Reversible dark-induced senescence of soybean root nodules

Nodule senescence was induced in intact soybean [Glycine max. (L.) Merr., cv Woodworth] plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod.     

Diazotrophic growth of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata under microaerobic conditions in the dark

Diazotrophy of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata was not obligatorily linked to photosynthesis. In the dark R. acidophila grew with dinitrogen as sole nitrogen source at a dissolved oxygen tension of 15 Torr (= 2.0 kPa); the doubling time was 8 h. Acetylene reduction by whole cells was more sensitive to oxygen in the light than in the dark. 16.5 mg N₂ were fixed per g lactic acid consumed. R. capsulata synthesized nitrogenase and fixed dinitrogen in the dark at a dissolved oxygen tension of less than one Torr (= 0.13 kPa). The doubling time of this bacterium was 16 h and 10.5 mg N₂ were fixed per g lactic acid consumed.Abbreviation kPa kilopascal     

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.     

Nitrogen fixation and ammonia switch-off in the photosynthetic bacterium Rhodopseudomonas viridis.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.     

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non‐heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro‐oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark‐mediated nitrogenase in the absence of DCMU nor light‐mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non‐axenic cultures of L. nodulosa. RT‐PCR demonstrated that this gene was expressed only in non‐axenic cultures. Western blotting showed that the Fe‐protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.     

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.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

A pellet method to demonstrate nitrogen fixation by free-living rhizobia

Summary A method is described to demonstrate nitrogen fixation by free-living Rhizobium cells. After aerobic growth in a nutrient solution, the bacteria are centrifuged. Acetylene reduction by the rhizobial cells in the pellet can be measured within a few days. Hydrogen gas frequently stimulates acetylene reduction.     

Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of ¹⁴C₂H₂ to slurries resulted in the formation of ¹⁴CO₂ and the transient appearance of ¹⁴C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C₂H₂ to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C₂H₂.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora.

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Short-term influence of nitrate on acetylene reduction, net photosynthesis and nodule respiration in black alder (alnus glutinosa L. gaertn.) seedlings

The use of nitrogen-fixing trees such as black alder (Alnus glutinosa L. Gaertn.) as forest silvicultural tools has recently been recognized. The potential benefit of black alder in silvicultural practices may be reduced by nitrate fertilization. Fifteen-month-old, nodulated, black alder rooted cuttings were fertilized for 6 days with 0, 7.5 or 15 mM NO₃ to determine the influence of nitrate on acetylene reduction, nodule respiration and net photosynthesis. Acetylene reduction, net photosynthesis and nodule respiration were measured on the second, fourth and sixth days of nitrate application. Nitrate treatment significantly reduced acetylene reduction and nodule respiration by day 4. Acetylene reduction was 75% lower and nodule respiration 36% lower for the 15 mM NO₃ treatment when compared to that of the control treatment. By day 6, net photosynthesis and nodule respiration were significantly reduced by 29 and 59%, respectively, for seedlings treated with 15 mM NO₃. This study suggests that nitrate fertilization has a profound influence on nitrogenase activity and that nitrogen-fixing tree species may respond to nitrate fertilization by shifting photosynthetic rates.     

Nitrogen fixation in the stag beetle, Dorcus (Macrodorcus) rectus (Motschulsky) (Col., Lucanidae)

Abstract:  Stag beetles are xylophagous insects that feed mainly on dead wood. They play an important role in the decomposition of dead wood in forest ecosystems. Most dead wood contains 1% nitrogen at most. It is suspected that stag beetles can utilize atmospheric nitrogen. We show that the larvae of Dorcus ( Macrodorcus ) rectus exposed to nitrogen reduce acetylene to ethylene in a time-dependent fashion. No reaction was detected with the dead wood or autoclaved larvae, suggesting that living larvae use the reaction for fixing nitrogen. Acetylene reduction to ethylene by larvae increased with incubation time. This effect was not seen using decayed wood only, autoclaved wood only or autoclaved larvae. Acetylene reduction by the larva proceeded at 1.25 ± 0.37 nmol acetylene/h/g (fresh wt), corresponding to the fixation of 0.25  μ g nitrogen per day per larva.     

Hydrogenase activity in nitrogen-fixing methane-oxidizing bacteria

Hydrogenase activity in cells of the nitrogen-fixing methane-oxidizing bacterium strain 41 of the Methylosinus type increased markedly when growth was dependent upon the fixation of gaseous nitrogen. A direct relationship may exist between hydrogenase and nitrogenase in this bacterium. Acetylene reduction was supported by the presence of hydrogen gas.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO₃⁻ at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO₃⁻-N per hectare and harvested plants treated with 40 kilograms NO₃⁻-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N₂-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.     

Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae.

Acetylene reduction activity of intact rice plants was measured in closed assay chambers with plants grown in water culture. Acetylene was added to the liquid medium, and the ethylene formed was measured from both gas and liquid phases. After cutoff of mineral nitrogen supply and inoculation of fresh soil, rice plants grown from the seedling stage in water culture exhibited acetylene reduction activity after a lag period. However, rice plants grown in a paddy field and transferred to water culture were more suitable for N₂ fixation studies because of their higher, less variable acetylene reduction activity. The time course of acetylene reduction was monitored by continuous circulation of gas between the gas phase and the liquid phase, and the result showed an initial 2- or 3-h period of lower activity, followed by increased and almost constant activity up to 24 h. The effects on acetylene reduction activity of aeration, ammonium, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition are reported. Ammonium was inhibitive at 0.33 mM, and its depressive effect was alleviated by ammonium uptake by the plants.     

Implications of Competitive Inhibition in the Acetylene Reduction Assay for Dinitrogen Fixation

An earlier paper which analysed the implications of diffusionlimitation for acetylene reduction by intact legume noduleshas recently been criticized for ignoring competitive inhibitionof acetylene reduction by dinitrogen. Mathematical analysesof competitive inhibition show that the dependence of acetylenereduction rate on acetylene concentration under atmosphericN₂ is described adequately by an equation functionally equivalentto the Michaelis—Menten equation, despite competitiveinhibition. Therefore, no modification of the original analysisis required. However, competitive inhibition is shown to bea significant factor when experiments under atmospheric N₂ andunder argon are compared. Acetylene reduction, dinitrogen fixation, competitive inhibition, diffusion limitation, legume nodules     

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL     

Nitrogenase. VI. Acetylene reduction assay: Dependence of nitrogen fixation estimates on component ratio and acetylene concentration.

Acetylene reduction, an assay for nitrogenase activity (nitrogen:(acceptor) oxidoreductase, EC 1.7.99.2), Is dependent on the ratio of the two protein components of nitrogenase as well as on C2H2 concentration. As the component I : component II ratio (based on activity) is increased, the C2H2 reduction : N2 fixation ratio decreases to a minimum of 3.4 and then increases. The minimum is found at a ratio near 1 : 1. At a component I : component II ratio of 20 : 1, the C2H2 reduction : N2 fixation ratio is 5.3. Acetylene exhibits substrate inhibition in assays for nitrogenase activity. Both the apparent Km and Ki for acetylene vary as a function of the relative concentrations of components I and II present in the assay. When the more labile component II is limiting in the assay and "saturating" levels of C2H2 (above 0.1 atm) are used, N2-fixation capacity may be greatly under-estimated.