Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mol·min^-1·mg^-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.Abbreviations FTHFS formyltetrahydrofolate synthetase - kb kilobase - H₄-folate tetrahydrofolate - SDS sodium dodecyl sulfate A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Atlanta, GA, 1987 (C. R. Lovell, A. Przybyla and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, K126, p. 223).     

Cloning of the glutamine synthetase gene fromspirulina platensis

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.     

Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence

While considerable progress has been achieved in plant CDPK studies in the past decade, there is relatively no information about the potential substrates of CRKs. In this report, a yeast two-hybrid screen was performed with truncated form of AtCRK3 as bait to identify its interacting proteins in an effort to dissect its physiological roles. One gene encoding cytosolic glutamine synthetase AtGLN1;1 was isolated. Further analyses indicated that AtGLN1;1 could interact specifically with AtCRK3 and the kinase domain of AtCRK3 and the catalytic domain of AtGLN1;1 were responsible for such interaction, respectively. Furthermore, in vitro and in vivo co-immunoprecipitation results strongly supported that they could physically interact with each other. Phosphorylation assays revealed that AtGLN1;1 could be specifically phosphorylated by AtCRK3 in vitro. All the results demonstrate that AtGLN1;1 may be a substrate of AtCRK3. In addition, both AtGLN1;1 and AtCRK3 could be induced by natural or artificially induced leaf senescence, implying that such interaction may be involved in the regulation of nitrogen remobilization during leaf senescence.     

A discovery of novel Mycobacterium tuberculosis pantothenate synthetase inhibitors based on the molecular mechanism of actinomycin D inhibition

Mycobacterium tuberculosis pantothenate synthetase is a potential anti-tuberculosis target, and a high-throughput screening system was previously developed to identify its inhibitors. Using a similar system, we screened a small library of compounds and identified actinomycin D (ActD) as a weak inhibitor of pantothenate synthetase. A new method was established to discover more effective inhibitors by determining the molecular mechanism of ActD inhibition followed by structure-based virtual screening. The molecular interaction of inhibition was determined by circular dichroism and tryptophan fluorescence quenching. The structure-based search and virtual screening were performed using the Molecular Operating Environment (MOE) program and SYBYL 7.5, respectively. Two inhibitors were identified with an IC₅₀ for pantothenate synthetase that was at least ten times better than that of ActD.     

Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property

The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.     

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

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

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Biochemical and molecular characterization of transgenic<Emphasis Type="Italic"> Lotus japonicus</Emphasis> plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS₁) or in the chloroplast (GS₂). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS₁ gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS₁ polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.Abbreviations GS Glutamine synthetase - UTR Untranslated region     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

Some properties of glutamine synthetase from Anabaena cylindrica

Some properties of the biosynthetic and -glutamyltransferase activities of glutamine synthetase (EC 6.3.1.2) from Anabaena cylindrica are described, including requirement for divalent cations, pH optimum and K_m for substrates. The -glutamyl-transferase reaction was inhibited by L-glutamate, ammonia and ATP. The inhibition by L-glutamate and ammonia was competitive for L-glutamine and non-competitive for hydroxylamine. Both the biosynthetic and the -glutamyltransferase activities of the desalted enzyme were much more sensitive to inactivation by treatments such as urea, hydroxylamine and incubation at 50° C than the preparation which contained a divalent cation. The effects of some substrates of these reactions on protection against thermal denaturation and hydroxylamine were examined. An interpretation of these results in terms of the sequence of binding of substrates both in the biosynthetic and the -glutamyltransferase reactions are discussed.     

CpG oligodeoxynucleotides promote phospholipase D dependent phagolysosome maturation and intracellular mycobacterial killing in M. tuberculosis infected type II alveolar epithelial cells

CpG oligodeoxynucleotides have been previously shown to enhance antimycobacterial response in human monocytes/macrophages. The present study reports evidences showing the capability of CpG oligodeoxynucleotides to induce (i) host phospholipase D (PLD) activation, (ii) PLD dependent reactive oxygen intermediate production, (iii) PLD dependent phagolysosome maturation and (iv) PLD dependent intracellular mycobacterial killing in type II alveolar epithelial cells. These are the first evidences showing that alveolar epithelial cells may represent efficient effecter cells during primary innate antimycobacterial immune response.