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.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

Nutritional Requirement for the Expression of Nitrogenase Activity by Rhizobium sp. in Agar Culture

The effect of various carbon sources, nitrogen sources, vitamins and trace elements on the ability of three strains (32H1, CB627, CB744) of a slow-growing Rhizobium sp. to develop nitrogenase activity in agar culture was studied. Strains 32H1 and CB627 developed nitrogenase but showed differences with respect to the nature and concentrations of carbon sources required for optimum activity. Strain 32H1 had less specific requirements than CB627 in this respect and could sustain high nitrogenase activity over a wider range of phosphate concentration (5 to 60 mmol/1) in the medium than CB627. There were only minor differences between these two strains with respect to the nitrogen source [glutamine, asparagine, histidine or (NH₄SO₄] required in the medium for nitrogenase induction, and nitrogenase activity in both strains was unaffected by changes in the concentration of vitamins or trace elements supplied. Strain CB744 did not develop nitrogenase activity under any of the conditions tested. Adenosine 3', 5'-cyclic monophosphate (1 mmol/1) was found to accelerate derepression of nitrogenase synthesis in agar cultures of strains 32H1 and CB627.     

Expression of asparagine synthetase in rice (Oryza sativa) roots in response to nitrogen

The expression of asparagine synthetase (AS; EC 6.3.5.4) in response to externally supplied nitrogen was investigated with respect to enzyme activity and protein levels as detected immunologically in rice ( Oryza sativa ) seedlings. The asparagine content was very low in leaves and roots of nitrogen-starved rice plants but increased significantly after the supply of 1 m M NH₄⁺ to the nutrient solution. While neither AS activity nor AS protein could be detected in leaves and roots prior to the supply of nitrogen, levels became detectable in roots but not in leaves within 12 h of the supply of 1 m M NH₄⁺ or 10 m M glutamine. Other nitrogen compounds, such as nitrate, glutamate, aspartate and asparagine had no effect. Methionine sulfoximine completely inhibited the NH₄⁺-induced accumulation of AS protein but did not affect the glutamine-induced accumulation of the enzyme. The results suggested that glutamine or glutamine-derived metabolites regulate AS expression in rice roots.     

Short-term effect of ammonia on nitrogenase activity of Anabaena variabilis (ATCC29413)

Abstract Intact filaments of the cyanobacterium Anabaena variabilis switch off nitrogenase activity very rapidly upon addition of NH₄Cl when incubated in an alkaline environment (pH 10.0) permitting a fast NH₃-influx into the cells. When assayed in cell-free extracts (prepared from ammonia-treated filaments), nitrogenase remains inhibited in the presence of an ATP-regenerating system. Furthermore, l -methionine- d,l -sulfoximine, an inhibitor of glutamine synthetase, added to the filaments, prevents inactivation of nitrogenase by ammonia, showing that ammonia is not the compound directly responsible for nitrogenase switch-off.     

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.     

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.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

AMMONIUM ION INHIBITION OF EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM HIPPOCAMPAL SLICES

Abstract— The effect of pathophysiological levels (2-5 m m ) of ammonium chloride on the efflux of endogenous and exogenous [¹⁴C]glutamate from hippocampal slices was studied. The evoked release of glutamate which occurs dring tissue depolarization with 56 m m -KCl was greatly reduced when the tissue had been exposed to NH₄Cl for 40–80 min. This effect was seen whether or not glutamine (0.5 m m ) was present in the incubation medium. The effect was completely reversible. The spontaneous efflux and the evoked release of [¹⁴C]glutamate was, on the contrary, completely unaltered after exposure of the slice to ammonium ions. Nigher (20–36 m m ) amounts of NH₄Cl evoked a release of [¹⁴C]glutamate from the crude mitochondrial fraction, as did high concentrations of KCl. The results are discussed in relation to the compartmentation of glutamate metabolism and the pathogenesis of hepatic coma.     

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.     

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.     

Ecophysiology of associative nitrogen fixation in a rhizosphere model in pure and mixed culture

Abstract A gradostat (multistage chemostat) was used as a model of the rhizosphere. Investigations of the influence of NH₄Cl and O₂ gradients on a diazotrophic rhizosphere bacterium in pure culture and in mixed culture with non-diazotrophic strains were carried out. The diazotrophic isolate was able to grow on N₂ and NH₄Cl simultaneously. The diazotrophic isolate could successfully compete with the non-diazotrophic isolates in the presence and absence of NH₄Cl in most experiments. Only minor amounts of nitrogen were transferred to the non-fixing organisms. A concept of transfer of nitrogen to non-fixing organisms is proposed.     

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.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

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 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.     

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.     

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.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.