Nitrogenase activity,amino acid pool patterns and amination in blue-green algae

Summary The free amino acid pools in the nitrogen-fixing blue-green algae Anabaena cylindrica, A. flos-aquae and Westiellopsis prolifica contain a variety of amino acids with aspartic acid, glutamic acid and the amide glutamine being present in much higher concentrations than the others. This pattern is characteristic of that found in organisms having glutamine synthetage/glutamate synthetase [glutamine amide-2-oxoglutarate amino transferase (oxido-reductase)] as an important pathway of ammonia incorporation. Under nitrogen-starved conditions the level of acetylene reduction (nitrogen fixation) and the glutamine pool both increase but the free ammonia pool decreases, suggesting that ammonia rather than glutamine regulates nitrogen fixation.Glutamine synthetase has been demonstrated in Anabaena cylindrica using the -glutamyl transferase assay and also using a biosynthetic assay in which P_i release from ATP during glutamine synthesis was measured. The enzyme (-glutamyl transferase assay) is present in nitrogen-fixing cultures and activity is higher in aerobic than in microaerophilic cultures. Ammonium-grown cultures have lowest levels of all and activity in the presence of nitrate-nitrogen (150 mg nitrogen 1^-1) is lower than in aerobic cultures growing on elemental nitrogen. Ammonium-nitrogen and nitrate-nitrogen have no effect on glutamine synthetase in vitro. Glutamate synthetase also operates in nitrogen-fixing cultures of Anabaena cylindrica.     

Interrelationship between hydrogen peroxide,ammonia, glutamine,hydroxylamine and nitrite metabolism in the cyanobacterium Phormidium uncinatum

On transition from nitrogen starvation to ammonia or ammonia/glutamine sufficiency Phormidium uncinatum produces high amounts of H₂O₂, which is consumed by several oxidative reactions catalyzed by thylakoid membrane bound enzymes. These include: oxidation of glutamine to free hydroxylamine, of ammonia to nitrite, of bound hydroxylamine to nitrite, and dismutation of free hydroxylamine to ammonia and nitrite. A possible role of these transformations for detoxification is discussed.Non-standard abbreviations FCCP p-trifluormethoxy carbonylcyanide phenylhydrazone - DCMU dichloromethyl urea     

The control of glutamine synthetase level in Lemna minor L.

Summary The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM -aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC₅₀ value for this species is 7.465?mM total ammonia (Upper 95% CL?=?8.498?mM and Lower 95% CL?=?6.557?mM) or 0.51?mg/L as unionized ammonia (NH₃) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07?mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1?mM (or 0.007?mg/l NH₃) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC₅₀ and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.     

THE ROLE OF GLUTAMINE SYNTHETASE IN THE INCORPORATION OF AMMONIUM IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

The K_m for ammonia for glutamine synthetase and glutamate dehydrogenase was measured in enzyme extracts from Skeletonema costatum (Grev.) Cleve. At similar physiological pH and temperature the half-saturation constant for glutamine synthetase was 29 μM, whereas for GDH it was 28mM. On the basis of relative enzymic activity, as well as substrate affinity, it is suggested that glutamine synthetase is the enzyme primarily responsible for the incorporation of ammonium into the amino acid pool, when extracellular nitrogen is at ecological concentrations.     

Diel variation in ammonia excretion,glutamine levels,and hydration status in two species of terrestrial isopods

Terrestrial isopods (suborder Oniscidea) excrete most nitrogen diurnally as volatile ammonia, and ammonia-loaded animals accumulate nonessential amino acids, which may constitute the major nocturnal nitrogen pool. This study explored the relationship between ammonia excretion, glutamine storage/mobilization, and water balance, in two sympatric species Ligidium lapetum (section Diplocheta), a hygric species; and Armadillidium vulgare (Section Crinocheta), a xeric species capable of water-vapor absorption (WVA). Ammonia excretion (12-h), tissue glutamine levels, and water contents were measured following field collection of animals at dusk and dawn. In both species, diurnal ammonia excretion exceeded nocturnal excretion four- to fivefold while glutamine levels increased four- to sevenfold during the night. Most glutamine was accumulated in the somatic tissues (body wall). While data support the role of glutamine in nocturnal nitrogen storage, potential nitrogen mobilization from glutamine breakdown (162 mol g^–1 in A. vulgare) exceeds measured ammonia excretion (2.5 mol g^–1) over 60-fold. This may serve to generate the high hemolymph ammonia concentrations seen during volatilization. The energetic cost of ammonia volatilization is discussed in the light of these findings. Mean water contents were similar at dusk and dawn in both species, indicating that diel cycles of water depletion and replenishment were not occurring.     

The mechanism of ammonia assimilation in nitrogen fixing bacteria

Summary Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium glutamine glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium glutamate glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of -ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N₂-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH₃-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K _m for ammonium predominates in the N₂-grown cell.Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.Abbreviation DON 6-diazo-5-oxo-l-norleucine     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln ^-) and l-methionine d,l-sulfoximine (MSX) resistant (MSX ^r) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, -keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N₂ or NH ₄ ⁺ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

Transformation of Lotus corniculatus Plants with Escherichia coli Asparagine Synthetase A: Effect on Nitrogen Assimilation and Plant Development

Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagine synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagine is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher than that of transgenic plants. While glutamine levels in transgenic plants are about 3–4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO₂ conditions.     

The re-assimilation of ammonia produced by photorespiration and the nitrogen economy of C3 higher plants

Photorespiration involves the conversion of glycine to serine with the release of ammonia and CO₂. In C₃ terrestrial higher plants the flux through glycine and serine is so large that it results in the production of ammonia at a rate far exceeding that from reduction of new nitrogen entering the plant. The photorespiratory nitrogen cycle re-assimilates this ammonia using the enzymes glutamine synthetase and glutamine:2-oxoglutarateaminotransferase.     

Evidence for adenylylation/deadenylylation control of the glutamine synthetases of Rhodospirillum tenue and Rhodocyclus purpureus

The phylogenetically related phototrophic bacteria Rhodospirillum tenue and Rhodocyclus purpureus modulate activity of their glutamine synthetases by adenylylation/deadenylylation. Evidence for covalent modification includes the inhibitory effect of Mg²⁺ on the activity of glutamine synthetase extracted from cells of either species grown on excess ammonia, and the lack of Mg²⁺ inhibition of activity of the enzyme isolated from N₂-(R. tenue) or glutamine (R. purpureus)-grown cells. In addition, snake venom phosphodiesterase treatment of glutamine synthetase from either species grown on excess ammonia relieved Mg²⁺ inhibition of the enzyme (as measured via the -glutamyl transferase assay), and changed the cation specificity from Mn²⁺ to Mg²⁺ (in the biosynthetic assay).     

Metabolic regulation of ammonia emission in different senescence phenotypes of Nicotiana tabacum

In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH₄⁺, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH₄⁺ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH₄⁺ content.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Effect of NO3 on N2 fixation and nitrogenous solutes of xylem in two nodulated West African geocarpic legumes,Kersting's bean (Macrotyloma geocarpum L.) and Bambara groundnut (Vigna subterranea L.)

Nodulation, nitrogen (N₂) fixation and xylem sap composition were examined in sand cultured plants of Bambara groundnut (Vigna subterranea L.) and Kersting's bean (Macrotyloma geocarpum L.) inoculated with Bradyrhizobium strain CB756 and supplied via the roots for a 4 week period from the third week onwards with different levels of (¹⁵N)-nitrate (0–15 mM). The separate contributions of nitrate and N₂ to plant nitrogen were measured by isotope dilution. Increasing levels of nitrate inhibited nodule growth (measured as dry matter or nodule N) of both species parallel with decreased dependence on symbiotically-fixed N. Specific nodule activity (N₂ fixed g nodule dry⁻¹ d⁻¹ of nodules) was reduced progressively with time in V. subterranea at higher (5 or 15 mM) levels of NO₃, but this was not so for M. geocarpum. Root xylem bleeding sap of both species showed ureides (allantoin and allantoic acid) as predominant (>90%) solutes of nitrogen when plants were relying solely on atmospheric N. Levels of ureide and glutamine decreased and those of asparagine and nitrate in xylem increased with increasing level of applied nitrate. Relative levels of xylem ureide-N were positively correlated (R²=0.842 for M. geocarpum and 0.556 for V. subterranea), and the ratio of asparagine to glutamine in xylem exudate negatively correlated (R²=0.955 for M. geocarpum and 0.736 for V. subterranea) with plant reliance on nitrogen fixation. The data indicate that xylem sap analyses might be useful for indirect field assays of nitrogen fixation by the species and that Kersting's bean might offer some potential as a symbiosis in which N₂ fixation is relatively tolerant of soil N.     

Seasonal variation of leaf glutamine synthetase isoforms in temperate deciduous trees strongly suggests different functions for the enzymes

Changes in the activities of leaf glutamine synthetase (GS) isoforms were followed in four temperate deciduous trees from full leaf expansion to senescence (May to November). In the early part of the season, total GS activity was high in all species, with values ranging from 90 to 200 μmol h⁻¹ g⁻¹ fw. During this early period this activity comprised only the activity of the chloroplastic (GS₂) isoform in all species. These high GS₂ activities are consistent with the role of GS₂ in the re-assimilation of photorespired ammonia. The early high values also coincided with high nitrate reductase activity in one of the species, the highly nitrophilous species Sambucus nigra, with values of up to 16μmol h⁻¹ g⁻¹ fw. This indicates that GS₂ is also important in the assimilation of ammonia produced from nitrate reduction. From mid- to late-season, the cytosolic isoform (GS₁) was detected in all four species and became increasingly more active in comparison to GS₂. By the time of senescence it was the dominant enzyme of the two forms in both S. nigra and Carpinus betulus. The results provide strong support for recent findings that GS₁ is an important enzyme for the mobilization of nitrogen for translocation or storage.     

Oxidative and hydrolytic transformations of hydroxylamine compounds in cyanobacteria

Ammonia-incubated cyanobacteria liberated H₂O₂, accumulated hydroxylamine compounds and nitrite and catalyzed dismutation of hydroxylamine as well as oxidations of ammonia, glutamine, and oximes. Ethyl acetohydroximate-adapted Phormidium released excess H₂O₂ and phototrophically metabolized the oxime via hydrolysis and dismutation to nitrite and ammonia, which were consumed by nitrite reductase and glutamine synthetase. Added ammonia stimulated H₂O₂ production and oxime metabolism via glutamate dehydrogenase pathway.Abbreviations EAH Ethyl acetohydroximate - GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase