The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

The potential for diazotrophy in iron-and sulfur-oxidizing acidophilic bacteria

Acetylene reduction was observed with ferrousiron-oxidizingThiobacillus ferrooxidans, as expected from previous studies with this bacterium. Acetylene reduction was also found during the growth ofT. ferrooxidans on tetrathionate. OnlyLeptospirillum ferrooxidans, one of several other phylogenetically diverse, ferrous-iron-and/or sulfur-oxidizing acidophilic microorganisms, also reduced acetylene. A reduction of the oxygen concentration in the culture atmosphere was necessary to alleviate inhibition of nitrogenase activity. DNA sequences homologous tonif structural genes were found in both organisms. Diazotrophic growth ofL. ferrooxidans was inferred from an increase in iron oxidation in ammonium-free medium when the oxygen concentration was limited and from apparent inhibition by acetylene under these conditions.     

In vivo and in vitro studies on ATP and electron donors to nitrogenase in Rhodospirillum rubrum

The photosynthetic bacterium, Rhodospirillum rubrum (ATCC 11170), was tested for its ability to fix nitrogen (acetylene reduction) under aerobic and dark-anaerobic conditions. Whole cells reduced acetylene under darkanaerobic conditions if pyruvate was supplied. Reactions of the cells were inhibited less by oxygen in the dark than in the light, and the cells were capable of acetylene reduction in the presence of low levels of oxygen (0.6%) in the dark. Crude extracts of R. rubrum reduced acetylene if pyruvate and Coenzyme A were added; ferredoxin from R. rubrum greatly increased the pyruvate-driven activity in crude extracts. It was not possible to demonstrate light-driven acetylene reduction in crude extracts unless a reductant (dithionite) was added.Abbreviations Fld flavodoxin - DTT dithiothreitol     

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.Addition of sodium molybdate, a specific inhibitor of sulphate reduction inhibited acetylene reduction activity in Z. noltii colonised sediments by > 80% as measured by both slurry and whole core techniques irrespective of the light regime, throughout the year inferring that sulphate reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. A mutualistic relationship between Z. noltii and nitrogen fixing SRB in the rhizosphere, based on the exchange of organic carbon and fixed nitrogen is proposed. In uncolonised sediments sodium molybdate initially severely inhibited acetylene reduction rates, but the level of this inhibition declined over the course of the year. These data indicate that the nitrogen fixing SRB associated with the Zostera roots and rhizomes were progressively replaced by an aerobic population of nitrogen fixers associated with the decomposition of this recalcitrant high C:N ratio organic matter.Acetylene and sulphate reduction rates in the seagrass beds showed distinct summer maxima which correlated with a reduced availability of NH ₄ ⁺ in the sediment and the growth cycle of Z. noltii in the Bassin. Overall, these data indicate that acetylene reduction (nitrogen fixation) activity in the rhizosphere of Z. noltii was regulated both by release of organic carbon from the plant roots and maintenance of low ammonium concentrations in the root zone due to efficient ammonium assimilation.Nitrogen fixation rates determined from acetylene reduction rates measured by the whole core technique ranged from 0.1 to 7.3 mg N m^–2 d^–1 in the Z. noltii beds and between 0.02 and 3.7 mg N m^–2 d^–1 in uncolonised sediments, dependent upon the season. Nitrogen fixation in the rhizosphere of Z. noltii was calculated to contribute between 0.4 and 1.1 g N m^–2 y^–1 or between 6.3 and 12% of the annual fixed nitrogen requirement of the plants. Heterotrophic nitrogen fixation therefore represents a substantial local input of fixed nitrogen to the sediments of this shallow coastal lagoon and contributes to the overall productivity of Z. noltii in this ecosystem.     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

Nitrogen fixation and nitrate utilization by marine and freshwater Beggiatoa

Four newly isolated marine strains of Beggiatoa and five freshwater strains were tested for nitrogen fixation in slush agar medium. All strains reduced acetylene when grown microaerobically in media containing a reduced sulfur source and lacking added combined nitrogen. The addition of 2 mmol N, as nitrate or ammonium salts, completely inhibited this reduction. Although not optimized for temperature or cell density, acetylene reduction rates ranged from 3.2 to 12 nmol·mg prot^-1 min^-1. Two freshwater strains did not grow well or reduce acetylene in medium lacking combined nitrogen if sulfide was replaced by thiosulfate. Two other strains grew well in liquid media lacking both combined nitrogen and reduced sulfur compounds but only under lowered concentrations of air. All freshwater strains grew well in medium containing nitrate as the combined nitrogen source. Since they did not reduce acetylene under these conditions, we infer that they can assimilate nitrate.     

Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn²⁺ significantly inhibited activity. The K _m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.     

Nitrogen fixation by hydrogen-utilizing bacteria

Seventeen strains of nitrogen-fixing bacteria, isolated from different habitats on hydrogen and carbon dioxide as well as on other substrates, morphologically resembled each other. All strains, including Mycobacterium flavum 301, grew autotrophically with hydrogen. The isolate strain 6 was sensitive to oxygen when dependent on N₂ as nitrogen source, a consequence of the sensitivity of its nitrogenase towards oxygen. At the same time, strain 6 was sensitive to hydrogen when growing autotrophically on N₂ as nitrogen source, but hydrogen did not affect acetylene reduction by these cells.Abbreviations MPN Most probable number - BS medium basal salts medium     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

The effects of soybean cultivar,rhizobium strain and nitrate on plant growth,nodule mass and acetylene reduction rate

Summary The effects of twelve strains ofBradyrhizobium japonicum and ten cultivars of soybean (Glycine max (L.) Merr.) on plant and nodule weights, and acetylene reduction rates (33 to 41 days) were measured in the presence and absence of 6mM nitrate. No interactions between strains and cultivars were observed. Strain by nitrate interactions were found for plant and nodule weights, and acetylene reduction rates per gram of nodule. Cultivar by nitrate interactions were found for nodule weights, acetylene reduction rates per plant and per gram of nodule. Blackhawk with all strains, and all cultivars with strains 110 and CB 1809, seemed to be able to grow as well in the absence of nitrate (utilizing nodule fixation) as in its presence. The problems of identifying strains and cultivars with especially good nitrogen fixing ability in the presence of nitrate are discussed.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

The influence of shading on associative N2 fixation

Summary The effect of reduced solar radiation on associative N₂-fixation and plant parameters was studied in three field experiments (1978–80). Gahi-3 pearl millet (Pennisetum americanum (L.) K. Monch.) field plots were shaded with saran shade cloth that reduced solar radiation by 50% and 75%. Acetylene reduction activity (ARA) was reduced by shading in one of the three experiments. The two non-responding experiments were conducted on a wall-drained, low-activity site (ARA means ranging 17–68 n moles ethylene core^–1 h^–1), the responding experiment was conducted on a poorly drained, high-ARA site.Shading affected the plants drastically, reducing fresh weight and dry matter yields up to 46% (50% shade) and 57% (75% shade). Shading also reduced dry matter percentage from 19.6 (no shade) to 15.3 (75% shade) and increased nitrogen content from 0.6% (no shade) to 1.53% (75% shading). However, shading did not affect protein yield. Inoculation withAzospirillum brasilense had no measurable effect on yield or acetylene reduction in the first two experiments.In the third experiment, shading reduced mean ARA of inoculated plots over 100% but had no significant effect on control plots. Inoculation significantly increased ARA in the nonshaded plots but not in shaded plots. Acetylene reduction activity was high, with means ranging between 208 and 465 n moles ethylene evolved core^–1 h^–1. Soil moisture and millet growth stage also affected acetylene reduction activity.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Metabolism of acetylene by Nocardia rhodochrous.

A Nocardia rhodochrous strain capable of utilizing acetylene as its sole source of carbon and energy exhibited slow growth on low concentrations of acetaldehyde. Resting cells incubated with acetylene formed a product identified as acetaldehyde, but attempts to demonstrate acetylene hydrase activity in cell-free extracts were unsuccessful. Acetaldehyde dehydrogenase in N. rhodochrous was found to be NAD+ linked and nonacylating, converting acetaldehyde to acetate. Specific activities of acetaldehyde dehydrogenase, acetothiokinase, and isocitrate lyase were enhanced in cells grown on acetylene and ethanol as compared with cells grown on alternate substrates. These results suggest that acetylene is catabolized via acetaldehyde to acetate and eventually to acetyl coenzyme A. Acetylene oxidation in N. rhodochrous appears to be constitutive and is not inhibited in the presence of either ethylene, nitrous oxide, or methane.     

Response ofPanicum maximum Jacq. to inoculation withAzospirillum brasilense

Summary The effects on plant dry weight and acetylene reduction activity after applyingAzospirillum brasilense (strain 13t) to guineagrass,Panicum maximum Jacq., grown in clay pots under greenhouse conditions, are reported and discussed.     

Properties of heterocysts isolated with colloidal silica

A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and Na₂S₂O₄ reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13% of the activity of the intact filaments was accounted for in the isolated heterocyst preparation.Isolated heterocysts took up O₂ in a light-independent process; O₂ uptake with added NADP⁺ was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway.     

Nouvelle méthode pour mesurerin situ l'activité nitrogénase par réduction de l'acétylène

Résumé Les auteurs décrivent un dispositif qui permet de mesurer l'activité nitrogénasein situ par réduction de l'acétylène chez les plantes fixatrices d'azote, sans perturber leur physiologie et sans les détruire. Le système racinaire est isolé de l'atmosphère extérieure par une substance mucilagineuse déposée sur le sol autour des plantes testées. Les gaz acétylène ou éthylène sont injectés ou prélevés directement dans le sol au niveau des nodosités. Avec ce nouveau dispositif les valeurs de l'activité réductrice d'acétylène obtenues surTrigonella foenum-graecum L. etMedicago sativa L. cultivés en plein champ, sont très supérieures à celles mesurées sur ces mêmes végétaux enfermés dans une enceinte en polyéthylène. Cette méthode peu onéreuse, permet de détecter des activités réductrices faibles; elle est utilisable sur des jeunes plantes et également sur des individus ramifiés à la base.

---

New method forin situ measuring of nitrogenase activity by acetylene reduction

Summary A non-destructive method using a special device for measuringin situ acetylene reducting activity by nitrogen fixing plants is described. Plant roots are isolated from external atmosphere with a mucilaginous material laid on the soil around the plants. Acetylene or ethylene is directly injected into or taken from the soil around the nodules. Using this device the values of acetylene reducing activity ofTrigonella foenum graecum L. andMedicago sativa L. are much higher than those obtained with the same plants placed under polyethylene bags. This method is not expensive and allows the detection of low enzyme activities. It doesn't perturb plant physiology and can be used for young plants as well as for plants with ramified stems at their base.

     

The influence of temperature and nitrate on vegetative growth and nitrogen accumulation by nodulated soybeans

Summary Inoculated soybeans [Glycine max (L.) Merrill] were grown in controlled environments to evaluate the relationship between temperature and applied NO₃−N on growth rates, N accumulation, and acetylene reduction activity during the vegetative growth stage. Soybeans were grown at day/night temperatures of 22/18 and 26/22°C in sand culture with daily applications of 21.4 mM (high) and 2.1 mM (low) NO₃−N in a complete nutrient solution for durations of 14, 21, and 42 days after emergence and with an N-free solution. Dry matter and N accumulation were greater at 26/22 than 22/18°C. In general, both increased as the level and duration of applied NO₃−N was increased. These increases were attributable to an abbreviation in the interval between emergence and onset of rapid growth. The presence and assimilation of NO₃−N, even at the high level, did not inhibit development of functional nodules. Neither mass nor acetylene reduction activity of nodules was reduced by high NO₃−N; however, the root mass was increased by NO₃−N more than the nodule mass. There was an interaction between temperature and NO₃−N on specific nodule activity as measured by acetylene reduction. The specific nodule activity was unaffected by NO₃−N at 22/18°C, but at 26/22°C the specific activity was lower in the absence of NO₃−N than when NO₃−N was present. Apparently, rapid early growth at 26/22°C depleted cotyledonary reserves of N before nodules became active and, thereafter, the plants were unable to develop adequate leaf area to support nodule development and functioning. This result has implications in N fertilization of late-planted soybeans. Paper number 6637 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina, 27650. The research was supported in part by a grant from the North Carolina Soybean Producers Association and by USDA-SEA-CR grant 701-15-26.