Structural Gene For Glutamine Synthetase Research Articles

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.

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation.

The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression. A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region. Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots. P(II) is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.

Diel variability in transcription of the structural gene for glutamine synthetase (glnA) in natural populations of the marine diazotrophic cyanobacterium Trichodesmium thiebautii

Abstract A 360-bp fragment of the Trichodesmium thiebautii glutamine synthetase gene (glnA) was amplified from DNA isolated from oceanic populations of this ecologically important, diazotrophic cyanobacterium. The cloned fragment showed high homology to the type 1 glutamine synthetase (GS) enzymes of other cyanobacteria and was used to probe the in situ, temporal variability in transcription of Trichodesmium GS. Three distinct phases in the diel pattern of glnA mRNA abundance were observed. These appear to be correlated with the temporal variability in carbon and nitrogen fixation rates and the effect that these physiological processes have on the size of cellular pools of the primary end-products of N assimilation. Our data show that Trichodesmium thiebautii GS is regulated at the level of transcription and support the hypothesis that GS expression in this organism is subject to similar controls as those established for heterocyst-forming cyanobacteria.

Glutamine synthesis in Streptomyces — a review

The synthesis of glutamine synthetase (GS), a key enzyme in ammonium (NH 4 + assimilation, is regulated by nitrogen availability in several Streptomyces strains. In addition, the enzymatic activity of the GS enzyme is post-translationally regulated by adenylylation. Nitrogen regulation of GS synthesis is mediated at the transcriptional level in S. coelicolor, and transcription of the GS structural gene ( glnA) requires a positive regulatory protein, GlnR. The amino acid sequence of the GlnR protein is similar to that of the Escherichia coli positive regulatory proteins, OmpR and PhoB, which belong to the family of bacterial two-component regulatory systems. DNA encoding a GSII-like enzyme has been cloned from S. viridochromogenes and S. hygroscopicus, but the role of this GS isoenzyme in NH 4 + assimilation in Streptomyces is unclear.

Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2)

Six Streptomyces coelicolor mutants that required glutamine for growth at the wild-type rate on all nitrogen sources (Gln-) were isolated. The phenotypes of all six mutants were similar. The glutamine synthetase (GS) levels were 20- to 100-fold lower in extracts of the Gln- mutants than in extracts of their parents. The reduced levels of GS activity in the Gln- mutants were not due to adenylylation of the GS protein, because GS activity in Gln- extracts did not increase after snake venom phosphodiesterase treatment. No transcripts of the GS structural gene (glnA) could be detected in RNA isolated from the Gln- mutants in primer extension experiments. All six gln mutations mapped adjacent to adeA. S. coelicolor chromosomal DNA complementing the Gln- mutants was isolated from a library of S. coelicolor chromosomal DNA constructed in the low-copy-number S. coelicolor plasmid pIJ922. Subcloning experiments showed that a 1.45-kb DNA fragment could complement all six Gln- mutants. This DNA fragment did not hybridize with either the cloned S. coelicolor glnA gene or the cloned S. viridochromogenes GSII gene in Southern blots. Since glnA transcription was restored in the Gln- mutants containing the complementing DNA, the gln mutations appear to lie in one or more closely linked genes that are required for glnA transcription in S. coelicolor.

Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense

The complete nucleotide sequence of the open reading frame (ORF) located upstream of the glnA structural gene for glutamine synthetase (GS) in Azospirillum brasilense Sp7 was determined. This ORF, which codes for a 12 kDa protein, was identified as glnB, the structural gene for the PII protein, a component of the adenylylation cascade involved in the regulation of GS activity in some gram-negative bacteria. Transcription analysis and mRNA mapping of glnB and glnA of A. brasilense was performed with bacteria grown under different physiological conditions. The glnA gene can be transcribed either as a glnB-A mRNA of 2.4 kb or as a glnA mRNA of 1.5 kb. Differential expression of the two mRNAs was found to depend on the nitrogen source. The glnB-A mRNA was the major transcript under nitrogen fixation conditions, while the synthesis of the glnA mRNA was almost completely abolished. The glnA mRNA was predominantly produced in NH4(+)-containing medium. Transcription start site analysis revealed the presence of three different types of nitrogen-regulated promoters. GlnB-A mRNA was transcribed selectively from tandem promoters. One of them is similar to the NtrA-dependent promoter and the other to the Escherichia coli sigma 70 promoter. The synthesis of glnA mRNA was regulated by a promoter, which was repressed (or non-activated) only under conditions of nitrogen fixation, when moleuclar nitrogen was the sole nitrogen source. The transcriptional initiation site in front of glnA is not preceded by a canonical E. coli sigma 70 promoter. A sequence reminiscent of the NtrA-dependent promoter consensus, except for a fundamental mismatch, was found at positions -33 to -21. This sequence overlapped a putative "weak" NtrC-binding site, similar to those identified in enteric bacteria. From these results, it is postulated that glnA mRNA is controlled by a novel type of nitrogen-regulated promoter.

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

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, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath)

The structural gene (glnA) encoding the ammonia-assimulation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli glnA mutants was demonstrated. In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent Mr 60,000, as determined by PAGE. Expression of the M. capsulatus (Bath) glnA gene in E. coli was regulated by nitrogen levels in an Ntr+ but not an Ntr- background. The nucleotide sequence of the M. capsulatus (Bath) glnA gene and flanking sequences was determined. This gene, of 1407 bp, encoded a polypeptide of Mr 51717 containing 468 amino acids. The 5' leader region contained three putative promoters. Promoters P1 and P3 resembled the canonical -10 -35 E. coli-type promoter. Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms. A potential NtrC-binding site was also determined, flanking the Pribnow box at P1. Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M. capsulatus are made.

Regulation of transcription of the glnALG operon of Escherichia coli by protein phosphorylation

The transcription of glnA, the structural gene for glutamine synthetase in enteric bacteria, is regulated by the phosphorylation and dephosphorylation of an effector protein, NR I. In its phosphorylated form the effector activates the intiation of transcription at promoters specific of σ 54, rather than the abundant σ 70. The ability of NR I-phosphate to stimulate the formation of open promoter-σ 54 RNA polymerase complexes is enhanced by specific binding sites, located in the case of glnA 100 and 130 base pairs upstream from the transcriptional start site. These sites can be moved more than 1000 base pairs upstream or downstream without losing their effectiveness. The phosphorylation and dephosphorylation of NR I-NR I-phosphate is catalyzed by the modulator protein NR II. Its activity is controlled by an intracellular signal, the ratio of glutamine to 2-ketoglutarate, which is generated by glutamine synthetase in response to the environmental stimulus, the availability of lack of ammonia. The signal is transduced to the modulator by means of 2 additional proteins: uridylytransferase and P II.

Regulation of glutamine synthetase in Streptomyces coelicolor

Glutamine synthetase (GS) in Streptomyces coelicolor was shown to be regulated at two levels. First, the S. coelicolor GS protein is subject to a posttranslational covalent modification which is likely to involve adenylylation. Adenylylation is important in regulating GS activity both after sudden changes in ammonium availability and during steady-state growth. Since higher levels of adenylylated GS were seen in S. coelicolor mutants deficient in glutamate synthase than in wild-type cells, glutamine or a metabolite derived from glutamine is likely to be involved in the metabolic signal that regulates GS adenylylation. Second, the GS structural gene (glnA) is transcriptionally regulated in response to nitrogen availability during steady-state growth. Transcription of the glnA gene occurred from the same promoter during vegetative growth, stationary phase, and sporulation. The nucleotide sequence of this promoter has significant homology with the -10, but not the -35, region of the consensus sequence of Streptomyces vegetative promoters. The glnA gene is transcribed as a monocistronic mRNA.

Nucleotide sequence and expression of the glutamine synthetase structural gene, glnA, of the archaebacterium Methanococcus voltae

The sequence of a 2,746-bp DNA fragment of Methanococcus voltae carrying the glnA gene for glutamine synthetase (GS), was established. A 1,338-bp open reading frame (ORF), encoding a 446-amino-acid polypeptide of 50,142 Da, was defined as glnA on the basis of its similarity to other glnA genes and on the ability of a DNA fragment carrying this ORF to complement an Escherichia coli Gln − mutant. No sequence homology was found between sequences flanking the M. volae glnA gene and other eubacterial glnA genes. In M. voltae, the gene was transcribed as a monocistronic unit and GS synthesis was partially repressed at high ammonia concentrations. At the amino acid sequence level, the highest similarity was found with GS of Bacillus subtilis and Clostridium acetobutylicum.

Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli.

It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI. We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII. In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI. NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction.

Identification of the Klebsiella pneumoniae glnB gene: Nucleotide sequence of wild-type and mutant alleles

The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein PII, has been cloned and sequenced. The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene. The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln+ suppressor of this mutation were also determined. The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of PII, and the suppression was shown to be caused by Tn5 insertion in glnB. The 3' end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB. Potential homologues of glnB encoding polypeptides extremely similar in sequence to PII were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.

Sequence of the Bacillus subtilis glutamine synthetase gene region

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.

Azospirillum : genetics of nitrogen fixation and interaction with plants

Bacteria of the genus Azospirillum are free-living diazotrophs that were isolated from the rhizosphere and from the roots of grasses. Genetic analysis of nitrogen fixation was essentially initiated in A. brasilense Sp7, where genetic tools and mutants are available. A DNA region covering 25 kb and containing the nitrogenase structural genes ( nif HDK), nif E and another nif cluster has been cloned. In addition, the structural gene for glutamine synthetase, which might be involved in nif regulation, was cloned and sequenced. To identify bacterial genes involved in the root colonization process,DNA-DNA hybridization was performed with Rhizobium nodulation ( nod and hsn ) genes. Homology was detected in both cases and clones containing DNA homologous to hsn genes were isolated. Azospirillum contains large plasmids. Preliminary experiments suggest that the hsn homologous region is located on the 90 MDa plasmid of strain Sp7.

Purification and Regulation of a Cloned Thiobacillus ferrooxidans Glutamine Synthetase

Summary: Glutamine synthetase (GS) (EC 6.3.1.2) was purified from an Escherichia coli glnA deletion strain containing the Thiobacillus ferrooxidans GS structural gene. The apparent Mr of the cloned T. ferrooxidans GS subunit was approximately 60000. This indicates a particle Mr for the undissociated enzyme of 720000, assuming the enzyme is the typical dodecamer. Electron microscopy of purified GS revealed characteristic disc shaped molecules with central holes. The cloned T. ferrooxidans GS was regulatedby Mg2+ or Mn2+,adenylylation, and nitrogen source but was not affected by feedback modifiers. The GS has a γ-glutamyltransferase isoactivity point of pH 7.71.

Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH 4 + ) by Rhodopseudomonas capsulata B100 supported the presence of an NH 4 + transport system. Experimentally NH 4 + was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent Km's of 1.7 μM and 11.1 μM (pH 6.8, 30°) and a Vmax at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q10 quotient was calculated to be 1.9 at 100 μM NH 4 + , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH 4 + , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH 4 + at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH 4 + as the nitrogen source, remained derepressed for nitrogenase in the presence of NH 4 + , and displayed rates of NH 4 + uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH 4 + transport is tightly coupled to assimilation.

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.