Influence of nitrogen source on growth and nitrogenase activity inAzotobacter vinelandii

Growth and nitrogenase activity were studied in cultures ofAzotobacter vinelandii growing with dinitrogen, ammonium sulfate, aspartic acid or yeast extract. Nitrogenase activity was measured by means of the C₂H₂ reduction test.In the presence of ammonium sulfate nitrogenase is completely repressed. After exhaustion of ammonia its activity is restored following a diauxic lag period of 30 min. With aspartic acid nitrogenase activity is partially repressed, and growth yield is higher than in the culture growing with N₂ only. This is due to simultaneous use of dinitrogen and aspartate. Fluctuations of nitrogenase activity occurring during exponential growth and the mechanism of their regulation are discussed.Abbreviations NA nitrogenase activity - BNF Burk's nitrogen free medium     

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MB_o) when the concentration of this mediator in the medium is increased up to 72 M. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NR_o) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H₂ uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H₂ evolved/mg cell protein/min from the nitrogenase and 160 nM H₂-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H₂ evolution and for H₂ uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Degradation of the nitrogenase proteins during encystment of Azotobacter vinelandii

Nitrogenase activity in cell-free extracts of Azotobacter vinelandii declines during encystment. Upon germination a rapid increase in activity is observed, which is suppressed by rifampicin, suggesting that de novo biosynthesis of the nitrogenase proteins is required. The decline of activity during encystment is accompanied by disappearance of both nitrogenase proteins from cell extracts, indicating irreversible proteolysis. Total proteinase activity does not change significantly during encystment.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Free and glucosylated phenolics, phenol β-glucosyltransferase activity and membrane permeability in cucumber roots affected by derivatives of cinnamic and benzoic acids

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.01, 0.1 and 0.5 mM solutions of derivatives of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) as stress factors. In cucumber roots phenolics (free and glucosylated), phenol β-glucosyltransferase (E.C. 2.4.1.35) activity as well as membrane permeability were examined. The most intensive glucosylation took place in the first hour of stress duration in roots treated with 0.01 mM ferulic and p-coumaric acids and with 0.01 and 0.1 mM p-hydroxybenzoic and vanillic acids. At these concentrations a high phenol β-glucosyltransferase activity was found. The deterioration of capacity for phenolic glucosylation as well as the decrease of the phenol β-glucosyltransferase was observed at the higher concentrations. It was associated with increased membrane permeability.     

Growth kinetics and nitrogenase induction inFrankia sp. HFPArI 3 grown in batch culture

Summary Kinetics of growth and nitrogenase induction inFrankia sp. Ar13 were studied in batch culture. Growth on defined medium with NH ₄ ⁺ as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH ₄ ⁺ arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 nM C₂H₄·mg protein^–1·min^–1) was sufficient to support growth on N₂ and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K₂ HPO₄/KH₂PO₄, 0.1 mM CaCl₂ and MgSO₄. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10 g protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO₂ and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.     

Regulatory properties of the nitrogenase fromRhodopseudomonas palustris

Ammonium salts, glutamine, asparagine, and urea cause an immediate inactivation (switch-off) of light-dependent acetylene reduction in intact cells of the photosynthetic bacteriumRhodopseudomonas palustris. This effect is reversible showing the same kinetic pattern of inactivation and reactivation with all effector compounds. Its duration depends on the amount of effector added to the cells. Both nitrogenase components are found catalytically active in a cell-free preparation after enzyme switch-off in vivo. Involvement of the ammonia assimilating system in this regulatory mechanism is indicated by the following observations: ammonia uptake during the switch-off period, resumption of acetylene reduction after disappearance of ammonia from the outer medium, and persistence of enzyme switch-off with dihydrogen and thiosulfate as electron donors in the absence of an additional carbon source. Nitrogenase activity in crude extracts is non-linear with time and is stimulated by manganese ions. After resolution of nitrogenase into its MoFe-protein and Fe-protein these properties are lost, indicating the presence of an activating factor. Nitrogenase ofR. palustris cross reacts reciprocally with the complementary proteins ofAzotobacter vinelandii, but not with those ofClostridium pasteurianum.Abbreviations CCCP m-chlorocarbonyl cyanide phenyl hydrazone - DNP 2,4-dinitrophenol - EPR electron paramagnetic resonance - HEPES N-2-hydroxyethylpiperazine--2-ethane sulfonic acid - NOQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - TES N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid     

Growth and nitrogenase activity of Azotobacter vinelandii on soil phenolic acids

M oreno , J., de la R ubia , T., R amos -C ormenzana , A. & V ela , G.R. 1990. Growth and nitrogenase activity of Azotobacter vinelandii on soil phenolic acids. Journal of Applied Bacteriology 69 , 850–855.
Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii were studied in soil suspensions supplemented with p -hydroxybenzoic, vanillic, p -coumaric and ferulic acids. Nitrogenase activity was detected only when the microorganism was cultured on p -hydroxybenzoic acid, suggesting that this compound could be utilized as a carbon source by A. vinelandii for the maintenance of its biological activities under certain environmental conditions.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.     

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. -ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.Abbreviations HEPES [4-(2-hydroxyethyl)-1-piperazine-ethane; sulfonic acid] - MOPS [3-(N-morpholino) propane sulphonic acid] - MSX Methionine sulfoximine     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Comparative physiology of nitrogenase activity and vesicle development for Frankia strains CpI1, ACN1 ag,EAN1pec and EUN1f

The relationship between nitrogen fixation and development of a specialized cell structure, called the vesicle, was studied using four Frankia isolates. Nitrogenase activity was repressed in all four strains during growth with ammonia. Strain CpI1 formed no vesicles during NH₄ growth. Strains ACN1 ^ag , EAN1_pec and EUN1_f produced low numbers of vesicles in the presence of ammonia. Following transfer to nitrogen-free media, a parallel increase in nitrogenase activity and vesicle numbers occurred with all four isolates. Appearance of nitrogenase activity was more rapid in those strains that possessed some vesicles at the time of shift to N₂ as a nitrogen source. The ratio of vesicle numbers to level of nitrogenase activity varied widely among the four strains and in response to different growth conditions and culture age of the individual strains. Optimum conditions of temperature, carbon and energy source, nitrogen source and availability of iron and molybdenum were different for each of the four strains. Those conditions that significantly reduced nitrogenase activity were always associated with decreased numbers of vesicles.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Cluster analysis of genes for nitrogen fixation from several diazotrophs

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.     

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.     

Utilization of some phenolic compounds byAzotobacter chroococcum and their effect on growth and nitrogenase activity

Azotobacter chroococcum was isolated from straw-amended soil and found to utilize 4-hydroxybenzoic acid, resorcinol, pyrocatechol and vanillic acid as sole carbon source. Growth and nitrogenase activity ofA. chroococcum were supported by 8, 6 and 4 mmol/L of 4-hydroxybenzoic acid, resorcinol and pyrocatechol, respectively. The generation time of 1.71 h in 4-hydroxybenzoic acid did not significantly differ from the generation time of 1.64 h, observed when grown in mannitol. 4-Hydroxybenzoic acid was utilized rapidly. However, the decomposition of other tested phenolic compounds set in only slowly. It was concluded that this isolate has good potential to utilize some phenolic compounds released during biodegradation of plant wastes.     

The utilization of short-chain monocarboxylic acids as carbon sources for the production of gamma-linolenic acid by Mucor strains in fed-batch culture

The Fischer-Tropsch reaction water, which contains C₂ to C₅ monocarboxylic acids, generated as a co-product of the Sasol industrial oil-from-coal process, constitutes a potential cheap carbon substrate for the production of gamma-linolenic acid (GLA) by selced Mucor species. Three strains of Mucor were each grown in an air-lift reactor operated in a fed-batch, pH-stat mode under N-limitation with a mixture of C₂ to C₅ monocarboxylic acids as both pH titrant and carbon source. The production of GLA from this substrate was evaluated. Growth typically resulted in the rapid assimilation of acetic, n-butyric and n-valeric acids. Although propionic, iso-butyric and iso-valeric acids were assimilated to varying degrees, these acids accumulated in the culture. Mucor circinelloides CBS 203.28 gave the best results in that it assimilated 36% to 100% of each acid, had a biomass yield coefficient of 0.3 (calculated on acids utilized), and contained 28% crude oil, 84% of which comprised neutral lipids with a GLA content of 14.4%, giving 33 mg GLA/g biomass. GLA accumulation coincided with a decrease in the stearic-acid content of the neutral-lipid fraction. The results were comparable with previous results obtained with acetic acid and glucose as sole carbon sources, demonstrating the feasibility of producing GLA from the above mixture of organic acids.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

Organic acids and prolonged nitrogenase activity by non-growing,free-living Rhizobium japonicum

Rhizobium japonicum 61-A-101 grew and fixed nitrogen more effectively on media containing an organic acid and a pentose sugar than on media containing only one of these carbon sources. Peak specific activities in the range 10–15 nmol C₂H₄ · h^-1 · mg protein^-1 were found for these organisms in a spot of growth about 1 cm diameter on agar surfaces exposed to air. Increasing concentrations of the organic acids (succinate or malonate) in a medium containing arabinose resulted in longer lasting activity. The inclusion of a third carbon source, glycerol, gave activity which remained at the maximum from about the 8 to the 18 day after inoculation although no growth of the bacteria occurs during the last 8 or 10 days. At low concentration of organic acid l-arabinose was a much better carbon source for supporting nitrogenase activity of these organisms that the d-form. Both organic acids affected the morphology of the bacteria. Higher concentrations, especially of malonate, gave swollen and distorted cells. When bacteria growing on organic acid-containing agar plates were suspended and plated after appropriate dilution on yeast extract — mannitolglycerol agar there was heterogeneity of colony form, with up to 90% microcolonies after growth on high malonate concentrations. The effects of malonate may be correlated with characteristics of the bacteroid form inside the nodule which contains relatively high concentrations of organic acids, especially malonate.