The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.
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.
Sanangelantoni
A M
AM
Dipartimento di Genetica e Microbiologia A. Buzzati-Traverso, Università di Pavia, Italy.
Forlani
G
G
Ambroselli
F
F
Cammarano
P
P
Tiboni
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D015183:Restriction Mapping; D016415:Sequence Alignment; D012910:Snake Venoms / Q000201:enzymology; D013696:Temperature
D004269:DNA, Bacterial; D012910:Snake Venoms; D010727:Phosphoric Diester Hydrolases; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium