Properties of a Soluble Nitrogenase in Azotobacter

A nitrogenase system that remains in the supernatant fluid after centrifuging for 3 hr at 180,000 x g can be extracted from Azotobacter vinelandii by osmotic lysis of the bacteria. This nitrogenase preparation is oxygen-labile and appears to be similar, though not identical, to that obtained from Clostridium pasteurianum. The particulate characteristic and oxygen stability of previously described preparations are likely due to the method of cell disruption, e.g., in the French pressure cell. The data support a nitrogenase model system in the intact cell in which oxygen-labile enzymes are protected from oxygen by the extensive internal membranous system which Azotobacter synthesize only when they fix nitrogen.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.     

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.     

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, mu) of 0.15.h-1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15.h-1. At a lower growth rate (D = 0.09.h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 +/- 0.12). Cellular levels of flavodoxin and FeS protein II were largely constant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 microM oxygen, respectively. After 2 h of incubation, no significant decrease in the cellular levels of Av1 and Av2 could be observed. This suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.     

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.     

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N₂ fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.     

The role of cellulose-decomposing fungi in nitrogenase activity ofAzotobacter chroococcum

Azotobacter chroococcum was grown on cultures containing five carbon sources alone and also in co-cultures with three cellulolytic fungi (Aspergillus niger, Penicillium funiculosum andTrichoderma harzianum). In the absence of fungal species, nitrogenase activity was relatively low. The best nitrogenase activity was recorded in cultures containing faba bean straw followed by that in cultures having wheat straw, sugar cane leaves, carboxymethyl cellulose (CMC) or cellulose. In co-cultures with fungi,Azotobacter showed substantial nitrogenase activity on all tested substrates.Azotobacter —Trichoderma association showed the highest nitrogenase activity.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Regulation of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii grown under nitrogen or oxygen limitation.

Azotobacter beijerinckii was grown in ammonia-free glucose/mineral salts media in chemostat culture under oxygen or nitrogen limitation. Selected enzymes of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism were monitored in relation to oxygen supply for both steady and transition states. Two dissolved oxygen concentrations were used for the nitrogen-limited steady state to investigate the possible effects of respiratory protection of nitrogenase on these enzymes. The levels of NADH oxidase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase increased markedly on relaxation of oxygen limitation while pyruvate dehydrogenase and citrate synthase were relatively unaffected. beta-Ketothiolase and acetoacetyl-CoA reductase levels decreased as oxygen limitation was relaxed. Respiratory activity, as measured by the QO2 value, increased with oxygen supply rate. Imposition of oxygen limitation on a nitrogen-limited culture caused an immediate increase in the NADH/NAD ratio but this rapidly readjusted to its previous steady-state value. These changes are discussed in relation to respiratory protection of nitrogenase and poly-beta-hydroxybutyrate metabolism in A. beijerinckii.     

Selective inactivation of nitrogenase in Azotobacter vinelandii batch cultures.

When the exhaustion of sucrose or sulfate or the induction of encystment (by incubation in 0.2% beta-hydroxybutyrate) leads to termination of growth in Azotobacter vinelandii batch cultures, the nitrogenase levels in the organisms decreased rapidly, whereas glutamate synthase and glutamine synthetase levels remained unaltered. Glutamate dehydrogenase activities were low during the whole culture cycle, indicating that ammonia assimilation proceeds via glutamine. Toward depletion of sucrose or during induction of encystment, slight secretion of ammonia with subsequent reabsorption was occasionally observed, whereas massive ammonia excretion occurred when the sulfate became exhausted. The extracellular ammonia levels were paralleled by changes in the glutamine synthetase activity. The inactivation of the nitrogenase is explained as a result of rising oxygen tension, a consequence of a metabolic shift-down (reduced respiration) that occurs in organisms entering the stationary phase.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

Nitrogenase in synchronized Azotobacter vinelandii OP.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor)oxidoreductase)(EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 x 10(-3)M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.     

固氮蓝藻与棕色固氮菌固氮酶组分的交叉互补功能

本文报告了藻菌之间固氮酶组分的交叉互补试验。初步结果证明:固氮蓝藻(Anabacnaazotica水生686)的钼铁蛋白与棕色固氮菌(Azotobacter vinelandii)的铁蛋白之间存在着明显的互补功能。但这种蓝藻的铁蛋白在非细胞形态下很不稳定,易于失活。本实验为不同生理类型和不同进化程度的固氮生物之间固氮酶组分的交叉互补研究提供了新的资料。       

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.