Summary: The nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined. The B. fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated M
r of 82827. The apparent M
r of the GS subunit determined by SDS-PAGE was approximately 75000. A single mRNA transcription start point was identified upstream of the B. fragilis glnA open reading frame. The B. fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes. The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B. fragilis is a hexamer. Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B. fragilis GS. The GS of B. fragilis is not regulated by adenylylation and lacks the adenylylation site. It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.

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Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis.

Publisher Summary This chapter provides an overview of glutamine synthetase (GS) from Escherichia coli ( E. coli ). GS in E. coli and other gram-negative bacteria is subject to rigorous cellular control. This is because glutamine produced by the enzyme serves as a source of nitrogen atoms in the biosynthesis of all amino acids, purines and pyrimidine nucleotides, of glucosamine-6-phosphate, p -aminobenzoic acid, and of nicotinamide derivatives. GS links the assimilation of NH 3 with biosynthetic pathways leading to the formation of proteins, nucleic acids, complex polysaccharides, and different coenzymes. To accommodate such diverse functions, GS is regulated by varying the enzyme level through protein synthesis, irreversible inactivation, and degradation, by cumulative feedback inhibition on the enzymic activity, and by a closed bicyclic cascade that is capable of sensing changes in the concentration of many metabolites simultaneously and integrating their effects such that the catalytic activity of the enzyme can be adjusted to meet the everchanging demand for glutamine.

[29] Glutamine synthetase from Escherichia coli

We examined the regulation of nitrogen metabolism in four classes (glnA, glnB, glnF, and glnG) of Gln- auxotrophs of Klebsiella pneumoniae. These studies indicate that glutamine synthetase does not directly mediate the physiological response to NH4+ in this organism. We present evidence suggesting that the effect of NH4+ on the expression of genes involved in nitrogen metabolism involves the products of the glnF and glnG genes.

Regulation of nitrogen metabolism in glutamine auxotrophs of Klebsiella pneumoniae.

Identification of a 28 kDa secretory protein from rat olfactory epithelium as a thiol-specific antioxidant.

SUMMARY: 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 M
r of approximately 75000, 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.

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Expression and purification of glutamine synthetase cloned from Bacteroides fragilis.

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.

S L Streicher

Papers

Purification of glutamine synthetase from a variety of bacteria.