Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: evidence of bistability

In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h-1 was described (Müller et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h-1. For each steady state the specific activities of GS and glutamate dehydrogenase (GDH)--the other key enzyme of the two NH3 assimilation routes in E. coli--and in addition the remaining NH3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH3 concentration are found. The higher state of GS is connected with a lower GDH activity and NH3 concentration. With rising D the GS activities decrease whereas GDH activities and NH3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. It describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH3 concentration on the other. It is shown that the specific uptake rate of the limiting NH3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Isolation, characterization and complementation analysis of nirB mutants of Escherichia coli deficient only in NADH-dependent nitrite reductase activity

Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities. These NirB- strains synthesize cytochrome c552 and grow normally on anaerobic glycerol-fumarate plates. The defects map in a gene, nirB, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB. Complementation studies established that nirB+ and cysG+ can be expressed independently. The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4). The nirB gene is apparently defective in the previously described nirD mutant, LCB82. The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c552 was detected. Strains carrying a nirE, nirF or nirG mutation gave normal rates of nitrite reduction by glucose, formate or NADH.     

Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point

Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15)N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT) strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.     

Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase

Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.     

Biochemical and genetic characterization of nirB mutants of Escherichia coli K 12 pleiotropically defective in nitrite and sulphite reduction

Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth. With one exception these mutants require cysteine for growth. Cytochrome C552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation. The defective gene is located between the crp and aroB genes at minute 73 on the E. coli chromosome. Mapping and reversion studies indicate the nirB is identical to the previously described cysG gene. It is suggested that the product of the cysG+ (nirB+)?gene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide.     

Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli.

The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH ₊ ⁴ (i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH ₊ ⁴ concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH ₊ ⁴ in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH ₊ ⁴ and were maximum when ammonia was limiting, suggesting that at low NH ₊ ⁴ levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH ₊ ⁴ levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS Glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.     

Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Nitrite reduction in Escherichia coli: genetic analysis of nir mutants.

Summary Five mutants of Escherichia coli impaired on nitrite reduction were studied. All have lost NADH-nitrite reductase activity but have retained the capacity to synthesize all or part of their cytochrome c₅₅₂. Three genes, nir C, nir D, and nir E were mapped at 26, 72.5 and 49.5 min, respectively. Another gene, nir F was tentatively localized around 52 min.     

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.