocyst formation and uptake hydrogenase activity

Abstract The metronidazole-resistant ( Mtn-R ) mutant strain of N. muscorum produced drug-resistant NADPH: ferredoxin (Fd) oxidoreductase and showed derepression of heterocyst formation and uptake hydrogenase activity in NH₄⁺-medium. The observation of NH₄⁺-repression in regulation of nitrogenase activity alone in the mutant strain suggests, that heterocyst formation and nitrogenase activity are regulated by two separate NH₄⁺-repression control systems, one specific for heterocyst and uptake hydrogenase and the other for nitrogenase. The partial drug-resistant NADPH: Fd oxidoreductase enzymatic activity seems to be the reason for drug-resistant growth of the cyanobacterium in N₂-medium and NH₄⁺-medium.     

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.     

Nitrogenase from Rhodospirillum rubrum. Relation between 'switch-off' effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios.

Nitrogenase activity of 'membrane-free' extracts, produced from nitrogen-starved Rhodospirillum rubrum to which 4 mM NH4+ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this 'switch-off/switch-on' effect and the function of the membrane component is discussed. Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein rations. The ATP/2E- values are 4-5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.     

Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain. To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen-free medium after growth on NH4Cl (depression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen-free medium. No nitrogenase activity could be detected until t = 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane-bound polypeptides of Mr 29,000 and 30,000. Analysis with respect to changes in membrane-bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29,000 in the NADPH dehydrogenase complex.     

Effects of NaCl on growth and nitrogen fixation and assimilation of inoculated and KNO3 fertilized Vicia faba L. and Pisum sativum L. plants

Faba bean (Vicia faba L. var. minor cv. Alborea) and pea (Pisum sativum L. cv. Lincoln) plants, inoculated with Rhizobium leguminosarum biovar. viciae strain GRA19, were treated with salt (100 mM NaCl) and/or nitrate (8 mM KNO₃) to test whether plants grown with inorganic-nitrogen are more tolerant to salinity than plants entirely reliant upon fixed nitrogen. According to the growth inhibition recorded, pea plants dependent on dinitrogen fixation proved more tolerant to salt stress than those N-fertilized, in contrast to results obtained for faba bean plants. This study therefore confirms that plants dependent on nitrogen fixation are not always more sensitive to salinity than are N-fertilized plants. Nitrate addition did not reduce the specific nitrogenase activity in pea, but did in faba bean. However, nodulation was inhibited in both legumes. The specific nitrogenase activity was more affected by salt treatment in N-fertilized plants for both legumes. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADH-GOGAT) was inhibited by salt stress both in N-fixing and in N-fertilized pea and faba bean plants.