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The Glutamine Synthetase from Azotobacter vinelandii: Purification, Characterization, Regulation and Localization
Abstract
The glutamine synthetase (EC 6.3.1.2) from the N2-fixing bacterium Azotobacter vinelandii was purified to homogeneity by heat treatment, ammonium sulfate precipitation and ion-exchange chromatography. The following molecular parameters were determined: molecular weight 640 000, subunit molecular weight 53 000, partial specific volume 0.710 cm3/g, isoelectric point 4.6, amino acid composition. Most of the molecules are composed of 12 identical subunits but active oligomers of other degrees of polymerization, apparently aggregates with 8, 10 and 24 subunits, were also detected to a lesser extent. The enzymatic activity is regulated via adenylylation-deadenylylation cycles: liberation of AMP was detected upon treatment of the adenylylated form with phosphodiesterase along with a change in the catalytic properties. Adenylylation in vivo is specifically induced by high extracellular ammonia levels. The Km values for the Mg2+-dependent formation of glutamine were independent of the degree of adenylylation for glutamate and ATP, but varied for ammonia. Furthermore the catalytic activity is regulated by several nitrogenous feedback inhibitors. The degree of inhibition in some cases was dependent on the substrate concentrations: the sensitivity towards glycine, alanine and serine decreased with a decreasing ammonia level, while the sensitivity towards ADP or AMP increased with a decreasing ATP concentration. Part of the enzyme (about 30%) seems to be attached to the plasma membrane while the main fraction is found in the cytosol.
... Activity was measured at both pH 7.27 and pH 7.9, the isoactivity points for the native E. coli and K. pneumoniae GS enzymes, respectively . The isoactivity point for the A. vinelandii enzyme, pH 8.5, was not used as both the adenylylated and unadenylylated forms of the enzyme have low activity at this pH (Kleinschmidt and Kleiner, 1978; Siedel and Shelton, 1979). As shown inTable I, similar levels of transferase activity were obtained with both plasmids in either background. ...
... Though this activity was much higher than that in the mutants, it was significantly lower than that found in either of the glnA+ parental strains or in either mutant carrying a plasmid, pGE1O (Espin et al., 1981), containing the glnAntrBC region of K. pneumoniae. The low activity from pLV50 and pAT512 may reflect a lowered level of glnA expression ; however, the transferase assay is known to underestimate the activity of unadenylylated GS in A. vinelandii (Kleinschmidt and Kleiner, 1978; Siedel and Shelton, 1979). A number of subclones complemented ET8556 for growth on arginine (Figure 2)Figure 402 3, specific fragments of pLV50 DNA hybridized to both probes . ...
The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non-enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.
... One of these pathways is comprised of glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT), which together form the GS-GOGAT cycle (Fig. 5A). In this pathway, GS catalyzes the condensation of glutamate and ammonia to form glutamine, converting one molecule of ATP to ADP in the process (52). Next, GOGAT transfers the amino group from glutamine to a-ketoglutarate (aKG), forming two molecules of glutamate and converting one molecule of NAD(P)H to NAD(P) 1 (53). ...
Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol.
... The adenylylation level, and thus the activation state of GS ( Fig. 1), can be estimated by the ratio of its glutamine biosynthetic and γ-glutamyl transferase activities. The deadenylylated form of GS, which accumulates in cells deprived of ammonium, has a more prominent glutamine biosynthetic activity, and the adenylylated form of GS, which accumulates in ammonium cultivated cells, presents an enhanced γ-glutamyl transferase activity (Ambrosio et al. 2017;Kleinschmidt and Kleiner 1978). ...
There is an increasing interest in the use of N2-fixing bacteria for the sustainable biofertilization of crops. Genetically-optimized bacteria for ammonium release have an improved biofertilization capacity. Some of these strains also cross-feed ammonium into microalgae raising additional concerns on their sustainable use in agriculture due to the potential risk of producing a higher and longer-lasting eutrophication problem than synthetic N-fertilizers. Here we studied the dynamic algal cross-feeding properties of a genetically-modified Azotobacter vinelandii strain which can be tuned to over-accumulate different levels of glutamine synthetase (GS, EC 6.3.1.20) under the control of an exogenous inducer. After switching cells overaccumulating GS into a noninducing medium, they proliferated for several generations at the expense of the previously accumulated GS. Further dilution of GS by cell division slowed-down growth, promoted ammonium-excretion and cross-fed algae. The final bacterial population, and timing and magnitude of algal N-biofertlization was finely tuned in a deferred manner. This tuning was in accordance with the intensity of the previous induction of GS accumulation in the cells. This bacterial population behavior could be maintained up to about 15 bacterial cell generations, until faster-growing and nonammonium excreting cells arose at an apparent high frequency. Further improvements of this genetic engineering strategy might help to align efficiency of N-biofertilizers and safe use in an open environment.
Key points
• Ammonium-excreting bacteria are potential eutrophication agents
• GS-dependent deferred control of bacterial growth and ammonium release
• Strong but transient ammonium cross-feeding of microalgae
... In this diazotroph, the glnA gene product plays an important role in ammonium assimilation, while it appears not to be required for expression of the nif regulon (19). In A. vinelandii, GlnA (Avin_45850) is a type I GS, and thus, its activity is expected to be regulated by the adenylyltransferase GlnE (Avin_44890) (20,21) (Fig. 1), but little is known about the role of this enzyme in the regulation of ammonium assimilation and nitrogen fixation. ...
Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance.
IMPORTANCE
In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.
Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium Azotobacter vinelandii emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development.
Assimilatory NAD(P)H-nitrate reductase (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by affinity chromatography on blue Sepharose. The specific activity of the purified enzyme is in the range of 72 to 80 units/mg of protein. The electronic spectrum of the native enzyme shows absorption maxima at 278, 414 (Soret), 532 (beta), 562 (alpha), and 669 nm and shoulders at 455 and 484 nm, with an A278/A414 ratio of 2.56. The reduced enzyme shows absorption maxima at 424 (Soret), 528 (beta), 557 (alpha),and 669 n. The enzyme complex (Mr = 467,400) is composed of eight similar subunits (Mr = 58,750) and contains 4 molecules of FAD, 4 heme groups, and 2 atoms of molybdenum. Labile sulfide and nonheme iron were not detected. Electron micrographs show the eight subunits arranged alternately in two planes, and an 8-fold rotational symmetry was deduced from highly magnified images processed by optical superposition.
The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under special growth conditions both the single and the double mutant strains consistently released very high levels of ammonium (> 20 mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N2 and CO2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N2 from the air into crops.
N2-fixing organisms are able to use a variety of nitrogen sources, notably the inorganic Compounds NH
4+, NO
3- and N2. Although the N2-fixing strains discovered so far are scattered among many species differing widely in their physiology (see Stewart, 1977a), the major pathways for the assimilation of inorganic nitrogen ??? leading to glutamine, glutamate, and carbamyl phosphate ??? are rather similar in all species. A general pattern of major and minor pathways will be outlined in the first part.
The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. In this study we compared the effect of controlling the maximum activation state of the Azotobacter vinelandii glutamine synthase by a point mutation at the active site (D49S mutation) and impairing the ammonium-dependent homeostatic control of nitrogen-fixation genes expression by the ΔnifL mutation on ammonium release by the cells. Strains bearing the single D49S mutation were more efficient ammonium producers under carbon/energy limiting conditions and sustained microalgae growth at the expense of atmospheric N2 in synthetic microalgae-bacteria consortia. Ammonium delivery by the different strains had implications for the microalga's cell-size distribution. It was uncovered an extensive cross regulation between nitrogen fixation and assimilation that extends current knowledge on this key metabolic pathway and might represent valuable hints for further improvements of versatile N2-fixing microbial-cell factories.
Proteins with known isoelectric points(pl), as determined by isoelectric focusing, are tabulated. When available, the native molecular weight and the submit molecular weight and stoichiometry are reported. For each entry, the source and, when applicable, the organ of origin and/or subcellular location are given. A previous table [P.C. Righetti and T. Caravaggio, J. Chromatogr., 127 (1976) 1–28] convered the years from 1966 (the intoduction of isoelectric focusing) to 1975. The present compilation spans the years 1976–1979 and contains approximately three times as many references and entries (>900).
Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration
on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated
into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate
Polyacrylamide gel electrophoresis was 100,000. TheK
m formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were
essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.
When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.
The two forms of glutamine synthetase (GS-I, GS-II) isolated from etiolated soybean hypocotyls had similar molecular weights, but different elution properties from DEAE-cellulose, different apparent Km-values for glutamine and had different responses to inhibitors of GS. Alanine, the most inhibitory amino acid, resulted in a 35% inhibition at 1 mM (GS-I). Serine was a non-competitive inhibitor with respect to glutamine for GS-I. Increased phosphorylation of adenine nucleotides resulted in increased inhibition with ATP the most inhibitory (50% at 1 mM). CTP interacted with ATP synergistically and exhibited non-competitive inhibition with respect to glutamine for GS-II. Cumulative feedback inhibition was evident with amino acids and nucleotides for both enzymes. Pyrophosphate (5 mM) produced almost total inhibition of both activities with GS-II slightly more sensitive. These results indicate the involvement of amino acids and nucleotides in the feedback control of GS activity in soybean in a cumulative manner and the possible involvement of an energy charge system of regulation.
The major route of ammonia assimilation is the reaction which is catalysed by glutamine synthetase to give glutamine. Regulation and characterization of the enzyme in E. coli were done but not the localization. Cell-free extracts and purified cytoplasmic membranes using differential centrifugation and density gradient techniques, were assayed for the percentage activity of the enzyme. Glutamine synthetase was detected in all cell-free extracts of E. coli. 83% of the enzyme activity was found associated with the cytoplasmic membranes. Using monospecific glutamine synthetase antiserum and pronase, the major part of the enzyme was localized to the inside of the membranes and about 20% of the enzyme was found exposed to the outer surface.
Addition of MnCl2 but not of CaCl2, MgCl2 or ZnCl2, at low concentrations to nitrate-grown Azotobacter chroococcum cells enhanced the rate of assimilatory nitrate uptake. In the presence of Mn(II), however, the newly-reduced anion was excreted into the medium in the form of ammonium. When Mn(II) was added to a cell suspension in which the nitrate uptake activity had been inactivated by prior incubation with NH4Cl, stopping of assimilation of the ammonium present and resumption of nitrate uptake took place. If Mg(II) at a 100-fold concentration with respect to Mn(II) was then added, both NH4+ assimilation and NH4+ inhibition of nitrate uptake resumed. Cell suspensions that were preincubated with Mn(II) and assayed for glutamine synthetase (l-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2) activity in situ, showed an increase in the Mg(II)-dependent biosynthetic activity and a simultaneous decrease in the Mn(II)-dependent biosynthetic activity. It could be shown, however, that the Mg(II)-dependent glutamine synthetase biosynthetic activity, as assayed in situ, was significantly inhibited by Mn(II). These results suggest that Mn(II) exerts an external control on nitrate assimilation in Azotobacter, glutamine synthetase being the target for the effect of Mn(II) on this biological process.
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 μM 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.
Addition of ammonium salts to N2 fixing continuous cultures of Clostridium pasteurianum caused immediate stop of nitrogenase synthesis, while the levels of glutamine synthetase, glutamate dehydrogenase and asparagine synthetase remained constant. No evidence for an interconversion of the glutamine synthetase was found. The activities of glutamate synthase in crude extracts were inversely related to the nitrogenase levels. The intracellular glutamine pool rapidly expanded during nitrogenase repression and decreased as fast during derepression while the pool sizes of all other amino acids were not strongly related to the rate of nitrogenase formation. These investigations suggest glutamine as corepressor of nitrogenase synthesis.
A Tn5 insertional prototrophic mutant of Paracoccus denitrificans (UBM219) was generated which grew on high (>1 mM) but not low (<0.5 mM) ammonium as sole nitrogen source. It did not utilize nitrate and most amino acids except glutamate and aspartate. UBM219 showed more than 10-fold lower levels of ammonium (methylammonium) transport, aspartate and alanine aminotransferase, but more than 10-fold higher activities of glutamate dehydrogenase and glutamate synthase. This pleiotropy indicates a mutation in a regulatory gene affecting nitrogen metabolism in general. - Ammonia assimilation pathways and regulation in Paracoccus resemble the patterns in enterobacteria with the exception, that alanine is generated by amino transfer from glutamate to pyruvate.
Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg2+-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of 15N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.
1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.2.2. The holoenzyme with a Mr of 630,000 is composed of 12 subunits of Mr 52,000.3.3. A modified subunit of Mr 53,000 was also found by electrophoresis under denaturing conditions.4.4. It is shown that the Mr 53,000 species is the adenylylated subunit.5.5. The apparent Km values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.
This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.
We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product.
The gene encoding glutamine synthetase (GS), glnA, was cloned from Azotobacter vinelandii on a 6-kb EcoRI fragment that also carries the ntrBC genes. The DNA sequence of 1,952 bp including the GS-coding region was determined. An open reading frame of 467 amino acids indicated a gene product of Mr 51,747. Transcription of glnA occurred from a C residue located 32 bases upstream of an ATG considered to be the initiator codon because (i) it had a nearby potential ribosome-binding site and (ii) an open reading frame translated from this site indicated good N-terminal homology to 10 other procaryotic GSs. Sequences similar to the consensus RNA polymerase recognition sites at -10 and -35 were present at the appropriate distance upstream of the transcription initiation site. As expected from earlier genetic studies indicating that expression of A. vinelandii glnA did not depend on the rpoN (ntrA; sigma 54) gene product, no sigma 54 recognition sequences were present, nor was there significant regulation of glnA expression by fixed nitrogen. Repeated attempts to construct glutamine auxotrophs by recombination of glnA insertion mutations were unsuccessful, Although the mutated DNA could be found by hybridization experiments in drug-resistant A. vinelandii transformants, the wild-type glnA region was always present. These results suggest that glnA mutations are lethal in A. vinelandii. In [14C]glutamine uptake experiments, very little glutamine was incorporated into cells, suggesting that glutamine auxotrophs are nonviable because they cannot be supplied with sufficient glutamine to support growth.
This chapter focuses on the adenylylation of bacterial glutamine synthetase. The enzyme glutamine synthetase catalyzes the synthesis of glutamine from ammonia and glutamate in an ATP-dependent reaction. In bacteria and higher plants, it also participates in net synthesis of glutamate from ammonia and 2-oxoglutarate by functioning in a cycle with glutamate synthase. Physiological studies of Hölzer and colleagues indicated that the GS of Escherichia coli was very rapidly adenylylated when the organism was shifted from N-limited medium to a medium containing excess ammonium. Strains of S. typhimurium (glnE) that lack the ability to adenlylate GS show a large growth defect upon shift from N-limited media, media in which they have accumulated high levels of GS, to media containing a high concentration of NH4+. This growth defect appears to be because of excessive GS activity after the shift because it is eliminated in glnE strains that also synthesize abnormally low amounts of GS in both N-limited and NH4+, it is prolonged in glnE strains that also synthesize abnormally high amounts of GS in NH4+ excess media and the latter strains continue to show a growth defect even after adaptation to NH4+, and glnE strains have normal amounts of both of the glutamatebiosynthetic enzymes under N-limited conditions and after NH4+ upshift. At present, there appears to be no simple explanation for the distribution of this covalent modification. Although adenylylation of GS occurs widely among gram-negative bacteria, a more complete understanding of the physiological requirements for adenylylation of GS will contribute to understanding the basis for distribution of this covalent modification among the prokaryotes.
Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA- (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg- phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA+ Reg- phenotype. However, GlnA- RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA- RegC mutants accumulated chemically unaltered [14C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as gamma-glutamylmethylamide. GlnL Reg- mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA- strains is due to polar effects on glnL and glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt.
Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.
Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.
NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.
Glutamine synthetase (GS) was isolated from log phase cells and purified to a single protein as evidenced by gel electrophoresis. Protamine and ammonium sulfate precipitation and chromatography on DEAE-cellulose and Bio-Gel resulted in 380-fold purification. The enzyme was most sensitive to alanine (85% inhibition at 0.1 mM) but was also inhibited by glycine, arginine and serine. Combinations of inhibitory amino acids or nucleotides (AMP, ADP, ATP) exhibited cumulative inhibition. Cooperative inhibition was noted with CTP and any single nucleotide. Inhibition by CTP alone was uncompetitive with respect to glutamine. The enzyme was also regulated by the energy charge of the cell.
The glutamine synthetases from several Pseudomonas species were purified to homogeneity, and their properties were compared with those reported for the enzymes from Escherichia coli and other gram-negative bacteria. The glutamine synthetase from Pseudomonas fluorescens was unique because it was nearly precipitated quantitatively as a homogeneous protein during dialysis of partially purified preparations against buffer containing 10 mM imidazole (pH 7.0) and 10 mM MnCl2. The glutamine synthetases from Pseudomonas putida and Pseudomonas aeruginosa were purified by affinity chromatography on Affi-blue gel. Dodecamerous forms of the E. coli and P. fluorescens glutamine synthetases had identical mobilities during polyacrylamide gel electrophoresis. Their dissociated subunits, however, migrated differently and were readily separated by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulfate. This difference in subunit mobilities is not related to the state of adenylylation. Regulation of the Pseudomonas glutamine synthetase activity is mediated by an adenylylation-deadenylylation cyclic cascade system. A sensitive procedure was developed for measuring the average number of adenylylated subunits per enzyme molecule for the glutamine synthetase from P. fluorescens. This method takes advantage of the large differences in transferase activity of the adenylylated and unadenylylated subunits at pH 6.0 and of the fact that the activities of both kinds of subunits are the same at pH 8.45.
The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.
A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.
We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate.
The characteristics of soluble and membrane-bound glutamine synthetase (GS) from Rhodospirillum rubrum were compared with those of the enzyme located in situ (measured in detergent-treated cells). The results suggest that in vivo GS may be associated with, or bound to, the chromatophore membranes. GS was found to reversibly associate and dissociate from purified chromatophores as a function of the ionic strength of the buffer or the Mg2+ concentration. Solubilized GS was purified to homogeneity and found to be similar to the GS of enteric bacteria in that its molecular weight was about 600,000 and it had one type of subunit of 51,000 molecular weight. Removal of GS from the membrane had no effect on the Km values for the substrates of the biosynthetic reaction, but it did have a substantial effect on both its Mg2+ requirement (the Km increased 10-fold) and the sensitivity of the gamma-glutamyl transferase reaction to the inhibitor methionine sulfoximine (the I0.5 decreased from 1,500 to 60 microM). Both observations suggest that the active site of GS is influenced by its association with the membrane. GS activity was shown to respond to NH4+, phosphodiesterase, Mg2+, and adenylylation cofactors in a manner identical to that of the GS of the coliform bacteria, suggesting that the former may also respond to adenylylation and deadenylylation. Finally, R. rubrum GS was also inhibited by NH4+ by a newly observed, as yet undefined, system.
Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii. During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E. M. Barnes, Jr., and P. Zimniak, J. Bacteriol. 146:512-516, 1981). This metabolite has been isolated from A. vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound. Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake. The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A. vinelandii. The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase. In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide. Likewise, an antiserum against the glutamine synthetase of E. coli precipitated the transferase and both synthetic activities at similar titers. During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately. A partially purified preparation of glutamine synthetase from A. vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg). The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation. Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.
Azotobacter vinelandii was grown in oxygen-controlled continuous cultures under conditions of dinitrogen fixation. Different oxygen concentrations were adjusted with air. Cell-free extracts were employed to study the oxygen dependency of the intracellular distribution and activity of the following enzymes: nitrogenase, glutamine synthetase and glutamate synthase. Nitrogenase was localized exclusively in the soluble fraction. Its activity increased steeply when the oxygen concentration employed in growing the organism decreased from about 30% close to 0% air saturation. Glutamine synthetase was identified exclusively as a soluble enzyme. The degree of adenylylation of the enzyme increased from about one to about four parallel to nitrogenase activity when the oxygen concentration in the culture was lowered. Glutamate synthase was detected in both a soluble and a membrane-bound form. The sum of specific activities of both forms stayed constant irrespective of changes in the oxygen concentration. However, with increasing oxygen concentration, the proportion of the membrane-bound form increased up to two-thirds of the total activity.
Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [(L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH4+). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to approximately 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH4+ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH4+ -excess conditions and thereby to allow rapid growth.
Under N2-fixing conditions, Azotobacter vinelandii expresses a specific transport system for methylammonium (ammonium) [E. M. Barnes, Jr. and P. Zimniak (1981) J. Bacteriol. 146, 512-516]. This activity is decreased markedly by culture of cells in the presence of 10 mM ammonium or 2 mM methylammonium; in both cases, the Vmax values for methylammonium uptake were 25% of those of N2-fixing cells. Mixing experiments with assay medium indicate that transport activity is controlled by intracellular rather than extracellular metabolites. Glutamine synthetase activity of cells cultured with ammonium was 33% that of N2-fixing cultures, but activity was unaffected by incubation with methylammonium. Thus ammonium transport and ammonium fixation are regulated independently. When ammonium was removed from the medium, cells recovered over 90% of the initial transport activity after 1 h; this recovery was not affected by addition of chloramphenicol. The loss of uptake activity in cells incubated with ammonium or methylammonium correlated with over sixfold increases in intracellular levels of glutamine and gamma-glutamylmethylamide, respectively. Recovery of transport was accompanied by similar reductions in pools of these compounds. Over one-half of methylammonium transport activity could be blocked by direct addition of 10 mM glutamine or gamma-glutamylmethylamide to transport assays; these concentrations were similar to those observed in vivo. The glutamine analog, 6-diazo-5-oxo-L-norleucine, was the most potent inhibitor found (68% inhibition at 10 microM). These results indicate that the regulation of ammonium transport by ammonium and methylammonium is due to inhibition of the transporter by intracellular gamma-glutamyl amides rather than by repression of transporter synthesis.
The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential.
The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers.
Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2-oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.
A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.
GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridylylates two related proteins, PII and GlnK. The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC. Results presented here demonstrate that glnD is an essential gene in Azotobacter vinelandii. Null glnD mutations were introduced into the A. vinelandii genome, but none could be stably maintained unless a second mutation was present that resulted in unregulated activity of GS. One mutation, gln-71, occurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein. The second, created by design, was glnAY407F (MV75), altering the adenylylation site of GS. The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia coli glnE gene into MV72, a glnD(+) derivative of MV71, restored the regulation of GS activity. GlnK-UMP is therefore apparently required for GS to be sufficiently deadenylylated in A. vinelandii for growth to occur. The DeltaglnD GS(c) isolates were Nif(-), which could be corrected by introducing a nifL mutation, confirming a role for GlnD in mediating nif gene regulation via some aspect of the NifL/NifA interaction. MV71 was unexpectedly NtrC(+), suggesting that A. vinelandii NtrC activity might be regulated differently than in enteric organisms.
Helicobacter pylori causes gastroduodenal disease, which is mediated in part by its outer membrane proteins (OMPs). To identify OMPs of H. pylori strain 26695, we performed a proteomic analysis. A sarcosine-insoluble outer membrane fraction was resolved by two-dimensional
electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 10 to 100 kDa, were
visible on the gel in the alkaline pI regions (6.0 to 10.0). The proteome of the OMPs was analyzed by matrix-assisted laser
desorption ionization-time-of-flight mass spectrometry. Of the 80 protein spots processed, 62 spots were identified; they
represented 35 genes, including 16 kinds of OMP. Moreover, we identified 9 immunoreactive proteins by immunoblot analysis.
This study contributes to the characterization of the H. pylori strain 26695 proteome and may help to further elucidate the biological function of H. pylori OMPs and the pathogenesis of H. pylori infection.
The Escherichia coli AmtB protein is member of the ubiquitous Amt family of ammonium transporters. Using a variety of [14C]methylammonium-uptake assays in wild-type E. coli, together with amtB and glutamine synthetase (glnA) mutants, we have shown that the filtration method traditionally used to measure [14C]methylammonium uptake actually measures intracellular accumulation of methylglutamine and that the kinetic data deduced from such experiments refer to the activity of glutamine synthetase and not to AmtB. Furthermore, the marked difference between the K(m) values of glutamine synthetase calculated in vitro and those calculated in vivo from our data suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. The use of a modified assay technique allows us to measure AmtB activity in vivo. In this way, we have examined the role that AmtB plays in ammonium/methylammonium transport, in the light of conflicting proposals with regard to both the mode of action of Amt proteins and their substrate, i.e. ammonia or ammonium. Our in vivo data suggest that AmtB acts as a slowly conducting channel for NH3 that is neither dependent on the membrane potential nor on ATP. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face.
Hydrogenobacter thermophilus TK-6, a thermophilic and obligately chemoautotrophic bacterium, assimilates ammonium using glutamine synthetase (GS). GS was purified using three chromatography steps. The purified GS was found to belong to GS type I on the basis of its subunit composition and molecular weight. The Mg2+ -dependent activity of this GS significantly increased after incubation with phosphodiesterase, indicating that GS is subject to adenylyl/deadenylyl regulation, a posttranslational modification system reported mainly among enterobacteria. The degree of this posttranslational modification changed depending on growth phase, confirming that adenylyl/deadenylyl regulation functions in vivo. Interestingly, the Km for glutamate of H. thermophilus GS was significantly higher than those of other organisms, suggesting that GS activity is affected by intracellular glutamate concentration.
Glutamine synthetase, purified from rat liver, is homogeneous on acrylamide gel electrophoresis and on ultracentrifugation (s20,w, 15.0 S). The enzyme, which consists of eight subunits (subunit molecular weight, 44,000), resembles ovine brain glutamine synthetase in its physical properties, amino acid composition, and substrate specificity. Complete inhibition of the liver enzyme by methionine sulfoximine is associated with the binding of 4 moles of inhibitor per mole of enzyme. The enzyme is activated by α-ketoglutarate (in the presence of Mg⁺⁺ or Mn⁺⁺) and is inhibited by glycine, l-alanine, l-serine, and carbamyl phosphate (with Mn⁺⁺ only). Evidence is presented that inhibition by carbamyl phosphate is produced by the binding of this compound to the active site of glutamine synthetase. Thus, the enzyme can catalyze the synthesis of ATP from carbamyl phosphate and ADP. When glutamine synthetase is incubated with ADP, carbamyl phosphate, and glutamate, glutamine is formed. Enzyme inactivated by treatment with methionine sulfoximine and ATP did not utilize carbamyl phosphate. Liver glutamine synthetase also catalyzes ATP synthesis from ADP and acetyl phosphate; this reaction is competitively inhibited by glutamate. Glutamine synthetases from ovine brain, rat brain, and Escherichia coli also catalyze ATP synthesis from carbamyl phosphate and ADP.
A procedure is described for the purification of glutamine synthetase from Bacillus subtilis, grown under conditions of nitrogen limitation, to a nearly homogeneous state. Although the purified enzyme differs from the glutamine synthetase from Escherichia coli in catalytic properties and stability, the physical characteristics of these two enzymes are quite similar. The B. subtilis enzyme has a molecular weight of ∼600,000, a sedimentation coefficient at infinite dilution of 19.3 S, and is seen in electronmicrographs as a molecule composed of 12 subunits arranged in 2 superimposed hexagonal rings. Studies with the dissociated enzyme suggest that the subunit molecular weight is ∼50,000, in agreement with a dodecameric aggregate structure of the native enzyme which contains 12 subunits of similar size. Both Mn²⁺ and Mg²⁺ activate the enzyme in the biosynthesis of l-glutamine, but, unlike the E. coli system, the Mg²⁺-dependent activity is intrinsically less stable than the Mn²⁺-dependent activity. Differences between the B. subtilis and E. coli enzymes are apparent also in the amino acid compositions, in the susceptibility to digestion by carboxypeptidase A, in the C-terminal amino acid of the subunit polypeptide chains, in immunochemical properties, and in that the E. coli adenylylating enzyme system does not incorporate 5'-¹⁴C-adenylyl groups into the B. subtilis glutamine synthetase.
When the exhaustion of sucrose or sulfate or the induction of encystment (by incubation in 0.2% beta-hydroxybutyrate) leads to termination of growth in Azotobacter vinelandii batch cultures, the nitrogenase levels in the organisms decreased rapidly, whereas glutamate synthase and glutamine synthetase levels remained unaltered. Glutamate dehydrogenase activities were low during the whole culture cycle, indicating that ammonia assimilation proceeds via glutamine. Toward depletion of sucrose or during induction of encystment, slight secretion of ammonia with subsequent reabsorption was occasionally observed, whereas massive ammonia excretion occurred when the sulfate became exhausted. The extracellular ammonia levels were paralleled by changes in the glutamine synthetase activity. The inactivation of the nitrogenase is explained as a result of rising oxygen tension, a consequence of a metabolic shift-down (reduced respiration) that occurs in organisms entering the stationary phase.
Unified theory for gel electrophoresis and gel filtration: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers. This model has been generalized to apply to nonspherical molecules and to several gel types. The model provides equations for inter-relationships between mobility, partition coefficient, gel concentration, and molecular radius; it gives a non-Gaussian distribution of pore sizes as a function of gel concentration. The theory defines conditions for optimal separation and optimal resolution in gel filtration and gel electrophoresis. The difference in resolving power between the two fractionation methods is accounted for by the fact that gel filtration is a form of partition chromatography.
This chapter describes the assay, purification, and properties of glutamine synthetase. The synthesis of glutamine in microorganisms may be regarded as the first step in a highly branched pathway, which leads ultimately to the biosynthesis of a large number of different compounds, including tryptophan, adenylic acid, cytidylic acid, and glucosamine 6-phosphate. Glutamine synthetase from Escherichia coli is, therefore, of cardinal importance in biosynthetic metabolism and is a strategic target for cellular regulation. The purified enzyme may be assayed by measuring the production of inorganic phosphate in the biosynthetic reaction. Alternatively, a continuous recording of catalytic activity may be achieved by coupling the production of adenosine diphosphate (ADP) in the biosynthetic assay to the oxidation of diphosphopyridine nucleotide (DPNH) by the addition of phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase in excess. Preparations of purified glutamine synthetase may differ from one another in the amount of covalently bound adenosine monophosphate (AMP) residues that they contain. Mn2+ stabilizes the native dodecameric structure of the enzyme thus, protecting the enzyme from reacting with sulfhydryl reagents and other mild denaturants.
The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.
1.1. A method is presented for the determination of relatedness among proteins based upon statistical analysis of differences in amino acid composition.2.2. Analytical results of this method correlate closely with comparisons based upon the sequence of amino acids within the three families of related proteins tested: hemoglobins, light chains of myeloma proteins and cytochromes.3.3. The method is applied to immunoglobulins for which amino acid sequence data are not available and general conclusions are drawn regarding the evolutionary relationships of these immunoglobulins.
This chapter discusses the structure the glutamine synthetase of Escherichia coli. The glutamine synthetase from E. coli and that from other gram-negative microorganisms tested exist in two forms: an unmodified form and a form possessing covalently bound adenylyl groups (10-Ig). Under appropriate conditions, both forms of the enzyme are catalytically active; however, they differ significantly with respect to catalytic potential, pH optimum, divalent cation specificity, and susceptibility to feedback inhibition. Covalent modification of the E. coli enzyme is the basis of an elaborate mechanism for the fine control of glutamine synthetase activity in this organism. E. coli glutamine synthetase has a molecular weight of 600,000 and is composed of 12 apparently identical subunits. Electron microscopic examination of preparations negatively stained with sodium silicotungstate disclosed that the subunits are arranged in two hexagonal rings that lie one on top of the other in a face-to-face fashion. In hydrodynamic studies, the enzyme in solution behaves as a compact, essentially spherical particle. It was calculated from these data that the β parameter for describing the shapes of kinetic units in solution is 2.1 X 106 or about the same value as that for a spherical particle. The chapter also discusses the amino acid composition of E. coli glutamine synthetase. The composition is expressed in terms of the number of amino acid residues per 50,000 g since it is believed that all 12 subunits of the enzyme are identical. It is especially noteworthy that each subunit contains 5 cysteinyl, 15 tryosyl, 3-tryptophanyl, 15-16 methionyl, 26 lysyl, and 25 arginyl residues.
The primary steps of N2, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N2 as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2–3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthetase activity. An increase in the degree of adenylation correlates with a repression of nitrogenase synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.—After addition of nitrate ammonia appears in the medium, probably due to the action of a membrane bound dissimilatory nitrate reductase.—Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.
1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.
This chapter describes that enteric bacteria have evolved in a manner that has made glucose their preferred source of carbon and energy, and ammonia their preferred source of nitrogen. However, they have the ability to use a large variety of carbon compounds as substitutes for glucose and a large variety of nitrogen compounds as substitutes for ammonia. Some compounds, like the amino acid L-histidine, can substitute for both glucose and ammonia: their degradation serves to provide the cell with energy, as well as with carbon- and nitrogen-containing building blocks. The utilization of these alternative sources of energy—carbon and nitrogen—depends on enzymes not required by cells growing on glucose and ammonia. The chapter provides an overview of the classical and postclassical modes of regulation of their synthesis. The result of these regulatory mechanisms is that the production of these enzymes occurs only when they are required for growth.
The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.
The primary steps of N2, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N2 as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2-3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.--Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.
Previous attempts to demonstrate adenylylation/deadenylylation control of glutamine synthetase from photosynthetic organisms have given negative results; we have reexamined the enzyme of Rhodopseudomonas capsulata in this connection. Studies on other systems have shown that adenylylated and deadenylylated forms of glutamine synthetase respond differently to Mg2+ in the γ-glutamyltransferase assay, and this was observed with the R. capsulata enzyme when its adenylylation state was stabilized by addition of cetyltrimethylammonium bromide to cells before they were harvested for preparation of extracts. The relative activities of glutamine synthetase in the presence and absence of added Mg2+ is an index of adenylylation state and on the basis of this and other criteria, the enzyme in R. capsulata cells grown with excess NH+4 as N source is largely adenylylated, whereas the deadenylylated form predominates when the organism is grown on N2. The results of experiments on the effects of snake venom phosphodiesterase on response of partially purified glutamine synthetase (from R. capsulata cells grown on NH+4) to metal ion supplementation (Mg2+ and Mn2+) provided confirmatory evidence for adenylylation. Conditions are described for demonstration of regulation of R. capsulata glutamine synthetase adenylylation state, in vivo, by light intensity. When the N source for growth is glutamate, the extent of adenylylation decreases with increase of light intensity. In cells subjected to a sudden shift-down in light intensity, the extent of glutamine synthetase adenylylation increases rapidly; conversely, a shift-up in light intensity causes, after a lag, a decrease in the degree of adenylylation. The light intensity effects are interpreted to reflect regulation of adenylylation/deadenylylation by the ‘energy state’ of the cell.
Comparisons of the amino acid compositions of the nitrogenase proteins from different organisms and their correlation with cross-reactivities and taxonomical data suggest an evolution within bacterial genomes rather than within plasmids. Comparisons of the amino acid compositions of nitrogenases and other ATP-ases show similarities which might be due to the evolution of these ATP-ases from a common ancestral protein.
Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH4+ concentrations were investigated during the transition from an NH4+ free medium to one containing NH4+ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80–100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1–2 hrs the NH4+ remaining in the medium is absorbed too, indicating the induction or activation of a new NH4+ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH4+ salts leads to acidification of the culture. Anaerobiosis suppresses NH4+ transport.
A rise in the extracellular NH4+ level leads to a reversible rise in the glutamine synthetase activity, which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH4+ level of about 0.6 mM does not change when extracellular NH4+ is taken up and repression of nitrogenase starts.
A method to determine the dry weight (0.25-2 mg) of aqueous protein solutions within 1 h, using an electrobalance, is described. The drying of 50-200 mul solution pipetted onto a glass fiber disc is carried out in vacuo at 70 degrees C until the recorded dry weight becomes constant (within 25-40 min). It has been shown that the dried residue can subsequently be used for other purposes, such as quantitative amino acid analyses. The method is also suitable for the determination of moisture content in lyophilized protein samples.
The interaction of proteins with small ligands embodies the fundamental physicochemical principles that are operative in the physiological regulation of enzyme activity, the specific interactions among proteins and of proteins with nucleic acids and other macromolecules. This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity and ligand binding. Many parts of the compact protein structure can contribute toward the shift in energy and structure taking place on ligand binding. The energies of ligand–ligand interaction, although small are sufficient to shift the covalent equilibria in which proteins take part, to a significant extent, and this may be found responsible for the interconversion of chemical and osmotic energies in metabolism. While, the ligand correlation because of the existence of free-energy coupling among the bound ligands is a molecular property to be explained by a study of the isolated protein, the enhancement of the effects by the simultaneous changes in ligand concentration is a system property that requires consideration of other entities. The consideration of ligand–protein interactions provides with a model for the relative importance to be assigned to molecules in the organic functions. Thus, study of the interactions of proteins and small ligands provides the basis for the understanding of biological specificity at the molecular level.
Antisera prepared against adenylylated and unadenylylated Escherichia coli glutamine synthetase cross-reacted with the glutamine synthetases from a number of gram-negative bacteria and one gram-variable species as demonstrated by immunodiffusion and inhibition of enzyme activity. In contrast, the antisera did not cross-react with the glutamine synthetases from gram-positive bacteria (with one exception) nor with the synthetases of higher organisms. Modification of the various glutamine synthetases by covalent attachment of adenosine 5'-monophosphate (or other nucleotides) was tested for by determining whether or not snake venom phosphodiesterase altered catalytic activity in a manner similar to its effect on adenylylated E. coli glutamine synthetase. Only the activity of the glutamine synthetases from gram-negative bacteria grown with specific levels of nitrogen sources could be altered by snake venom phosphodiesterase. In addition, a relative order of antigenic homology between cross-reacting enzymes was suggested based on the patterns of spur formation in the immunodiffusion assay.
Glutamine synthetase (EC 6.3.1.2) has been purified from Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 630 000, and that of a component obtained on treating the enzyme with 4 M urea or 1% sodium dodecyl-sulfate plus 10 mM 2-mercaptoethanol 540 000 or 500 000, respectively, suggesting that the enzyme consists of 12 subunits. The enzyme requires divalent cations for activity, Mg2+ being the most effective activator. The pH and temperature optima in the presence of Mg2+ or Mn2+ are 7.3 or 6.5 and 70 or 75 °C, respectively. The enzyme is inactivated on exposure to 70 °C, but the inactivation is partially protected by Mg2+ (Mn2+ or glutamate) and completely by Mg2+ (Mn2+), NH4Cl and glutamate. Thermodynamic quantities for the enzyme reaction show a conformational transition at 58 °C. Glycine, alanine, serine, tryptophan, histidine, AMP and CTP inhibit the enzyme in the presence of Mg2+ or Mn2+. The inhibition of the Mg2+- or Mn2+-activated enzyme by these compounds seems to be cumulative, except for the combined effects of amino acids on the Mn2+-activated enzyme. Circular dichroism analyses of the enzyme show an α-helix, ß-structure and unfolded conformation. Addition of Mg2+ or Mn2+ results in an increase of the α-helix content accompanied by a decrease of the unfolded conformation content.
Glutamine synthetase seems to act as a positive control element for
nitrogenase synthesis in a way similar to that already proposed for
histidase synthesis in Klebsiella. Studies along lines opened up by
these findings may provide answers to many of the problems in
establishing nitrogen fixation in non-leguminous plants.
Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.
A statistical method for quantifying the relatedness among proteins was used to perform 2926 paired comparisons of amino-acid composition among 77 contractile and membrane-associated proteins from diverse species and sources. Relatedness of amino-acid compositions correlates with homology of amino-acid sequence. A high degree of relatedness was detected among K(+)-dependent membrane ATPase of Streptococcus faecalis, coupling factors F(1) and CF(1) from mitochondria and chloroplasts, outer fiber protein of cilia, ciliary dynein, tubulin, various actins, and myosin subfragment S-1. Heavy meromyosin and tropomyosin were related to each other but not to the first group of proteins. Differences in the degree of methylation may account for some differences in physiological function. Because of their diverse sources, the high degree of relatedness among these proteins is more compatible with evolution from common ancestral genes than with convergent evolution. Squid axon filarin, molluscan paramyosin, and bacterial flagellins appear to be unrelated either to each other or to any of the other proteins studied. Existence of persistent homologies among so many diverse proteins implies conservation of genetic information during evolution by utilization of codons for preferred amino-acid sequences in various proteins.
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 α-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.
In the first 15–30 see after addition of 10 mM NH4+ to Escherichia coli cells, one observes a 20-fold increase in the concentration of glutamine, an approximate one-third reduction in the stationary concentration of glutamate, an approximate 90% decrease in the concentration of ATP and a decrease in the activity of glutamine synthetase to a few percent of the initial value. In the subsequent 30 see, glutamine synthetase remains inactive, glutamine concentration decreases, glutamate concentration increases 2 to 3-fold, and the ATP concentration rises slowly. The above-mentioned changes after addition of NH4+, together with previous observations on properties of purified glutamine synthetase and adenylyl transferase, make the following sequence of events probably: NH4+ enters the cells and is quickly incorporated into glutamate yielding glutamine, with a concomitant utilization of ATP. The accumulated glutamine stimulates the adenylyl transferase resulting in an inactivation of glutamine synthetase. Thus, 15 sec after addition of NH4+, further synthesis of glutamine is prevented. Consequently, glutamate concentration increases, glutamine concentration decreases, and ATP concentration increases. On the basis of these findings and conclusions, the following biological functions of NH4+ inactivation of glutamine synthetase are discussed: (a) prevention of a too high level of glutamine and (b) prevention of a sustained decrease in the ATP concentration which would be harmful to the cell.
The partial specific volume of a solute is a characteristic parameter that can be used in investigations of protein associations and changes in conformation, as well as in studies on protein solvent interactions and various other intermolecular interactions. It provides information needed for the determination of particle mass by means of ultracentrifugation and small-angle X-ray scattering. The precision density measurement in the apparatus described in the chapter is based on the determination of the natural frequency of an electronically excited, mechanical oscillator, its effective mass being composed of its own unknown mass and the well-defined, but also unknown, volume of the sample under investigation. To assure that this volume be well defined, the oscillator is made of a hollow, U-shaped glass tube that can be filled with the liquid sample. The mode of vibration is that of a bending-type oscillator. The positions of its vibrating nodes, which in fact determine the limits of the volume of sample taking part in the motion, is kept stable by the abrupt change in the cross-section of the glass tubes.
The inactivation of glutamine synthetase in vivo on addition of ammonium ions to the culture medium was studied in different microorganisms. Only those belonging to the family of Enterobacterioceae showed inactivation. The transfer activity of the enzyme was much less affected than the synthetic one. As in Escherichia coli , the glutamine synthetase of Salmonella typhimurium could be inactivated in vitro in a system requiring glutamine, ATP, Mg ²⁺ and an enzyme.
The inactivating enzymes of E. coli and S. typhimurium have been separated and cross‐tested against their synthetases. The inactivating enzymes inactivated both glutamine synthetases in the in vitro inactivation process. In contrast, synthetases of microorganisms which did not exhibit in vivo inactivation were not affected by these inactivating enzymes.
The fact that only glutamine synthetases of Enterobacteriaceae could be inactivated suggests that this regulatory mechanism is limited to a group of organisms possibly derived from the same ancestor.
The glutamine synthetase from E. coli has a molecular weight of 600,000 and is composed of 12 apparently identical subunits that are arranged in two superimposed hexagonal layers. The activity of the enzyme is modulated by the covalent attachment of one 5′-adenylyl group to each subunit of the enzyme. Adenylylation is catalyzed by a specific ATP:adenylyltransferase that catalyzes a transfer of the adenylyl moiety from ATP to a particular tyrosyl hydroxyl group of each subunit. Adenylylation is accompanied by a decrease in catalytic potential, conversion from an Mg²⁺-dependent to Mn²⁺-dependent enzyme, a shift in the pH optimum from 8.0 to 6.9 and an increase in susceptibility to feedback inhibition by CTP, AMP, histidine and tryptophan.
Evidence is presented that the dilution effect on nitrogenase from Azotobacter vinelandii can be overcome by the addition of an optimal amount of Component I (iron-molybdenum protein) or Component II (iron protein); and this optimized activity parallels the activity obtained by applying the dilution-factor correction. The synthesis of both of the nitrogenase components, after exhaustion of ammonia from the medium, seems to be coordinate. The degradation of both of the nitrogenase components after repression was found to be coordinate and neither component was found to be in excess at any time after repression. For the initial one-half generation the nitrogenase activity falls at approximately the same rate as the increase in cell mass, suggesting simple dilution. After this point, however, activity falls more rapidly and more than 95 % of the activity is lost in two generations.
We have identified the products of four of the six genes involved in bacteriophage T4 tail fibre assembly by sodium dodecyl sulphate-acrylamide gel electrophoresis of tail fibre mutant lysates and particles purified from them. Two large polypeptides, a 150,000 molecular weight species which is the product of gene 34 (P34), and a 120,000 molecular weight species which is the product of gene 37 (P37), are the major structural components of the fibres. Two smaller polypeptides, the products of genes 38 and 57, act in the conversion of the large structural polypeptide chains into morphological and antigenic half fibres. P38, molecular weight 26,000, does not appear to be a structural protein of the phage. In its absence, P37 is synthesized but remains unassembled. P57 plays a pleiotropic role in phage assembly: in its absence, P37 and P34 are both synthesized, but neither is assembled into fibres, and P12, a 60,000 molecular weight protein of the baseplate, is not incorporated into baseplates. The state of these unassembled polypeptide chains from 38- and 57- lysates can be distinguished from their state in wild-type lysates by two criteria: (a) they are soluble in sodium dodecyl sulphate at room temperature, whereas normal fibres and phages require heating for solubilization, and (b) they are concentrated in the low-speed pellet fractions of the lysates, suggesting that they are either aggregated, or bound to the cell envelope.A gene 36 amber mutation depressed the synthesis of P37 and a gene 37 amber mutation depressed the synthesis of P38, suggesting that these three genes are cotranscribed.These findings allow the formulation in greater detail of the early stages of the fibre assembly pathway.
The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be due to a color change in the cells from reddish brown to dark brown. Spectrophotometric examination of extracts from ammonia- and N2-grown cells did not reveal any components responsible for this color difference, but this result may reflect only the presence of interfering substances in the crude extract.
A method has been found which distinguishes between a size isomer family of proteins (e.g. bovine serum albumin polymers) and a charge isomer family of proteins (e.g. lactate dehydrogenase isoenzymes), utilizing disc gel electrophoresis. When the log of protein mobility relative to the dye front was plotted versus acrylamide gel concentration, size isomeric proteins gave a family of nonparallel lines extrapolating to a common point in the vicinity of 0% gel concentration; charge isomeric proteins gave a parallel family of lines. Proteins differing in both charge and size gave non-parallel lines intersecting at gel concentrations other than 0% gel concentration. The slope of such a plot is related to molecular weight. The molecular weight-slope relation was established utilizing 17 well-characterized proteins as standards. From this relation, it is possible to determine the molecular weight of a protein with an average precision of ±4%. This molecular weight method can be applied to a single protein in a mixture of proteins provided a specific detection test is available. This method should find uses in distinguishing between size and charge isomer families of proteins, for the rapid, easy, and accurate determination of protein molecular weights, and as a valuable aid in indicating the procedures to be used in enzyme purifications.
Glutamine synthetase from sheep brain has been isolated by a new procedure, which gives much higher yields of the enzyme than have been obtained previously. The new procedure may be conveniently carried out in the laboratory and may be adapted to a 20-fold scale-up for use in a pilot plant. The amino acid composition of the enzyme has been determined. A single N-terminal amino acid, arginine, was found. Close to 65 peptides were obtained after digestion of the enzyme with trypsin and about 16 peptides were found after treatment of the enzyme with cyanogen bromide. These findings are consistent with the view that the eight subunits of the enzyme are identical. The data indicate that 12-14 of the 15 half-cystine residues/enzyme subunit found on amino acid analysis are cysteine residues. Titration with 5,5′-dithiobis(2-nitrobenzoate) indicates that one sulfhydryl group per subunit reacts readily with this reagent and that the other enzyme sulfhydryl groups react more slowly and are presumably buried. The enzyme is inactivated by treatment with N-ethylmaleimide and protected from such inactivation by ATP + Mg2+.
A rapid method for the determination of tryptophan in proteins is presented. It is based on ab- sorbance measurements at 288 and 280 mp of the protein dissolved in 6 M guanidine hydrochloride. Blocked tryptophanyl (N-acetyl-L-tryptophanamide) and tyrosyl (glycyl-L-tyrosylglycine) compounds were selected as C urrent methods of protein amino acid analysis do not give quantitative values for tryptophan and conse- quently the amino acid compositions, which are other- wise complete, fail to report tryptophan values. The principal reason for this situation is that the standard procedure of protein hydrolysis in strong acid results in the destruction of tryptophan (Hill, 1965). Therefore a second procedure is required to measure tryptophan. Alkaline hydrolysis is less destructive but does not give quantitative recoveries generally (Spies and Chambers, 1949). Enzymatic hydrolysis of proteins can give quanti- tative yields of tryptophan but this method may not be generally valid (Hill and Schmidt, 1962).
A rapid and convenient colorimetric method for the determination of small amounts of protein, based on the biuret reaction and readings at 330 mμ, is described. The "micromethod" allows the determination of 10 to 200 mg per cent protein with an acurassy of ± 1.5 mg per cent. An "ultra micro" modification allows determinations of 10 to 400 micrograms protein. The method is applied for the determination of total protein in 1 ml of cerebrospinal fluid. © 1953 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Quantitative determination of amino acids is made simpler and more rapid by an instrument for automatically recording the ninhydrin color value of the effluent from ion exchange columns. The influent buffer, freed of air, is pumped at a constant rate through a column of sulfonated polystyrene resin. The effluent is met by a capillary stream of ninhydrin reagent delivered by a second pump. The color is developed by passing the mixture of reagent and effluent through a spiral of capillary Teflon tubing immersed in a boiling water bath. The absorbance of the resulting solution is measured continuously at 570 and 440 mμ as it flows through a cylindrical glass cell of 2-mm. bore. The peaks on the recorded curves can be integrated with a precision of 100 ± 3% for loads from 0.1 to 3.0 μmoles of each amino acid. A hydrolyzate of a protein or peptide may be analyzed in less than 24 hours. The more complex mixtures characteristic of blood plasma, urine, and mammalian tissues can be analyzed in 2 days. The instrument is applicable in principle to detection of ninhydrin-positive constituents in the effluent from various types of Chromatograph columns.
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