Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

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.     

Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.     

Bacterial type I glutamine synthetase of the rifamycin SV producing actinomycete, Amycolatopsis mediterranei 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 of a DNA sequence that complements glutamine auxotrophy in Saccharomyces cerevisiae

Glutamine (gln) requiring mutants of Saccharomyces cerevisiae have been isolated. They synthesize small amounts of glutamine synthetase (GS), which is more thermolabile than the enzyme from the parental strain. The gln auxotrophy was complemented in transformation experiments using an S. cerevisiae gene library constructed in the plasmid vector YEp13. The transformants were mitotically unstable and synthesized almost tenfold higher amounts of GS than wild-type cells. This activity was as thermoresistant as that from the wild-type strain. A recombinant plasmid was isolated from one of the transformants and partially mapped. Upon reintroduction into the auxotrophic strain, the transformation frequency to gln prototrophy was the same as that for the marker LEU2 gene. The evidence presented suggests that we have cloned the structural gene for GS from S. cerevisiae.     

Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in Klebsiella aerogenes.

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.     

Regulation of nitrogenase activity in Anabaena variabilis by modification of the Fe protein

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.     

Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity.

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.