A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated.

Fisher, Richard A.; Tuli, Rakesh; Haselkorn, Robert

doi:10.1073/pnas.78.6.3393

Cited by 89 publications

(57 citation statements)

References 25 publications

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“…Although the GSs from different micro-organisms exhibit structural similarities, their activities are regulated by different mechanisms, such as divalent-cationinduced conformational changes, feedback inhibition by various end-products, including glutamine, and covalent modification of the enzyme subunits by an adenylylation-deadenylylation system (Tyler, 1978). In general, the GSs from enteric bacteria and Streptomyces (Reitzer & Magasanik, 1987;Streicher 8z Tyler, 1981 ;Fisher & Wray, 1989) are subject to adenylylation unlike other Gram-positive bacteria (Deuel & Prusiner, 1974;Janssen et al, 1988), cyanobacteria (Fisher et al, 1981), and the archaeote Methanobacterium ivanovi (Bhatnagar et al, 1986). The insensitivity of the T. maritima GS transferase activity to SVP treatment indicates that it is not regulated by an adenylylation-deadenylylation mechanism, a conclusion also supported by the low degree of sequence conservation (4 out of 18 amino acids) around the tyrosine residue which functions as the adenylylation site in E. coli.…”

Section: Discussionmentioning

confidence: 99%

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

Sanangelantoni

Forlani

Ambroselli

et al. 1992

Journal of General Microbiology

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The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb Hind111 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 50088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolbus 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 (590000 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.

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Section: Discussionmentioning

confidence: 99%

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

Sanangelantoni

Forlani

Ambroselli

et al. 1992

Journal of General Microbiology

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“…To test the specificity of the DNA-binding activity of CalA, we used 4 other DNA fragments from the upstream regions of glnA, hetC, hetR and ntcA (denoted as U glnA , U hetC , U hetR and U ntcA , respectively) to compete with the fragment B2. glnA encodes glutamine synthetase (Fisher et al 1981), hetC encodes an ABC-cassette peptide transporter that affects cell division state transition during heterocyst differentiation (Khudyakov and Wolk 1997;Xu and Wolk 2001), hetR encodes the master regulator in heterocyst differentiation that shows both serine-type protease and transcriptional regulator activities (Zhou et al 1998;Huang et al 2004;Shi et al 2006), ntcA encodes a homo-dimeric transcriptional regulator that globally controls nitrogen metabolism ). These four DNA fragments have been shown to interact with the regulator NtcA or HetR Huang et al 2004;Laurent et al 2005;Muro-Pastor et al 1999).…”

Section: Specificity Of the Dna-binding Activity Of Calamentioning

confidence: 99%

CalA, a cyAbrB protein, binds to the upstream region of ftsZ and is down-regulated in heterocysts in Anabaena sp. PCC 7120

2010

Arch Microbiol

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A protein of cyAbrB family of regulators, Alr0946 (CalA), was identified by DNA affinity chromatography using DNA fragments from the promoter region of ftsZ in Anabaena/Nostoc sp. PCC 7120. Electrophoretic mobility shift assays showed that the protein could bind to upstream regions of ftsZ (as biotin-labeled DNA fragments) and that the binding was inhibited by the same DNA fragments and a fragment upstream of hetC but not by 3 fragments upstream of glnA, hetR or ntcA. Both transcriptional fusion with gfp (green fluorescence protein) and Western blot analysis indicated that the expression of calA was down-regulated in heterocysts relative to vegetative cells. An insertional mutant of calA could not be completely segregated. Over-expression of calA in Anabaena/ Nostoc caused no change in growth, heterocyst differentiation or expression of ftsZ. CalA as a DNA-binding protein is essential for the growth of Anabaena/Nostoc and may be required for the expression of ftsZ in vegetative cells.

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“…Three cyanobacterial glnA genes have been reported to be expressed in E. coli and to complement a glnA mutant (4,12,21). However, only the gene from Anabaena sp.…”

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Characterization of cis elements that regulate the expression of glnA in Synechococcus sp. strain PCC 7942

1995

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The upstream noncoding region of the Synechococcus sp. strain PCC 7942 (hereafter referred to as Synechococcus 7942) glnA gene was fused to the cat gene in order to study the expression of glnA both in Synechococcus 7942 and in Escherichia coli. The lack of cat expression in E. coli indicated that the glnA promoter was not recognized by E. coli RNA polymerase. The fused construct was integrated into the Synechococcus 7942 chromosome at a neutral site. Expression of the cat reporter gene was regulated under various nitrogen conditions in a way similar to that of the glnA gene. A deletion introduced at the binding site of the NtcA regulatory protein abolished derepression of the glnA promoter during growth in nitrate and under nitrogen starvation. Deletion of the sequence between the transcription and translation start sites of glnA prevented the repression observed during growth in ammonium. These results indicate that the glnA promoter is subject to complex regulation that involves sequences upstream and downstream from the transcription start site.

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A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated.

Abstract: The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3

Cited by 89 publications

References 25 publications

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

CalA, a cyAbrB protein, binds to the upstream region of ftsZ and is down-regulated in heterocysts in Anabaena sp. PCC 7120

Characterization of cis elements that regulate the expression of glnA in Synechococcus sp. strain PCC 7942

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