Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Isolation and characterization of a metronidazole-resistant (Mtn-R) mutant strain of Nostoc muscorum affected in ammonium regulation of heterocyst formation and uptake hydrogenase activity

Abstract The metronidazole-resistant ( Mtn-R ) mutant strain of N. muscorum produced drug-resistant NADPH: ferredoxin (Fd) oxidoreductase and showed derepression of heterocyst formation and uptake hydrogenase activity in NH₄⁺-medium. The observation of NH₄⁺-repression in regulation of nitrogenase activity alone in the mutant strain suggests, that heterocyst formation and nitrogenase activity are regulated by two separate NH₄⁺-repression control systems, one specific for heterocyst and uptake hydrogenase and the other for nitrogenase. The partial drug-resistant NADPH: Fd oxidoreductase enzymatic activity seems to be the reason for drug-resistant growth of the cyanobacterium in N₂-medium and NH₄⁺-medium.     

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.     

Nodulation and nitrogenase activity in nitrogen-fixing woody plants stimulated by CO2 enrichment of the atmosphere

The responses of three species of nitrogen-fixing trees to CO₂ enrichment of the atmosphere were investigated under nutrient-poor conditions. Seedlings of the legume, Robinia pseudoacacia L. and the actinorhizal species, Alnus glutinosa (L.) Gaertn. and Elaeagnus angustifolia L. were grown in an infertile forest soil in controlled-environment chambers with atmospheric CO₂ concentrations of 350 μl ⁻¹ (ambient) or 700 μl ⁻¹. In R. pseudoacacia and A. glutinosa , total nitrogenase (N₂ reduction) activity per plant, assayed by the acetylene reduction method, was significantly higher in elevated CO₂, because the plants were larger and had more nodule mass than did plants in ambient CO₂. The specific nitrogenase activity of the nodules, however, was not consistently or significantly affected by CO₂ enrichment. Substantial increases in plant growth occurred with CO₂ enrichment despite probable nitrogen and phosphorus deficiencies. These results support the premises that nutrient limitations will not preclude growth responses of woody plants to elevated CO₂ and that stimulation of symbiotic activity by CO₂ enrichment of the atmosphere could increase nutrient availability in infertile habitats.     

Nitrogen fixation by Rhodopseudomonas palustris OU 11 with aromatic compounds as carbon source/electron donors

Abstract A purple non-sulfur anoxygenic phototrophic bacterium, Rhodopseudomonas palustris (ATCC 51186; DSM 7375), grew fixing N₂ using aromatic compounds as the sole carbon source/electron donor. Benzoate, cinnamate and benzyl alcohol were used as electron donors for N₂ fixation, while aniline and nitrobenzene supported poor growth under N₂ atmosphere (in the absence of any other combined nitrogen in the medium) but did serve as sole carbon source/e⁻ donor in the presence of ammonium chloride as nitrogen source.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

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.     

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.     

Seasonal patterns of nitrogen fixation in termites

1. Termite nitrogenase activity was highest in autumn and spring (≈ 3 μg N₂ fixed termite fresh mass (g)^–1 day^–1) and lowest in winter and summer (≈ 0·8 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
2. The nitrogenase activity of worker termites was significantly higher than all other castes (1·58 ± 0·27 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
3. Worker termites constituted the largest proportion of all the castes throughout the study period (≈ 90%).
4. The localized input of fixed nitrogen by termites may reach 15·3 mg N log^–1 day^–1 and 5·6 g N log^–1 year^–1.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract: Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N₂). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N₂ fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N₂ because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N₂-fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N₂ generated within soil crusts and marine microbial mats.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2-FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N₂ fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin-containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

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.     

Identification and study of growth and nitrogenase activity of nitrogen fixing cyanobacteria from tropical soil

Identification of cyanobacteria species has been performed on samples coming from two different harvest areas. The most important fixing belongs to Scytonema genus. The other genus identified are Nostoc and Lyngbia. Moreover, these cells are living closely with non-fixing cyanobacteria as well as with bacteria. The growth of cells as well as nitrogenase activity has been studied on a semi-axenic strain of Scytonema, a nitrogen fixing cyanobacterium, isolated from soil crusts. The cell growth is relatively show in liquid medium depleted in combined nitrogen. The growth rate increases when nitrates are supplied to cells. A release of ammonium is observed in medium during cell culture. This release exhibits several maxima and minima during cell growth. The heterocyst cells disappear within four days when filaments are growing in nitrates supplied medium. On the contrary, the heterocyst frequency increases up to more 5% in a nitrogen depleted medium. The heterocyst frequency reaches a maxima after 4 days of culture, then decreases later on. Nitrogenase activity changes during cells growth too. The maximum activity is observed after 5 to 6 days of culture to decrease after even though the cells are still in their exponential phase of growth. Nitrogenase activity increases with light intensity, what indicate a possible relation between photosynthetic and nitrogenase activities.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Genetic factors in Rhizobium affecting the symbiotic carbon costs of N2 fixation and host plant biomass production

The effect of genetic factors in Rhizobium on host plant biomass production and on the carbon costs of N₂ fixation in pea root nodules was studied. Nine strains of Rhizobium leguminosarum were constructed, each containing one of three symbiotic plasmids in combination with one of three different genomic backgrounds. The resulting strains were tested in symbiosis with plants of Pisum sativum using a flow-through apparatus in which nodule nitrogenase activity and respiration were measured simultaneously under steady state conditions. Nodules formed by strains containing the background of JI6015 had the lowest carbon costs of N₂ fixation (7.10–8.10 μmol C/μmol N₂), but shoot dry weight of those plants was also smaller than that of plants nodulated by strains with the background of B151 or JI8400. Nodules formed by these two strain types had carbon costs of N₂ fixation varying between 11.26 and 13.95 μmol C/μmol N₂. The effect of symbiotic plasmids on the carbon costs was relatively small. A time-course experiment demonstrated that nodules formed by a strain derived from JI6015 were delayed in the onset of nitrogenase activity and had a lower rate of activity compared to nodules induced by a strain with the background of B151. The relationship between nitrogenase activity, carbon costs of N₂ fixation and host plant biomass production is discussed.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

In vivo nitrate reductase activities in different organs of lucerne plants: Effects of combined nitrogen during first vegetative growth and after shoot harvest

Nitrate reductase (EC 1.6.6.1–3; NR) activity was evaluated in nodulated lucerne ( Medicago sativa L. cv. Europe) grown aeroponically in both the presence and absence of applied nitrogen. Determination of in vivo NR activity was done with organ pieces in 0.1 M K⁺-phosphate, pH 7.5, 0.1 M KNO₃ and 1% n -propanol. NR activity was detected in all plant parts. Leaves accounted for 40% of the whole plant activity. Root activity was as high as leaf activity. Stem NR activity accounted for 14 to 20% of the total plant activity. NR activity was also detected in symbolically dependent plants grown without combined nitrogen. Nodule NR in symbolically dependent plants accounted for 17% of the tolal plant aclivity. When nitrate was present in the nulrienl medium, NR increased 5-fold as compared lo N₂-dependenl plants. Varying levels of nitrale (1.65 to 4 m M ) had no influence on leaf or stem activities. However, root NR activity seemed to be related to the nitrale concentration in the nulrient medium. Throughoul inilial vegelative growth, in vivo NR and nitrogenase (acelylene reduction) increased simultaneously. After shoot harvest, nitrogenase (acetylene reduction) aclivity drastically decreased with reduction of photosynthate supply, whereas NR increased in all organs, especially in N₂-dependenl plants.