Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Nitrogen fixation by the unicellular cyanobacterium Gloeothece. Nitrogenase synthesis is only transiently repressed by oxygen

Abstract The extent of recovery of nitrogenase activity of Gloeothece transferred from an atmosphere of O₂ to air depended on the duration of exposure to O₂. Activity recovered at increasing rates after up to 24 h exposure to O₂ and a lag before detection of activity, present after short (1 h) exposure times, disappeared with longer exposures. Synthesis of nitrogenase de novo was implicated, since chloramphenicol, tetracycline, or repressive levels of NH⁺₄, prevented recovery of activity. Specific radioimmunoassay of the rate of synthesis of the MoFe protein of nitrogenase under O₂ correlated well with the activity measurements, and indicate that a shift from air to O₂ only transiently represses nitrogenase synthesis.     

Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good "models" for studying the mechanism of nitrate inhibition of nitrogenase activity?

In soybeans ( Glycine max L. Merr.), high levels of soil nitrate inhibit N₂ fixation, and nitrate-tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short-term (i.e. 3-day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O₂ limitation of nodule metabolism that is characteristic of nitrate inhibition of N₂ fixation in soybean. In nts382, total nitrogenase activity (TNA = H₂ production in Ar:O₂) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate-treated symbioses showed less of an O₂ limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O₂ limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down-regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O₂-limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O₂ limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good “models” for studying the mechanism of nitrate inhibition of nitrogenase activity?

In soybeans ( Glycine max L. Merr.), high levels of soil nitrate inhibit N₂ fixation, and nitrate-tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short-term (i.e. 3-day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O₂ limitation of nodule metabolism that is characteristic of nitrate inhibition of N₂ fixation in soybean. In nts382, total nitrogenase activity (TNA = H₂ production in Ar:O₂) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate-treated symbioses showed less of an O₂ limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O₂ limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down-regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O₂-limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O₂ limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Ozone-induced ultrastructural changes in the plasma membrane of Chlorella sorokiniana

Abstract. Modifications in plasma membrane structure and permeability were observed in Chlorella sorokiniana following exposure to 0.2 gm⁻³(140 p.p.m.) O₃ for 30 min. Sixty-eight per cent of the cells were plasmolysed after 15 min O₃ exposure with disruption of organelles similar to that previously described in higher plants. Freeze-fracture exposed large areas of plasma membrane in 90% of the control cells and those exposed to O₃for short periods. After 20 min O₃ 90% of the cells cross-fracture, which indicates a change in molecular interactions in the membrane exposed to O₃ The earliest observed ultraslructural alteration is an aggregation of particles on the plasma membrane P face, statistically significant after 10 min O₃ Changes in ⁸⁶Rb influx occur during a similar time. After more extended exposure to O³ the plasma membrane P face shows regions of lipid phase transition to the crystalline state.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Protective mechanisms of nitrogenase against oxygen excess and partially-reduced oxygen intermediates

Diazotrophic systems have developed a number of strategies to protect nitrogenase (N₂ase; EC 1.18.6.1) from O₂ excess and active-oxygen species (AOS). Protection against O₂ excess is given by biochemical modifications of N₂ase, increased rates of low-efficiency respiration, temporal segregation of N₂ fixation and photosynthesis, physical barriers to O₂ diffusion, and hemoglobins. On the other hand, AOS may originate from oxidation of N₂ase components, ferredoxins, flavodoxins and hemoglobins; interaction among the AOS themselves, or between H₂O₂ and hemoglobins; and during reactions catalyzed by hydrogenase (EC 1.18.99.1), xanthine oxidase (EC 1.1.3.22) and uricase (EC 1.7.3.3). Active-oxygen species are scavenged enzymatically [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6). peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11)] or through non-enzymic reaction with low-molecular-weight compounds (ascorbate, α-tocopherol, glutathione).     

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Abstract The effect of certain nitrogen compounds on nitrogenase activity was studied in cells of Azospirillum brasilense strain Sp6, grown under microaerophilic conditions with nitrogenase fully derepressed. 0.5 mM NH₄Cl, 0.5 mM glutamine, 1.0 mM KNO₃ and 0.1 mM KNO₂ completely blocked nitrogenase activity. 1.0 mM asparagine, 1.0 mM aspartate, 1.0 mM histidine and 1.0 mM adenine did not caused no inhibition of nitrogenase; indeed asparagine, aspartate and histidine showed a slight stimulatory effect on N₂ fixation. The addition of 10 mM dl -methionine- dl -sulphoximine prevented the inhibitory effect of NH₄Cl and glutamine but did not counteract the effect of KNO₂. Rifampicin and chloramphenicol did not prevent the inhibition of nitrogenase by NH₄Cl.     

In search of the mechanism of nitrate inhibition of nitrogenase activity in legume nodules: Recent developments

The mechanisms involved in the inhibition of nitrogenase activity in legume nodules by nitrate is unclear. This paper reviews and evaluates proposed mechanisms of this inhibition. Emphasis is placed on recent developments, which suggest that nitrate causes an O₂ limitation of nitrogenase activity. Several mechanisms that involve a nitrate-induced increase in resistance to O₃ diffusion in the nodule cortex are discussed.     

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N₂ may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at ≈ 10–15 m depth in July and October had a δ¹⁵N of 0.5–1.5‰. These values closely reflect the ¹⁵N composition of atmospheric N₂, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at ≈ 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia . Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Interaction of elevated CO2 and O3 on growth, photosynthesis and respiration of three perennial species grown in low and high nitrogen

Seedlings of three species native to central North America, a C₃ tree, Populus tremuloides Michx., a C₃ grass, Agropyron smithii Rybd., and a C₄ grass, Bouteloua curtipendula Michx., were grown in all eight combinations of two levels each of CO₂, O₃ and nitrogen (N) for 58 days in a controlled environment. Treatment levels consisted of 360 or 674 μmol mol^-1 CO₂, 3 or 92 nmol mol^-1 O₃, and 0.5 or 6.0 m M N. In situ photosynthesis and relative growth rate (RGR) and its determinants were obtained at each of three sequential harvests, and leaf dark respiration was measured at the second and third harvests. In all three species, plants grown in high N had significantly greater whole-plant mass, RGR and photosynthesis than plants grown in low N. Within a N treatment, elevated CO₂ did not significantly enhance any of these parameters nor did it affect leaf respiration. However, plants of all three species grown in elevated CO₂ had lower stomatal conductance compared to ambient CO₂-exposed plants. Seedlings of P. tremuloides (in both N treatments) and B. curtipendula (in high N) had significant ozone-induced reductions in whole-plant mass and RGR in ambient but not under elevated CO_2. This negative O₃ impact on RGR in ambient CO₂ was related to increased leaf dark respiration, decreased photosynthesis and/or decreased leaf area ratio, none of which were noted in high O₃ treatments in the elevated CO₂ environment. In contrast, A. smithii was marginally negatively affected by high O_3.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

Effects of age, supra-ambient oxygen and repeated assays on acetylene reduction and root respiration in pea

An open flow-through gas system was used to investigate the effect of plant age on nitrogenase activity in relation to root respiration (measured as CO₂ release) and supra-ambient O₂ levels in 24- to 51-day-old, nodulated Pisum sativum L. cv. Bodil. The effect of assaying plants repeatedly was also studied. The respiratory efficiency of nitrogenase [mol CO₂ (mol C₂H₄)⁻¹] and the relative decline in nitrogenase (EC 1.7.99.2) activity in response to introduction of C₂H₂ in the gas stream were unaffected by plant age. In contrast, the nitrogenase-linked respiration as a proportion of total root respiration increased with time. Accordingly, the specific respiration linked-to growth and maintenace of the noduled root system decreased with time. C₂H₂ reduction and root respiration were increased by supra-ambient O₂ levels, but the tolerance to high O₂ concentrations seemed to decrease with plant age. Repeated C₂H₂ assays on the same plants decreased their rate of growth and N accumulation: in addition, nitrogenase activity and root respiration were somewhat negatively affected. The results indicate that results from experiments with plants of different ages cannot always be directly compared, and that repeated C₂H₂ assays on the same plants should be applied with caution in physiological work.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Hydrogen metabolism and energy costs of nitrogen fixation

Abstract The high energy costs of biological nitrogen fixation are partly caused by hydrogen production during the reduction of dinitrogen to ammonia. Some nitrogen-fixing organisms can recycle the evolved hydrogen via a membrane-bound uptake hydrogenase. The energetic aspects of hydrogen metabolism and nitrogen fixation are discussed.
Studies on both isolated nitrogenase proteins and nitrogen-fixing chemostat cultures show that energy limitation will result in a high hydrogen production by nitrogenase. In plant- Rhizobium symbiosis, the supply of oxygen or photosynthetate is the limiting factor for nitrogen fixation. In both cases, nitrogen fixation is energy-limited, and it is concluded that a large amount of hydrogen is produced during nitrogen fixation in these symbioses.
Hydrogen reoxidation yields less energy than the oxidation of endogenous substrates, and therefore expression of hydrogenase under oxygen-limited conditions is energetically unfavourable. Moreover, hydrogen reoxidation can never completely regain the energy invested during hydrogen production. The controversial reports of the effect of hydrogen reoxidation on the efficiency of nitrogen fixation are being discussed.
The determination of the energy costs of nitrogen fixation (expressed as the amount of ATP needed to fix 1 mol of N₂) using chemostat cultures is described. Calculations show that the nitrogenase-catalysed hydrogen production has more influence on the efficiency of nitrogen fixation than the absence or presence of a hydrogen uptake system.     

Measurements of photosynthesis and respiration in plants

Methods for measuring the rates of photosynthesis and respiration in plants are reviewed. Closed systems that involve manometric techniques, ¹⁴CO₂ fixation, O₂ electrodes and other methods for measuring dissolved and gas phase O₂ are described. These methods typically provide time-integrated rate measurements, and limitations to their use are discussed. Open gas exchange systems that use infra-red CO₂ gas analysers and differential O₂ analysers for measuring instantaneous rates of CO₂ and O₂ exchange are described. Important features of the analysers, design features of gas exchange systems, and sources of potential error are considered. The analysis of chlorophyll fluorescence parameters for estimating the quantum yield for O₂ evolution and CO₂ fixation is described in relation to new fluorescence imaging systems for large scale screening of photosynthetic phenotypes, and the microimaging of individual chloroplasts.