Influence of some environmental factors on nitrogen fixation in the rhizosphere of rice

Summary Nitrogen fixers make up a large percentage of the total microflora in the rhizosphere of lowland rice. There are more aerobic nitrogen fixers than there are anaerobic ones. When soil crumbs from the root zone were placed on a nitrogen free agar medium and inoculated at 0, 5, 10, and 21 percent oxygen concentration, colonies of aerobic nitrogen fixers reached their greatest diameter at 5 and 10 percent oxygen. In acetylene reduction assays rice plants grown in paddy fields and in solution culture were tested for the nitrogenase activities of their roots at different oxygen tensions. Nitrogenase activity was highest at 3 percent oxygen, lower at 0 percent, and far lower at 21 percent. When rice was grown in solution culture the redox potential of the nutrient solution strongly influenced nitrogenase activity. With declining redox potential, nitrogenase activity increased to a maximum value but dropped sharply as redox potential further decreased. Ten ppm of combined nitrogen as urea depressed nitrogenase activity on excised roots. Combined nitrogen applied to one part of the root system affected, to some extent, nitrogen fixation on other roots kept in a solution without nitrogen. Nitrogenase activity in a fertility trial with lowland rice, examined at several dates, showed no inhibitory effect of fertilizer nitrogen, however, presumably because the nitrogen concentration in the soil solution rapidly decreased. Instead, an overall stimulating effect of nitrogen dressing was noticeable. Diurnal fluctuations of nitrogenase activity in the rhizosphere, with a peak in the afternoon and low fixation rates after low solar radiation, suggest a photosynthetic effect on nitrogen fixation. re]19751208     

Oxygen Sensitive Nitrogen-Fixing Mutants of Anabaena

Two Anabaena mutants having heterocysts but incapable of fixing molecular nitrogen in air have been isolated by using ultraviolet radiation or NTG mutagenesis. Their vegetative cells differentiated into heterocysts at a higher frequency than that of the wild type. The phenotype of the mutants is stable and a low frequence of spontaneous reversion was observed. Under microaerobic condition the mutants cells can express the genetic information which encodes nitrogenase synthesis and were capable of utilizing nitrogen for growth with a low acetylene reductiop activity. The level of nitrogenase activity was correlated reciprocally with the content of cell phycocyanin and the light intensity. Both synthesis and activity of the mutant nitrogenase were very sensitive than wild type to the oxygen in vive. Introduction of 1% O2 (v/v) into the gas phase inhibited evidently acetylene reduction. Exposure of the mutant suspension to 20% O2 (v/v) resulted in total and irreversible denaturation of nitrogenase. Withdrawing of O2 in gas phase, the nitrogenase was synthesized de nero; The synthesis process was repressed by chloramphenical or ammonia. The nitrogenase activity of mutant cells increased significantly either by nitrogen- starvating to decrease the phycocyanin content or by lowering the light intensity. Specifically, during the anaerobic induction by treating the mutants filaments with diehloromethylurea which prevents photosynthetic oxygen production, the specific activity of mutant nitrogcnase was equivalent nearly to that of wild type. The ability to reduce 2, 3, 5-triphenyltetrazolium was lower in heterocysts and vegetative cells of mutants than in that of wild type. The results suggest that the oxygen sensitivity of nitrogen fixation by heterocystous bluegreen algal mutants may be duc to the defect of some enzymic systems which might play a role in scavenging oxygen toxity, so that the process of nitrogen fixation is inhibited by the active oxygen produced by vegetative cells. The mechanism of protecting nitrogenase from oxygen damage in blue-green algae is discussed.     

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.     

葡萄糖和氢对Gunnera／Nostoc固氮共生体固氮活力的影响

Ｇｕｎｎｅｒａ／Ｎｏｓｔｏｃ固氮共生体固氮相对效率（ＲＥ）可在０．２６～０．８０之间变动,而不是一个常数。外加１．５％葡萄糖液可使其固氮活力提高约１００％,同时也使组织的呼吸速率提高了近１６０％。加外源Ｈ２可使其固氮活力提高近１００％,但却使组织的呼吸速率降低了近５０％。正常生长条件下的组织净放Ｈ２量较低．而外源２％葡萄糖液可使组织净放Ｈ２量提高近２倍。外加５ｍｍｏｌ／Ｌ的ＮＨ１Ｃｌ溶液可使其固氮活力下降约７０％。故认为Ｇｕｎｎｅｒａ／Ｎｏｓｔｏｃ共生体固氮活力受碳水化合物供应状况及比代谢两者构成的“还原力库”或“电子库”的调节,在此“还原力库”中,Ｈ２代谢起到了一个“中间调节者”的作用。     

Nitrogenase Activity and Photosynthesis in Plectonema boryanum

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.     

The Relationships between Nitrogen Fixation and Several Kinds of Energy Metabolism by Gloeocapsa sp

Gloeocapsa sp., a species of anicellular blue-green alga, fixes dinitrogen mostly under light. The energy (ATP and reductant) needed for nitrogen fixation may be provided by photoreaction and aerobic catabolism. The nitrogenase activity (acetylene reduction) in vivo was decreased under the conditions of dark and inhibition of photo-phosphorylation or oxidative phosphorylation in the light. When photosystem Ⅱ was inhibited by the presence of DCMU, nitrogenase activities in both reactions of acetylene reduction and hydrogen evolution may be muchenhanced probably due to eliminating of the damage caused by the oxygen produced in the photolysis of water. The effects of the oxygen present in the atmosphere of the reaction systemand produced by the cells are different. It is shown that some trace oxygen seems to be required for nitrogen fixation by the energy supply of aerobic actabolism and oxidative phosphorylation. While the fixation of dinitrogen was inhibited by CO or no any reducible substrate was present, 70-100% of the energy accepted by nitrogenase was evolved as hydrogen. The algal cells also showed hydrogen uptake reaction, but no enhancement of nitrogen fixation by the hydrogen uptake was found.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

The Oxygen-Scavenging System Protecting Nitrogenase from Oxygen in Cells of Blue-Green Algae

There is a heat stable oxygen-scavenging system (OSS) associated with membrane which reduces oxygen endogenously in cells of blue-green algae. Addition of the OSS to cell suspension of heterocystous oxygen sensitive Anabaena mutant and non-heterocystous Pleetonema boryanum led to an increase in their nitrogenase activity by 10–100-fold higher than those under microaerobic condition and also could restore effectively their acetylene reduction activity at higher oxygen concentration since the oxygen presented was reduced effectively. The results suggest that the OSS possesses a function protecting nitrogenase from oxygen in cells. Furthermore, it was found that the efficiency of reducing oxygen of OSS from the Anabaena mutant and Plectonema was lower than those from Anabaena wild and Gloeocapsa in atm. oxygen level. This may be ralated with the susceptibility of nitrogen fixation to oxygen in the cells of Anabaena mutant and Plectonema. The present study firstly indicades the relationship between the heat stable OSS associated with membrane and the mechanism of protecting nitrogenase from oxygen in cells of blue-green algae. Activities of catalase, peroxidase and superoxide dismutase do not show obvious difference in cellfree extract of Anabaena wild and mutant. Methyl viologen can induce nitrogenase activity of Anabaena mutant by subverting a portion of electon flow to accelerate oxygen reduction.     

蓝藻Anabaena 7120光漂白细胞固氮的研究

蓝藻Anabaena 7120经光漂白后固氮活性明显下降,转入正常光照下又恢复活性。此种经光漂白的蓝藻细胞,其固氮活性对氧敏感度小,受分子氢的促进大些,而忍受CO_2和N_2抑制的浓度相对高些。其固氮活性为弱光和光合抑制剂减弱,而加入外源的碳水化合物则能提高它的固氮活性。当碳水化合物和光合抑制剂一起加入反应系统时,蓝藻光漂白细胞的固氮活性并不能受到促进。     

Increased Respiration Through Cytochrome d Enhances Microaerobic N2Fixation in Klebsiella Pneumoniae

Nitrogenase activity was increased in a Klebsiella pneumoniae strain (FN27) producing higher amounts of cytochrome d than the wild-type strain. The increased production of cytochrome d in FN27 showed a positive effect on nitrogenase activity in cells cultured with glucose as carbon source at 1 kPa oxygen but a negative effect at higher O₂concentrations. In cells cultured with pyruvate as carbon source, FN27 expressed higher activity of nitrogenase at all oxygen tensions tested when compared to the wild-type strain. This analysis shows that the over production of cytochrome d terminal oxidase improves nitrogen fixation in certain culture conditions.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Biological Control of the Resistance to Oxygen Flux in Nodules

Experiments have been conducted to investigate the responseof symbiotic nitrogen fixation in white clover to differentconcentrations of oxygen around the root. Over a wide rangeof oxygen concentrations (21–80 per cent) there was littlevariation in respiration and no evidence of damage to nitrogenase.It is suggested that, in the absence of respiratory protection,oxygen damage to nitrogenase is prevented by changes in theresistance to gaseous diffusion. Trifolium repens L, white clover, nitrogen fixation, nitrogenase, oxygen flux     

斯氏假单胞菌A1501固氮新基因PST1305的功能分析

摘要：【目的】研究斯氏假单胞菌A1501基因组“固氮岛”中PST1305基因在A1501生物固氮过程中所起的作用。【方法】利用同源重组与三亲接合的方法构建PST1305的非极性突变株。乙炔还原法测定固氮酶活。RT-PCR分析PST1305基因与其周围基因转录单元的关系,Real-Time PCR比较PST1305在最佳固氮与非固氮条件下表达水平的差异。【结果】突变株np1305的固氮酶活显著降低,功能互补菌株np1305Comp能基本恢复细胞的固氮作用。PST1305与其上游的nifB、fdxN、下游的nifQ等基因位于同一个转录单元,组成一个操纵子。基因芯片表明,PST1305基因在固氮比非固氮条件下表达量显著上调（约38.7倍）,Real-Time PCR验证支持这一结果。【结论】PST1305基因参与固氮过程,其突变会影响固氮酶的活性,该基因可能通过参与A1501固氮酶电子传递或者固氮酶的氧保护过程影响固氮效率。     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. II. Ionically adsorbed cells

Continuous culture studies of Azotobacter vinelandii cells immobilized by ionic adsorption to Cellex E anion exchange resin were conducted under oxygen-limited conditions for comparison to free-cell cultures. Immobilization had little effect upon the specific respiration and sucrose consumption rates as compared to free cells. However, maxima in specific nitrogen fixation rate and nitrogenase activity as a function of dissolved oxygen occurred at a C(O(2) ) value of approximately 0.005 mM as opposed to 0.02 mM for free cells. Further, in contrast to free-cell culture, most of the fixed nitrogen appeared in the medium rather than within intact cells. There were strong indications that reproduction of bound cells often resulted in cell lysis accounting for the fixed nitrogen content in solution.     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

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