Gene Encoding Glutamine Synthetase Research Articles

Phylogenomic analysis of glutamine synthetase gene family in Helianthus annuus L.

Glutamine synthetase is one of the predominant enzymes in nitrogen metabolism. In developing leaves, glutamine is mainly produced in chloroplasts by the activity of the GS2 isoenzyme. It catalyzes glutamine synthesis from glutamate and ammonia in an ATP-dependent reaction. The genes encoding glutamine synthetase play a crucial role in ammonia and glutamate detoxification, acid-base homeostasis, cell signaling, and cell proliferation. The gene family responsible for producing glutamine synthetase has been previously documented in model plants like Arabidopsis. Nevertheless, there has been no exploration into its existence and attributes in oilseed crops like sunflower (Helianthus annuus L.). This study thoroughly analyzes, gene structure, conserved motifs, chromosomal location, phylogenetic relationships, and expression patterns to identify the glutamine synthetase genes in H. annuus. Our findings unveiled 19 genes encoding glutamine synthetase within the H. annuus genome. These genes were distributed across 11 chromosomes of H. annuus. Furthermore, we examined the expression patterns of all the HaGS genes using RNA-seq datasets, specifically focusing on their response to biotic and abiotic stress conditions. Under biotic stress, H. annuus expresses genes for mycorrhizal fungi named Rhizoglomus irregulare at four days post inflorescence (dpi) and 16 dpi. Under abiotic stress, the effect of drought and hormones was investigated. In drought, one gene, HaGS6D, showed the highest expression in the leaf. Meanwhile, in roots, gene HaGS7B showed the highest expression. Under hormonal stress, the effects of auxin, brassinosteroid, and cytokinin were studied on the leaf. For auxin, the gene HaGS7C showed the highest expression. For brassinosteroid, the gene HaGS7D showed the highest expression; for cytokinin, the gene HaGS6E showed the highest expression. Thus, these findings can significantly contribute to our understanding of the arrangement of glutamine synthetase genes in H. annuus and offer valuable insights for developing of drought-resistant cultivars of this species.

Physiological and Molecular Responses of Camellia oleifera Seedlings to Varied Nitrogen Sources

Camellia oleifera Abel. is a unique woody edible oil tree species in China mainly distributed in red soil areas. Nitrogen is one of the important growth-limiting factors for C. oleifera, and the nitrogen form has a vital impact on the growth and development of the plants. Ammonium and nitrate are the two nitrogen forms that are maximally absorbed and utilized by the plants. Here, we used one-year-old seedlings of the main varieties of C. oleifera (‘Xianglin No. 1’, ‘Xianglin No. 27’, and ‘Xianglin No. 210’) to set up six experimental groups treated with different nitrogen forms ([NO3−:NH4+] 0:0, 10:0, 7:3, 5:5, 3:7, and 0:10) and investigated the effects of nitrogen on the vegetative growth and photosynthetic characteristics of the seedlings. This study showed that mixed nitrogen sources could significantly increase the seedling height, ground diameter, biomass accumulation, and photosynthesis efficiency. Transcriptome sequencing analysis led to the identification of 3561 differentially expressed genes in the leaves. Preliminary screening identified several key enzyme genes from nitrogen nutrient metabolic pathways that were differentially expressed among seedlings grown with different nitrogen forms, and their expression pattern changes were further characterized. All the results demonstrate that the same proportion of ammonium nitrate ratio promoted the expression of genes encoding glutamine synthetase and glutamate synthase, thereby improving nitrogen assimilation and utilization efficiency. This study could provide a theoretical basis for scientific and rational fertilization and the improvement of nitrogen utilization efficiency in C. oleifera seedlings.

Mepiquat chloride increases Cry1Ac protein content by regulating the carbon and amino acid metabolism of Bt cotton under high temperature and drought stress

The effects of mepiquat chloride (DPC) on Cry1Ac protein contents in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and high-efficiency cotton cultivation. This study was undertaken on the Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, China. The potted cotton plants were exposed to high temperature and drought stress, and 20 mg L−1 DPC and water (CK) were sprayed on cotton plants. Seven days after treatment, Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed. DESeq was used for differential gene analysis. Under the DPC treatment, in contrast with the water treatment (CK), Cry1Ac protein content increased by 4.7-11.9%. α-Ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased. Transcriptome analysis revealed that there were 7542 upregulated genes and 10449 downregulated genes for DPC vs. CK. GO and KEGG analyses showed that differentially expressed genes were mainly involved in biological processes, such as amino acid metabolism and carbon metabolism. Genes coding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated. DPC increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operating rate of the glycolytic pathway, the citric acid cycle. It also significantly up-regulated the genes encoding glutamate synthase and pyrrolidine-5-carboxylic acid dehydrogenase, and down-regulated the genes encoding glutamine synthetase as well as up-regulated the genes encoding glutamate oxaloacetate transaminase and N-acetylglutamate synthetase. Therefore, the synthesis ability of aspartic acid, glutamic acid, pyruvate, and arginine was increased by DPC, and then Cry1Ac protein contents were increased by regulating carbon and amino acid metabolism.

Magnesium promotes tea plant growth via enhanced glutamine synthetase-mediated nitrogen assimilation.

Acidic tea (Camellia sinensis) plantation soil usually suffers from magnesium (Mg) deficiency, and as such, application of fertilizer containing Mg can substantially increase tea quality by enhancing the accumulation of nitrogen (N)-containing chemicals such as amino acids in young tea shoots. However, the molecular mechanisms underlying the promoting effects of Mg on N assimilation in tea plants remain unclear. Here, both hydroponic and field experiments were conducted to analyze N, Mg, metabolite contents, and gene expression patterns in tea plants. We found that N and amino acids accumulated in tea plant roots under Mg deficiency, while metabolism of N was enhanced by Mg supplementation, especially under a low N fertilizer regime. 15N tracing experiments demonstrated that assimilation of N was induced in tea roots following Mg application. Furthermore, weighted gene correlation network analysis (WGCNA) analysis of RNA-seq data suggested that genes encoding glutamine synthetase isozymes (CsGSs), key enzymes regulating N assimilation, were markedly regulated by Mg treatment. Overexpression of CsGS1.1 in Arabidopsis (Arabidopsis thaliana) resulted in a more tolerant phenotype under Mg deficiency and increased N assimilation. These results validate our suggestion that Mg transcriptionally regulates CsGS1.1 during the enhanced assimilation of N in tea plant. Moreover, results of a field experiment demonstrated that high Mg and low N had positive effects on tea quality. This study deepens our understanding of the molecular mechanisms underlying the interactive effects of Mg and N in tea plants while also providing both genetic and agronomic tools for future improvement of tea production.

Characterization of curly branch of Gracilariopsis lemaneiformis (Rhodophyta) at morphological, physiological, and molecular levels

Gracilariopsis lemaneiformis is an important commercial macroalgae. However, G. lemaneiformis strains with curly branches (CB), located at tertiary order with different phenotype when compared with those with normal branches (NB), were found in the cultivation region. This study investigated CB of G. lemaneiformis. The results revealed that CB exhibited loosely arranged epidermal cells with decreased number of layers; thickened cell wall of medullary cells; reduced plastids with loose lamellas; and lower specific growth rate, pigment contents, chlorophyll fluorescence parameters, and nucleic acid contents when compared with NB. Genes involved in plastid, pyrimidine, and purine biosynthesis as well as nitrogen metabolism were enriched, whereas those encoding small heat shock proteins (sHSPs) in the vicinity of transposable elements (TEs) were evidently down-regulated in CB. The morphological changes in CB might be caused by the alteration at microstructural level. The photosystem II (PSII) in CB presented weak light energy utilization ability and electron transfer activity, compared with that in NB, and the photosynthetic structure of CB may be damaged, which further exerted negative effect on photosynthetic intensity. The reduced plastid structure may be one of the reasons for the decreased photosynthetic intensity of CB. Genes encoding glutamine synthetase involved in nitrogen metabolism were down-regulated in CB, resulting in decrease in nucleic acids synthesis. Under certain stress condition, TEs in the vicinity of sHSPs genes were activated, leading to a decrease in sHSPs, causing loss of algal flexibility to cope with changes in the external environment. These results provided valuable information about irregular branches formation in G. lemaneiformis.

Transcriptome analysis on responses of leaf photosynthesis and nitrogen metabolism of Larix gmelinii to environmental change

To reveal molecular mechanisms underlying photosynthesis responses of Dahurian larch (Larix gmelinii) to environmental changes, we used the high-throughput sequencing technology to sequence the transcriptome of larch leaves from four latitudinal sites with different environmental conditions, and compared differential expression genes (DEGs). The four sites from high- to low-latitude were Tahe (52°52' N), Songling (50°72' N), Heihe (49°22' N), and Dailing (47°08' N). A total of 282428811 clean reads were sequenced out, among which the abundace of DEGs were 16915, 18812, 28536, 20635, 29957 and 23617 for the Tahe-Songling, Tahe-Heihe, Tahe-Dailing, Songling-Heihe, Songling-Dailing, and Heihe-Dailing comparisons, respectively. The expression of nine Psb genes family (i.e., PsbB, PsbK, PsbO, PsbP, PsbQ, PsbS, PsbW, Psb27, and Psb28) encoding Photosystem Ⅱ and that of three genes (ATPF1A,atpA, ATPF1G, atpG, and ATPF1D, atpH) encoding F-type ATPase, which were involved in photosynthesis pathway, were significantly up-regulated with increasing environmental differences among the sites. A similar up-regulation pattern occurred for the expression of genes encoding glutamine synthetase (glnA, GLUL), nitrate reductase (NR), and carbonic anhydrase (cynT, can) that were involved in nitrogen metabolism pathway. The numbers of DEGs and up-regulated genes increased with the increases in environmental changes among the sites, resulting in inter-site divergence of photosynthetic capacity of larch trees.

Glutamine synthetase gene PpGS1.1 negatively regulates the powdery mildew resistance in Kentucky bluegrass.

Excessive nitrogen (N) application may induce powdery mildew (PM) in perennial grasses, but the resistance mechanisms to PM remain unclear. This study evaluated the physiological and molecular mechanisms of PM resistance affected by N supplies in Kentucky bluegrass (Poa pratensis L.). Cultivar 'Bluemoon' (N tolerant) and 'Balin' (N sensitive) were treated with low N (0.5mM), normal N (15mM), and high N (30mM) for 21d in a greenhouse. With increasing N levels, the disease growth was more severe in 'Balin' than in 'Bluemoon'. RNA-seq and weighted gene coexpression network analysis revealed that the PpGS1.1 gene encoding glutamine synthetase was a potential hub gene for PM resistance after comparisons across cultivars and N treatments. The N metabolism pathway was connected with the plant-pathogen interaction pathway via PpGS1.1. The expression of PpGS1.1 in rice protoplasts indicated that the protein was located in the nucleus and cytoplasm. Overexpression of PpGS1.1 in wild-type Kentucky bluegrass increased carbon and N contents, and the transgenic plants became more susceptible to PM with a lower wax density. The most differentially expressed genes (DEGs) for N metabolism were upregulated and DEGs for fatty acid metabolism pathway were downregulated in the overexpression lines. The results elucidated mechanisms of PM resistance in relation to N metabolism in Kentucky bluegrass.

Metagenomic Insights into the Effects of Seasonal Temperature Variation on the Activities of Activated Sludge.

It is well acknowledged that the activities of activated sludge (AS) are influenced by seasonal temperature variation. However, the underlying mechanisms remain largely unknown. Here, the activities of activated sludge under three simulated temperature variation trends were compared in lab-scale. The TN, HN3-H, and COD removal activities of activated sludge were improved as temperature elevated from 20 °C to 35 °C. While, the TN, HN3-H, COD and total phosphorus removal activities of activated sludge were inhibited as temperature declined from 20 °C to 5 °C. Both the extracellular polymer substances (EPS) composition (e.g., total amount, PS, PN and DNA) and sludge index of activated sludge were altered by simulated seasonal temperature variation. The variation of microbial community structures and the functional potentials of activated sludge were further explored by metagenomics. Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes were the dominant phyla for each activated sludge sample under different temperatures. However, the predominant genera of activated sludge were significantly modulated by simulated temperature variation. The functional genes encoding enzymes for nitrogen metabolism in microorganisms were analyzed. The enzyme genes related to ammonification had the highest abundance despite the changing temperature, especially for gene encoding glutamine synthetase. With the temperature raising from 20 °C to 35 °C. The abundance of amoCAB genes encoding ammonia monooxygenase (EC:1.14.99.39) increased by 305.8%. Meanwhile, all the enzyme genes associate with denitrification were reduced. As the temperature declined from 20 °C to 5 °C, the abundance of enzyme genes related to nitrogen metabolism were raised except for carbamate kinase (EC:2.7.2.2), glutamate dehydrogenase (EC:1.4.1.3), glutamine synthetase (EC:6.3.1.2). Metagenomic data indicate that succession of the dominant genera in microbial community structure is, to some extent, beneficial to maintain the functional stability of activated sludge under the temperature variation within a certain temperature range. This study provides novel insights into the effects of seasonal temperature variation on the activities of activated sludge.

Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA. However, the narrow dynamic range of repression of this method needs to be improved. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) technology was developed and was successfully applied in some unicellular cyanobacteria. The technology requires expression of nuclease-deficient CRISPR-associated protein 9 (dCas9) and a single guide RNA (sgRNA) that is complementary to a target sequence, to repress expression of the target gene. In this study, we employed CRISPRi technology for photosynthetic production of ammonium through repression of glnA, the only gene encoding glutamine synthetase that is essential for nitrogen assimilation in A. 7120. By strictly regulating dCas9 expression using the TetR gene induction system, we succeeded in fine-tuning the GlnA protein in addition to the level of glnA transcripts. Expression of sgRNA by the heterocyst-specific nifB promoter led to efficient repression of GlnA in heterocysts, as well as in vegetative cells. Finally, we showed that ammonium is excreted into the medium only when inducers of expression of dCas9 were added. In conclusion, CRISPRi enables temporal control of desired products and will be a useful tool for basic science.

Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena.

Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

Effects of NAD kinase 2 overexpression on primary metabolite profiles in rice leaves under elevated carbon dioxide

The concentration of carbon dioxide (CO2) in the atmosphere is projected to double by the end of the 21st century. In C3 plants, elevated CO2 concentrations promote photosynthesis but inhibit the assimilation of nitrate into organic nitrogen compounds. Several steps of nitrate assimilation depend on the availability of ATP and sources of reducing power, such as nicotinamide adenine dinucleotide phosphate (NADPH). Plastid-localised NAD kinase 2 (NADK2) plays key roles in increasing the ATP/ADP and NADP(H)/NAD(H) ratios. Here we examined the effects of NADK2 overexpression on primary metabolism in rice (Oryza sativa) leaves in response to elevated CO2. By using capillary electrophoresis mass spectrometry, we showed that the primary metabolite profile of NADK2-overexpressing plants clearly differed from that of wild-type plants under ambient and elevated CO2. In NADK2-overexpressing leaves, expression of the genes encoding glutamine synthetase and glutamate synthase was up-regulated, and the levels of Asn, Gln, Arg, and Lys increased in response to elevated CO2. The present study suggests that overexpression of NADK2 promotes the biosynthesis of nitrogen-rich amino acids under elevated CO2.

Indeterminate domain 10regulates ammonium‐mediated gene expression in rice roots

Indeterminate domain (IDD) genes are a family of plant transcriptional regulators that function in the control of development and metabolism during growth. Here, the function of Oryza sativa indeterminate domain 10 (OsIDD10) has been explored in rice plants. Compared with wild-type roots, idd10 mutant roots are hypersensitive to exogenous ammonium. This work aims to define the action of IDD10 on gene expression involved in ammonium uptake and nitrogen (N) metabolism. The ammonium induction of key ammonium uptake and assimilation genes was examined in the roots of idd10 mutants and IDD10 overexpressors. Molecular studies and transcriptome analysis were performed to identify target genes and IDD10 binding cis-elements. IDD10 activates the transcription of AMT1;2 and GDH2 by binding to a cis-element motif present in the promoter region of AMT1;2 and in the fifth intron of GDH2. IDD10 contributes significantly to the induction of several genes involved in N-linked metabolic and cellular responses, including genes encoding glutamine synthetase 2, nitrite reductases and trehalose-6-phosphate synthase. Furthermore, the possibility that IDD10 might influence the N-mediated feedback regulation of target genes was examined. This study demonstrates that IDD10 is involved in regulatory circuits that determine N-mediated gene expression in plant roots.

Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen‐use‐ and yield‐related traits in bread wheat

Glutamine synthetase (GS) plays a key role in the growth, nitrogen (N) use and yield potential of cereal crops. Investigating the haplotype variation of GS genes and its association with agronomic traits may provide useful information for improving wheat N-use efficiency and yield. We isolated the promoter and coding region sequences of the plastic glutamine synthetase isoform (GS2) genes located on chromosomes 2A, 2B and 2D in bread wheat. By analyzing nucleotide sequence variations of the coding region, two, six and two haplotypes were distinguished for TaGS2-A1 (a and b), TaGS2-B1 (a-f) and TaGS2-D1 (a and b), respectively. By analyzing the frequency data of different haplotypes and their association with N use and agronomic traits, four major and favorable TaGS2 haplotypes (A1b, B1a, B1b, D1a) were revealed. These favorable haplotypes may confer better seedling growth, better agronomic performance, and improved N uptake during vegetative growth or grain N concentration. Our data suggest that certain TaGS2 haplotypes may be valuable in breeding wheat varieties with improved agronomic performance and N-use efficiency.

Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.

Cholestasis is a marker for hepatocellular carcinomas displaying β‐catenin mutations

The Wnt/beta-catenin signalling pathway is activated in many human hepatocellular carcinomas (HCC). Identification of beta-catenin mutation relies mostly on sequence analysis and/or immunohistochemistry. beta-catenin mutation may also be detected by analysing the expression of its target genes. The GLUL gene encoding glutamine synthetase (GS), for example, appears to be a pertinent marker. The aim of this study was to correlate GS immunostaining and beta-catenin mutations with clinicopathological features in HCC. We found that GS immunostaining had a sensitivity of 90% for the detection of beta-catenin mutations, with 98% specificity, whereas beta-catenin immunostaining had a sensitivity of 63% with 98% specificity. We used the sensitive GS marker to characterize 190 HCC cases. Sixty-eight (36%) cases displayed Wnt/beta-catenin activation. In addition to their well-differentiated pattern, these tumours exhibited significant features such as a homogeneous microtrabeculo-acinar pattern, low-grade cellular atypia, and cholestasis. As these tumours exhibited cholestasis, we hypothesized that beta-catenin acts on specific bile synthesis and/or transport pathways. In conclusion, we propose that GS immunostaining and a cholestatic pattern are relevant criteria for the identification of HCC with beta-catenin mutations.

Genome organization of glutamine synthetase genes in rainbow trout (Oncorhynchus mykiss)

Unlike mammals, bony fish appear to possess multiple genes encoding glutamine synthetase (GS), the nitrogen metabolism enzyme responsible for the conversion of glutamate and ammonia into glutamine at the expense of ATP. This study reports on the development of genetic markers for each of the four isoforms identified thus far in rainbow trout (Oncorhynchus mykiss) and their genome localization by linkage mapping. We found that genes coding for GS01, GS02, GS03, and GS04 map to four different linkage groups in the trout genome, namely RT-24, RT-23, RT-08, and RT-13, respectively. Linkage groups RT-23 and RT-13 appear to represent distinct chromosomes sharing duplicated marker regions, which lends further support to the previous suggestion that GS02 and GS04 may be duplicate gene copies that evolved from a whole-genome duplication in the trout ancestor. In contrast, there is at present no further evidence that RT-24 and RT-08 share ancestrally homologous segments and additional genomic studies will be needed to clarify the evolutionary origin of genes coding for GS01 and GS03.

Bacterial Type I Glutamine Synthetase of the Rifamycin SV Producing Actinomycete, <italic>Amycolatopsis mediterranei</italic> U32, is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.

Cloning and Expression ofPseudomonas taetrolensY-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.

Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the gamma-glutamyl transferase (gamma-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.