Recombinant glutamine synthetase originating from high ammonium-assimilating Bacillus subtilis effectively reduces NH3 emissions and enhances the conversion into amino acids in liquid manure.

Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium

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.

谷氨酰胺合成酶(GS)是细胞中唯一可以从头合成谷氨酰胺的酶,在癌症代谢中扮演着重要角色。GS酶活缺陷突变往往导致严重的代谢疾病甚至是死亡。理解GS酶活降低对生理功能的影响可能为靶向治疗提供新的方向,同时对其内在机制的探索可以为治疗干预开辟新的途径。已报道的GS酶活缺陷突变包括R324C(第324位精氨酸突变为半胱氨酸)和R341C(第341位精氨酸突变为半胱氨酸)等,本研究新发现了GS的另一个酶活缺陷突变位点K241(第241位赖氨酸)。为了探究GS酶活缺陷突变带来的影响,本研究利用双组学技术对GS的酶活缺陷突变R324C和K241R(第241位赖氨酸突变为精氨酸)在肺癌细胞中的功能进行研究,通过对转录组和代谢组数据的整合揭示了GS酶活缺陷突变对一些重要生物过程的显著影响。GS的缺陷突变阻碍了细胞周期和多种氨基酸代谢途径。除了谷氨酰胺合成受到抑制,精氨酸-脯氨酸代谢、甘氨酸-丝氨酸-酪氨酸代谢以及天冬氨酸-谷氨酸-丙氨酸代谢在GS突变的细胞中表现出更强的活力,同时,氨酰-tRNA的生物合成途径也显著被激活。进一步的研究发现,GS酶活缺陷带来氨基酸代谢的改变源自谷氨酸的重新定向和相关代谢酶的表达变化,同时,氨酰-tRNA生物合成的激活源自谷氨酰胺在GS酶活缺陷细胞中带来的能量应激激活了特定蛋白的表达,如转录因子4(ATF4)。此外,细胞表型实验表明,GS酶活缺陷细胞的迁移能力低于野生型细胞。以上结果揭示了GS酶活缺陷突变体细胞中的代谢重编程现象,突出了癌细胞代谢的复杂性和适应性。

The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.

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.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

Ammonia emissions following liquid manure application impair human health and threaten natural ecosystems. In growing arable crops, where immediate soil incorporation of the applied liquid manure is not possible, best-available application techniques are required in order to decrease ammonia losses. We determined ammonia emission, crop yield and nitrogen uptake of winter wheat in eight experimental sites across Germany. Each individual experiment consisted of an unfertilized control (N0), broadcast calcium ammonium nitrate (CAN) application as well as four different techniques to apply cattle slurry (CS) and biogas digestate (BD). Fertilizer was applied to growing winter wheat at a total rate of 170 kg N ha−1 split into two equal dressings. The following application techniques were tested for both liquid manure types: (i) trailing hose (TH) application using untreated and (ii) acidified (~pH 6) liquid manure (+A), as well as (iii) a combination of open slot injection (SI) for the first dressing and trailing shoe (TS) application for the second dressing without and (iv) with the addition of a nitrification inhibitor (NI) for the first dressing. The highest ammonia emissions (on average 30 kg N ha−1) occurred following TH application of BD. TH application of CS led to significantly lower emissions (on average 19 kg N ha−1). Overall, acidification reduced ammonia emissions by 64% compared to TH application without acidification for both types of liquid manures. On average, the combination of SI and TS application resulted in 23% lower NH3 emissions in comparison to TH application (25% for the first application by SI and 20% for the second application by TS). Supplementing an NI did not affect ammonia emissions. However, decreasing ammonia emissions by acidification or SI did not increase winter wheat yield and nitrogen uptake. All organically fertilized treatments led to similar crop yield (approx. 7 t ha−1 grain dry matter yield) and above-ground biomass nitrogen uptake (approx. 150 kg ha−1). Yield (8 t ha−1) and nitrogen uptake (approx. 190 kg ha−1) were significantly higher for the CAN treatment; while for the control, yield (approx. 4.5 t ha−1) and above-ground biomass nitrogen uptake (approx. 90 kg ha−1) were significantly lower. Overall, our results show that reducing NH3 emissions following liquid manure application to growing crops is possible by using different mitigation techniques. For our field trial series, acidification was the technique with the greatest NH3 mitigation potential.

The glnA gene of Rhodobacter sphaeroides encoding glutamine synthetase (GS) has been cloned and sequenced. Molecular analysis revealed that there is a glnB gene upstream of glnA, in a single glnBA operon. A putative glnAp1-type promoter sequence, a consensus ntrC gene product binding site and a consensus upstream activator sequence were detected upstream of the glnB gene. The deduced amino acid sequences of the GS and GlnB proteins of R. sphaeroides showed strong homology with the same proteins from other Gram-negative bacteria. The sequence of the glnA gene isolated from glutamine auxotroph Gln83 was also determined. The glnA83 mutation was shown to result in premature termination of GS synthesis and formation of a 17 kDa C-truncated GS which could be complemented by a 5'-truncated glnA gene which encodes a 30 kDa N-truncated GS. This phenomenon is characteristic for interallelic complementation.

Mutations in a site, glnF, linked by P1-mediated transduction of argG on the chromosome of Klebsiella aerogenes, result in a requirement for glutamine. Mutants in this gene have in all media a level of glutamine synthetase (GS) corresponding to the level found in the wild-type strain grown in the medium producing the strongest repression of GS. The adenylylation and deadenylylation of GS in glnF mutants is normal. The glutamine requirement of glnF mutants could be suppressed by mutations in the structural gene for GS, glnA. These mutations result in altered regulation of GS synthesis, regardless of the presence or absence of the glnF mutation (GlnR phenotype). In GlnR mutants the GS level is higher than in the wild-type strain when the cells are cultured in strongly repressing medium, but lower than in the wild-type strain when cells are cultured in a derepressing medium. Heterozygous merodiploids carrying a normal glnA gene as well as a glnA gene responsible for the GlnR phenotype behave in every respect like merodiploids carrying two normal glnA genes. These results confirm autogenous regulation of GS synthesis and indicate that GS is both a repressor and an activator of GS synthesis. The mutation in glnA responsible for the GLnR phenotype has apparently resulted in the formation of a GS that is incompetent both as repressor and as activator of GS synthesis. According to this hypothesis, the product of the glnF gene is necessary for activation of the glnA gene by GS.

Glutamine synthetase plays an important role in ammonium tolerance of Myriophyllum aquaticum

Batch cultures of the phototrophic bacterium, Thiocapsa roseopersicina, were grown anaero­ bically in the light either on sulfide with various ammonia concentrations, N 2 or amino acids as nitrogen sources, or on several simple organic substrates in the absence of reduced sulfur com­ pounds using 6 mM NH4Cl as source of nitrogen. At high ammonia concentrations high activities of (NADPH-linked) glutamate dehydrogenase (GDH), but rather low transferase and no bio­ synthetic activity of glutamine synthetase (GS) were obtained, while under conditions of ammonia deficiency (growth with N 2 or glutamate) GDH activity was very low and both GS activities were strongly increased. Glutamate synthase (GOGAT) activity (NADH-dependent) showed little variation. These data indicate that at high NH+ concentrations ammonia is assimilated via GDH, under NHJ limitation, however, via the GS/GOGAT system. Glutamine as nitrogen source may be utilized via GOGAT as well as via an active glutaminase plus GDH. Ammonia, but not glutamine, seems to cause repression and inactivation of GS. Alanine and asparagine inactivate the enzyme inhibiting the biosynthetic, but not the transferase activity. These amino acids in part also influence the activities of GDH, GOGAT, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH). Cultures grown on acetate or pyruvate instead of sulfide showed increased GDH activities and high GS transferase activities possibly reflecting an increase of intracellular a-ketoglutarate concentration. On malate or fructose also increased GS transferase activities, but rather low GDH activities were observed. High biosynthetic GS activities and elevated GOGAT activities were found only in fructose-grown cells. On the organic substrates the ICDH activities always were somewhat higher than after lithoautotrophic growth. With the exception of acetate, the MDH activities were considerably elevated, especially on pyruvate. The different pathways of ar-keto-glutarate formation and their influence on the enzymes of ammonia assimilation are discussed.

Abstract: Pollution of air, soil and water caused by excessive ammonia (NH3) emission and deposition from animal manure is as an environmental concern. Gas-permeable membranes (GPM) may provide a solution for controlling NH3 emission to the environment by extracting it from liquid manure and potentially using the recovered NH3 as fertilizer. For this purpose, four lab-scale experiments were conducted to investigate the capture and recovery of NH3 from liquid manure by circulating an acid solution through a tubular GPM submerged into the liquid dairy manure. During these experiments, the depth of liquid manure in chambers of different dimensions and the tubular membrane parameters including diameter, length and pore size were held constant in order to study the effect of acid-filled membrane on NH3 extraction from different surface areas (1X, 2X, 4X, and 8X) of liquid manure. Results show that nearly 50% of the liquid manure NH3 measured prior to the start of each experiment from all but 8X chamber was captured in less than 20 days by acid-filled membranes. Also, NH3 extraction by the GPM system from liquid manure and NH3 gain in acidic solution were linearly correlated. The study showed that the experiment with the 4X chamber resulted in optimum NH3 extraction using the GPM system.

Expression of the cloned glnA gene [coding for glutamine synthetase (EC 6.3.1.2)] of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions. Mutations in the E. coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation. To test whether sequences within the B. subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B. subtilis glnA promoter with E. coli lacZ was constructed. beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B. subtilis glnA gene. Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase. Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression. DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation. These results suggest that the glnA gene of B. subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al. [Dean, D. R., Hoch, J. A. & Aronson, A. I. (1977) J. Bacteriol. 131, 981-987].

본 연구는 논에서 돈분 액비 활용시 담수 시기에 따른 암모니아 휘산량을 평가하였다. 담수 상태에서 돈분 액비 시용은 142.0 mg/<TEX>$m^2$</TEX> 의 암모니아 휘산량을 보인 반면, 돈분액비시용 1일 및 3일 후 담수시 암모니아 휘산량은 29.6, 25.4 mg/<TEX>$m^2$</TEX> 였다. 그리고 담수 상태에서 돈분액비 시용이 돈분액비 시용 후 담수에 비해 높은 암모니아 휘산량을 보인 것은 암모니아가 토양교질에 흡착되지 않고 수중에 높은 농도로 존재하기 때문인 것으로 나타났다. 따라서 논에서 돈비 액비 활용시 암모니아 휘산량 저감에 의한 질소 이용도 향상을 위해서는 돈분액비 살포 최소 1일 후 담수하는 것이 좋을 것으로 판단된다. BACKGROUND: Ammonia emissions from field-applied livestock manure are considered a threat to the environment worldwide. In Korea, a large amount of liquid manure was applied in the rice field before rice transplanting in order to reduce chemical fertilizer use. This study was conducted to provide the optimal flooding time after liquid manure application in an attempt to minimize ammonia emission. METHODS AND RESULTS: Ammonia emission from paddy field applied with liquid pig manure following different flooding time was measured using the dynamic chamber method. The five treatments used were : application of liquid pig manure to paddy field in flooding condition (F0T); one day (F1T) and three days (F3T) after flooding; without flooding (NF), and flooding without the application of liquid pig manure (control). Among the treatment, the highest ammonia emission was observed in F0T. The cumulative ammonia emission of F1T and F3T for 12 days were very similar and were about 4.7 times less than that of the F0T treatment. CONCLUSIONS: Ammonia emission in paddy field could be significantly reduced by liquid pig manure application after flooding rather than application of liquid pig manure in flooding condition. Therefore, flooding after liquid pig manure application would provide much more nitrogen for rice growth due to the reduction of ammonia emission.

A mouse 3T6 subline that grows in glutamine-free medium has been cloned and exposed to a regimen of increasing concentrations of the glutamine synthetase inhibitor, methionine sulfoxime. Cells selected for resistance to 700 microM methionine sulfoxime show a 75-fold increase in glutamine synthetase activity relative to the original subclone. Immune precipitation of extracts prepared from cells pulse-labeled with L-[35S] methionine indicates that the increase in enzyme activity reflects an increase in biosynthesis of glutamine synthetase. Results obtained from in vitro translation followed by immune precipitation suggests that the methionine sulfoxime-resistant cells are highly enriched in mRNA encoding glutamine synthetase. The increase in enzyme activity is lost upon culture of the cells in nonselective medium--a finding consistent with the observation of double minute chromosomes in only the drug-resistant cells. These data strongly support the notion that methionine sulfoxime treatment has resulted in selection of cells that have amplified the gene encoding glutamine synthetase.