Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

Compartmentation and flux characteristics of ammonium in spruce

Using ¹³NH ₄ ⁺ as a tracer, compartmental analyses for NH ₄ ⁺ were performed in non-mycorrhizal roots of intact Picea glauca (Moench) Voss. seedlings at four different concentration regimes of external NH ₄ ⁺ ([NH ₄ ⁺ ]_o), i.e. 0, 10, 100, and 1500 M. Three kinetically distinct compartments were identified, with half-lives of exchange of approximately 2 s, 30 s, and 14 min, assumed to represent surface adsorption, Donnan free space, and cytoplasm, respectively. No significant differences were found in half-lives of exchange with changes in [NH ₄ ⁺ ]_o. Influx was calculated to be 0.96 mol·g^–1·h^–1 in N-deprived plants (measured at 10 M [NH ₄ ⁺ ]_o), while under steady-state conditions it was 0.21 mol·g^–1h^–1 at 10 M [NH ₄ ⁺ ]_o, 1.96 mol·g^–1h·^–1 at 100 M [NH ₄ ⁺ ]_o, and 6.45 mol·g^–1·h^–1 at 1.5 mM [NH ₄ ⁺ ]_o. Efflux measured over the same range constituted approximately 9% of influx in N-deprived plants, 10% at 10 M, 28% at 100 M, and 35% at 1.5 mM [NH ₄ ⁺ ]_o. Cytoplasmic [NH ₄ ⁺ ] was estimated at 6 m M in N-deprived plants, 2 mM at 10 M [NH ₄ ⁺ ]_o, 14 mM at 100 M, and 33 mM at 1.5 mM. Free-space [NH ₄ ⁺ ] was 84 M, 50 M, 700 M, and 8 mM, respectively. In comparison with previously published data on fluxes and compartmentation of NO ₃ ^– in white-spruce seedlings, results of this study identify a pronounced physiological preference of this species for NH ₄ ⁺ over NO ₃ ^– as an inorganic N source in terms of uptake and intracellular accumulation. The significant ecological importance of this N-source preference is discussed.The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. P. Poon for essential assistance in experiments.     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

Effect of divalent cations on the short-term NH 4 + inhibition of nitrogen fixation in Azotobacter chroococcum

Incubation of Azotobacter chroococcum in the presence of micromolar concentrations of MnCl₂, but not MgCl₂, prevented nitrogenase activity from NH ₄ ⁺ inhibition. Mg(II), at a 100-fold concentration with respect to Mn(II), counteracted the protective effect of Mn(II) on nitrogenase activity. When Mn(II) was added to cells that had been given NH₄Cl, stopping of NH ₄ ⁺ uptake and recovery of nitrogenase activity took place, and a raise of NH ₄ ⁺ concentration in medium developed. Furthermore, incubation of A. chroococcum cells with 20 M Mn(II) under air, but not under an argon: oxygen (79%:21%) gas mixture, resulted in NH ₄ ⁺ excretion to the external medium. The Mn(II)-mediated uncoupling of nitrogen fixation from ammonium assimilation leads us to conclude that Mn(II) may act as a physiological inhibitor of glutamine synthetase.Abbreviations Hepes N-2-Hydroxyethylpiperazine-N-ethanesulfonic acid - Mops 3-(N-Morpholino)propanesulfonic acid     

Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis

Using the ammonium analogue ¹⁴CH₃NH ₃ ⁺ , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-dl-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous ¹⁴CH₃NH ₃ ⁺ or NH ₄ ⁺ because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.Abbreviations and symbol ATS/ATSs ammonium transport system/systems - Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-dl-sulphoximine - membrane potential     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

Different ammonium-ion uptake,metabolism and detoxification efficiencies in two Lemnaceae

¹⁵N-Nuclear magnetic resonance spectroscopy was used to follow nitrogen assimilation and amino-acid production in Wolffia arrhiza (L.) Hork. ex. Wimmer, clone Golan exposed to 4.0 mM ¹⁵NH₄Cl solutions for 24 h. The main ¹⁵N-labelled metabolites were asparagine and glutamine, as well as substantial amounts of unreacted, intracellular NH ₄ ⁺ . These results were compared with those of a previous study on Lemna gibba L. clone Hurfeish (Monselise et al., 1987, New Phytol. 10, 341–345) with regard to NH ₄ ⁺ uptake, assimilation and detoxification efficiencies. Both species, grown under continuous white light, were capable of preferential uptake of NH ₄ ⁺ in the presence of nitrate. Relative growth rates indicate that both species tolerate increased levels of NH ₄ ⁺ , up to 10^–2 mol · 1^–1, with L. gibba showing a slightly greater tolerance. No ¹⁵N-labelled free NH ₄ ⁺ was detectable in L. gibba, while in W. arrhiza excess NH ₄ ⁺ was found within the cells. This fact indicates that L. gibba is more efficient in detoxification than W. arrhiza, presumably because of inability of W. arrhiza to regenerate the NH ₄ ⁺ traps, glutamate and aspartate, rapidly enough. This is also evident from the observation that addition of -ketoglutarate to the medium caused nearly complete assimilation of intracellular NH ₄ ⁺ in W. arrhiza. In both plants, addition of -ketoglutarate increased both NH ₄ ⁺ uptake and assimilation. Addition of l-methionine dl-sulfoximine, an inhibitor of glutamine synthetase inhibited NH ₄ ⁺ assimilation, while addition of azaserine, an inhibitor of glutamate synthase, resulted in ¹⁵N incorporation into the glutamine-amide position only. These results are consistent with the glutamine synthetase-glutamate synthase pathway being the major route of NH ₄ ⁺ assimilation in the two plants under the conditions used.Abbreviations AZA azaserine (O-diazoacetyl-l-serine) - GOGAT glutamine oxoglutarate amine transferase=]glutamate synthase E.C. 1.4.7. and E.C. 1.4.1.13. - GS glutamine synthetase E.C. 6.3.1.2. - -KG -ketoglutarate=2-oxoglutarate - MSO l-methionine dl-sulphoximine - NMR nuclear magnetic resonance - RGR relative growth rate This article is dedicated to Professor Bernhard Schrader on the occasion of his 60th birthdayWe wish to thank Professor Robert Glaser for helpful discussions, and Mrs. Aliza Levkoviz and Mr. Gideon Raziel for skillful assistance.     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

Ammonium-stimulated,sodium-dependent uptake of glutamine in Bacillus pasteurii

The uptake of glutamine was studied in Bacillus pasteurii DSM 33. Only one uptake system was detected in the concentration range studied (between 1 and 100 M glutamine) which exhibited Michaelis-Menten saturation kinetics, with an apparent K _t of 10.7 (±3.5) M glutamine. The uptake was sodium-dependent (apparent K _t=0.2 mM Na⁺); none of several monovalent cations tested was able to replace sodium in the uptake reaction. Ionophores interfering with proton, sodium or potassium gradients across membranes strongly inhibited uptake of glutamine. Low uptake rates correlating with low potassium content and an acidic cytoplasm were measured in cells grown at high ammonium¹ concentrations. Ammonium and other permeant amines as well as potassium stimulated the uptake reaction in these cells, leading to an increase of up to 100-fold in V _max without affecting the affinity of the uptake system. In cells grown at low concentrations of ammonium, an alkaline cytoplasm and both high glutamine uptake activities and potassium content were measured; the uptake reaction was not further stimulated by permeant amines or potassium in such cells. Growth of the strain was inhibited by Tris at high concentrations; this inhibition was relieved by the addition of increasing amounts of ammonium.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide This work is dedicated to Prof. Dr. H. Kaltwasser on the occasion of his 60th birthday     

Evidence for the nitrate assimilation-dependent nitrite excretion in cyanobacterium Nostoc MAC

Nitrate uptake and nitrite efflux patterns in Nostoc MAC showed a rapid phase followed by their saturation. Nitrite efflux was maximum in nitrate medium whereas the cells incubated in N₂ and NH ₄ ⁺ media exhibited a decreased nitrite efflux activity. The simultaneous presence of NH ₄ ⁺ and nitrate significantly decreased nitrite efflux. L-Methionine-Dl-sulphoximine (MSX) prevented inhibition of nitrite efflux by NH ₄ ⁺ . In the dark there was negligible nitrite efflux, whereas illumination increased the rate of nitrite efflux significantly. The nitrite efflux system was maximally operative at pH 8.0, 30°C and a photon fluence rate of 50 mol m^-2. s^-1. These results confirm that (i) the nitrite efflux system in Nostoc MAC is dependent upon nitrate uptake and assimilation and is repressible by NH ₄ ⁺ ; (ii) NH ₄ ⁺ itself is not the actual repressor of nitrite efflux; a product of NH ₄ ⁺ assimilation via glutamine synthetase (GS) is required for repression to occur; (iii) the catalytic function of GS does not appear to be involved in nitrate assimilation-dependent nitrite efflux, and (iv) the optimum pH, temperature and illumination for maximum nitrite efflux were found to be 8.0, 30°C and 50mol m^-2. s respectively.B.B. Singh, P.K. Pandey and P.S. Bisen are with the Department of Microbiology, Barkatullah University. Bhopal 462026, India. S.Singh is with the Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon, India     

Ammonium (methylammonium) uptake by Alcaligenes eutrophus H16

The uptake of the radioactive ammoniumanalogue ¹⁴C-methylammonium¹ was measured in heterotrophically grown cells of Alcaligenes eutrophus H16 in order to study the mechanism of NH ₄ ⁺ uptake. MA gradients of up to 200 were built up by a substrate-specific and energy-dependent system which showed a K _m of 35–111 M and a V _max of 0.4–1.8 nmol MA/min per mg protein. The involved carrier exhibited a higher affinity towards NH ₄ ⁺ than towards CH₃NH ₃ ⁺ indicating that ammonium rather than MA was its natural substrate. Cold shock with hypotonic but not with hypertonic solutions caused the efflux of almost the entire accumulated MA. Osmotic shock did not affect the uptake reaction, suggesting that no periplasmic binding proteins were involved. Indirect observations indicate the membrane potential as driving force for MA uptake. High rates of uptake were observed in cells grown under nitrogen deficiency or with nitrate as nitrogen source. The uptake rate decreased during growth at high ammonium concentrations indicating that biosynthesis of nitrogenous compounds was supported by passive diffusion of NH₃. The data suggest that the formation of the carrier is subject to nitrogen control.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphe-nylhydazone - MA methylammonium - pCMB para-chlormercuribenzoate     

Ammonium and nitrate uptake rates and rhizosphere pH in non-mycorrhizal roots of Norway spruce [Picea abies (L.) Karst.]

Summary Relationships between root zone temperature, concentrations and uptake rates of NH ₄ ⁺ and NO ₃ ^– were studied in non-mycorrhizal roots of 4-year-old Norway spruce under controlled environmental conditions. Additionally, in a forest stand NH ₄ ⁺ and NO ₃ ^– uptake rates along the root axis and changes in the rhizosphere pH were measured. In the concentration (C_min) range of 100–150 M uptake rates of NH ₄ ⁺ were 3–4 times higher than those of NO ₃ ^– The preference for NH ₄ ⁺ uptake was also reflected in the minimum concentration (C_min) values. Supplying NH₄NO₃, the rate of NO ₃ ^– uptake was very low until the NH ₄ ⁺ concentrations had fallen below about 100 M. The shift from NH ₄ ⁺ to NO ₃ ^– uptake was correlated with a corresponding shift from net H⁺ production to net H⁺ consumption in the external solution. The uptake rates of NH ₄ ⁺ were correlated with equimolar net production of H⁺. With NO ₃ ^– nutrition net consumption of H⁺ was approximately twice as high as uptake rates of NO ₃ ^– In the forest stand the NO ₃ ^– concentration in the soil solution was more than 10 times higher than the NH ₄ ⁺ concentration (<100 M), and the rhizosphere pH of non-mycorrhizal roots considerably higher than the bulk soil pH. The rhizosphere pH increase was particularly evident in apical root zones where the rates of water and NO ₃ ^– uptake and nitrate reductase activity were also higher. The results are summarized in a model of water and nutrient transport to, and uptake by, non-mycorrhizal roots of Norway spruce in a forest stand. Model calculations indicate that delivery to the roots by mass flow may meet most of the plant demand of nitrogen and calcium, and that non-mycorrhizal root tips have the potential to take up most of the delivered nitrate and calcium.     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed ¹⁵N₂ and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to ¹⁵N₂, showed that highest initial ¹⁵N₂-labelling was into NH ₄ ⁺ . After 12 min little further increase in the NH ₄ ⁺ label occurred while that in the amide group of glutamine and in glutamate continued to increase. The ¹⁵N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of ¹⁵N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total ¹⁵N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which ¹⁴N₂ was added to cephalodia previously exposed to ¹⁵N₂, showed that the NH ₄ ⁺ pool rapidly became depleted of ¹⁵N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The ¹⁵N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the ¹⁵N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.Abbreviations MSX L-methionine-SR-sulphoximine - AOA amino-oxyacetate - HEPES N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid - Tris tris-(hydroxymethyl) methylamine - GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GPT glutamate-pyruvate aminotransferase - APT aspartate-pyruvate aminotransferase - ADH alanine dehydrogenase - GOT glutamate-oxaloacetate aminotransferase