Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L-methionine-DL-sulfoximine-induced NH₄⁺ release by ca 40%, while net CO₂ fixation and C₂H₂ reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH₄Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH₄Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO₂ in O₂) by about 80–90%. At pH 7.5, under high pO₂, NH₄Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH₄Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

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.     

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.     

COMBINATION OF DITHIOTHREITOL AND SULFIDE AS REDUCTANTS IN NITROGEN-FREE MINIMAL MEDIA FOR GROWTH OF ANAEROBIC RUMINAL BACTERIA AND PREVOTELLA RUMINICOLA STRAIN B14 ON AMMONIA

Cysteine is commonly employed as the medium reductant for ruminal bacteria, but many ruminal bacteria can use cysteine as a source of nitrogen as well as sulfur. The objective of the present study was to test a combination of dithiothreitol and sulfide as possible reductant substitutes for cysteine in anaerobic media containing ammonia as the nitrogen source. The type of reductant (cysteine versus dithiothreitol-sulfide) and ammonia concentration did not alter growth rates of Prevotella ruminicola strain B,4 (P>0.15). However, growth rates in dithiothreitol-sulfide reduced media varied tremendously between individual organisms ranging from 0.10 h⁻¹ for Ruminococcus flavefaciens to 1.6 h⁻¹ for Streptococcus bovis grown in 1 mM NH₃-N. At both 1 and 11 mM NH₄Cl, Str. bovis strain JB1 exhibited the greatest growth rate followed by Str. bovis strain C277. Megasphaera elsdenii strain T81 and Ruminococcus flavefaciens strain FD1 had the lowest growth rates at both NH₄Cl concentrations. Increasing NH₄Cl concentration from 1 to 11 mM resulted in increased growth rates for Ruminobacter amylophilus strains H18 and 70 and Str. bovis strain C277 (P<0.05), and decreased growth rates for S. ruminantium subsp. lactilytica strain HD₄ and Str. bovis strain JB1 (P<0.01). These results indicate that dithiothreitol and sulfide can be combined as reductants in nitrogen-free basal media for most ruminal bacterial species.     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids

A completely defined growth medium has been developed to determine the nitrogen requirements for several species of ruminal bacteria, and has revealed two strains which are impaired in de novo biosynthesis of certain amino acids. Using NH₄Cl as a sole nitrogen source, the medium supported growth of Butyrivibrio, Selenomonas, Prevotella and Streptococcus species. One strain of B. fibrisolvens (E14) and one strain of P. ruminicola (GA33) did not grow in the presence of NH₄Cl until the medium was supplemented with amino acids or peptides. For B. fibrisolvens strain E14, methionine was identified as the specific growth-limiting amino acid although methionine alone did not support growth in the absence of NH₄Cl. For P. ruminicola strain GA33, any individual amino acid other than methionine or cysteine could supplement the medium and support growth. Enzyme assays confirmed a lack of NADH and NADPH-dependent glutamate dehydrogenase (GDH) activities in this strain.     

Synthesis of glutamate by mitochondria – An anaplerotic function for glutamate dehydrogenase

The photorespiratory nitrogen cycle was initially thought to be a closed cyclic process. If this were true the loss of glutamate, glutamine, serine or glycine to other processes, such as protein synthesis or export from the leaves, would not be possible in a stoichiometric sense. However, recent studies with [¹⁵N]-labeled amino acids show that there are alternative sources of nitrogen for photorespiration, indicating that the nitrogen cycle is not a closed cyclic system. In addition recent work with ¹⁵NH₄Cl and [¹⁵N]-glycine and a metabolically competent mitochondria system has shown that glutamate is synthesized in the mitochondria. Hence the glutamate dehydrogenase (GDH, EC 1.4.1.2) in mitochondria could also be active in the reassimilation of NH₄. We would like to propose that one function of mitochondrial GDH is to synthesize glutamate from some of the NH₄ released by photorespiration and that this glutamate represents a reserve for use in biosynthetic reactions.     

SALT-DEPENDENT PROPERTIES OF HATCHING ENZYME FROM EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS

The effect of salts on hatching enzyme and protease from the embryo of the sea urchin, Hemicentrotus pulcherrimus , was investigated. The culture medium containing hatching enzyme secreted from the hatched blastula was dialyzed against Tris-HCl (pH 8.0) with or without salts. Both hatching enzyme and protease were activated and stabilized by CaCL₂, NaCI and KCI, while inhibited by MgCI₂. Protease activity was maximal at about 0.25 M NaCI. KCI, NH₄, CI and LiCI. Maximal activity of hatching enzyme was obtained at 0.5 M NaCl, KCI and NH₄ CI, while activity was inhibited by any concentration of LiC1. Among monovalcnt cations, the order of activation was NaCI, KCI > NH₄Cl. The activity of hatching enzyme was stabilized by dialysis against 1 M NaCI or KCI in the presence of CaCl.₂, but was rapidly lost by dialysis against lower concentrations of salts. Reactivation of hatching enzyme was not achieved by redialysis against I M NaCI. On the other hand, protease was reactivated by I M NaCI or KCI. From these results, hatching enzyme of the sea urchin may be called a moderate halophilic enzyme. It was assumed that at least two enzymes exist in the crude enzyme preparation and that they may have different functions.     

Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O₂ concentrations in the micromolar range. With increasing O₂ deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O₂. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K⁻ mutants, a deletion mutant in the nifK gene and the character of NH₄⁺-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term 'diazosomes' for them. Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions. Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Some kinetic properties of a purified glutamine synthetase from bacteroids of Glycine max

Abstract A purified glutamine synthetase was prepared from bacteroids of Rhizobium japonicum from nodules of Glycine max . For the biosynthetic assay the K _m values (mM) were l -glutamate 12.9, NH₄Cl 8.9 and ATP 14.3. When the enzyme was assayed by the γ-glutamyltransferase reaction the K _m values (mM) were l -glutamine 11.1 and hydroxylamine 3.3 compared with 7.7 and 1.2, respectively, for the purified enzyme from Rhizobium japonicum grown in culture. The enzyme prepared from bacteroids of Glycine max was 80% adenylylated.     

Glucose transport throughout fermentation by Saccharomyces cerevisiae NCYC 1108

The rate of fermentation of glucose by a polyploid strain of Saccharomyces cerevisiae growing in a defined salts medium depends on the availability of NH₄+⁺. Its decline after exhaustion of the nitrogen source corresponded with the ability of the cells to accumulate the glucose analogue 2-deoxyglucose. Addition of NH₄+⁺to a nitrogen-depleted culture stimulated both glucose utilization and 2-deoxyglucose uptake. Since stimulation was inhibited by cycloheximide, maintenance of glucose transport during fermentation is dependent on protein synthesis.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

The effect of nitrogen and phosphorus on starch accumulation and net photosynthesis in two variants of Panicum maximum Jacq.

Abstract. Two anatomical variants of Panicum maximum Jacq. were observed to accumulate an unusually large number of starch grains in the bundle sheath chloroplasts when grown under controlled environmental conditions in a nutrient medium containing a low level of nitrate nitrogen (20 mg N dm⁻³ as KNO₃). When these plants were placed under dark conditions the chloroplasts were destarched, but exhibited a marked distortion of the thylakoid membranes. Under a higher level of nitrate nitrogen supply (200 mg N dm⁻³ as KNO₃) the number of starch grains was markedly reduced compared to that observed above in both plant variants. When the nitrogen was supplied as ammonium nitrogen (200 mg N dm⁻³ as NH₄Cl) there was again a high level of starch in the bundle sheath chloroplasts, the level being only slightly lower than that observed at the low KNO₃ supply. An unusually large number of starch grains accumulated in the bundle sheath chloroplasts in the absence of added phosphorus in the nutrient medium, in the presence of the higher nitrate nitrogen level. It is suggested that the increased starch accumulation results from a reduced trans-location of Calvin cycle intermediates out of the chloroplasts into the cytoplasm and that both nitrate nitrogen and phosphorus may play an important role in this process. A good correlation between high net photosynthetic activity and low bundle sheath starch content was observed. Nutrient medium requirements favouring low starch content in chloroplasts also favoured high net photosynthetic rates.     

Elevated CO2 and nitrogen influence exudation of soluble organic compounds by ectomycorrhizal root systems

Root and mycelial exudation contributes significantly to soil carbon (C) fluxes, and is likely to be altered by an elevated atmospheric carbon dioxide (CO₂) concentration and nitrogen (N) deposition. We quantified soluble, low-molecular-weight (LMW) organic compounds exuded by ectomycorrhizal plants grown under ambient (360 p.p.m.) or elevated (710 p.p.m.) CO₂ concentrations and with different N sources. Scots pine seedlings, colonized by one of five different ectomycorrhizal or nonmycorrhizal fungi, received 70 μM N, either as NH₄Cl or as alanine, in a liquid growth medium. Exudation of LMW organic acids (LMWOAs), dissolved monosaccharides and total dissolved organic carbon were determined. Both N and CO₂ had a significant impact on exudation, especially of LMWOAs. Exudation of LMWOAs was negatively affected by inorganic N and decreased by 30–85% compared with the organic N treatment, irrespective of the CO₂ treatment. Elevated CO₂ had a clear impact on the production of individual LMWOAs, although with very contrasting effects depending on which N source was supplied.     

Nitrogen concentration, ammonium/nitrate ratio and NaCl interaction in vegetative and reproductive growth of peanuts

Peanuts ( Arachis hypogaea L. cv. Shulamit) grown with NO₃⁻ and saline water in hydroponics responded positively to addition of nitrogen (N) in their vegetative growth, but not in desert dune sand. In order to clarify these conflicting results, peanut plants were grown in a greenhouse pot experiment with fine calcareous sand. The nutrient solution contained 0 or 50 m M NaCl and 2 or 6 m M N in the form of Ca(NO₃)₂, NH₄NO₃ or (NH₄)₂SO₄. Three replicates were harvested after 48 days (beginning of reproductive stage) and three after 109 days (pod filling). In addition, gynophores were treated with 0, 50, 100, 150 or 200 m M NaCl outside the growth pot to check their sensitivity to salt. Shoot dry weight became greater with increasing NH₄⁺/NO₃⁻ ratio. Increasing the N concentration from 2 to 6 m M did not change shoot dry weight of the NH₄NO₃ or NH₄⁺-fed plants, but caused a reduction in shoot dry weight of NO₃⁻-fed plants. Shoot dry weight was not affected by increasing the NaCl concentration to 50 m M . Salt caused an increase in the number of gynophores per plant and a reduction of the mean pod weight. A NaCl concentration of 100 m M and above reduced gynophore vitality. It is concluded that the salt sensitivity of peanut plants resides mainly in the sensitivity of the reproductive organs.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.