Journal of General Microbiology (1992), 138, 383-393. Printed in Great Britain 383 The gZnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia col" ANNA M. SANANGELANTONI, GIUSEPPE FORLANI,~ FRANCO AMBROSELLI, PIERO CAMMARANO~ and ORSOLA TIBONI'" Dipartimento di Genetica e Microbiologia 'A. Buzzati- Traverso', Universita di Pavia, via Abbiategrasso 207, 27100 Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Universita di Roma 'LA Sapienza', Policlinico Umberto Pavia, Italy P, 1-00161, Roma, Italy (Received 10 September 1991 ; accepted 21 October 1991) ~ ~ ~ ~~~ 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. Sequencingof 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 significanthomology 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), estimatedby gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expectedfor 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. Introduction With the discovery of extremely thermophilic and hyperthermophilic Archaea and Bacteria, the upper limit of temperature compatible with life has been raised above 100°C (Huber et al., 1986; Zillig et al., 1990; Pledger & Baross, 1991). The strategies whereby proteins from these micro-organisms remain stable at tempera- tures equal to or higher than the boiling point of water are still largely undefined. It is also unclear how thermophilic enzymes succeed in combining the struc- tural rigidity required for stabilization with the flexibility required for catalytic function. Because proteins from thermophilic micro-organisms retain their heat-resist- ance upon expression in mesophilic hosts (Love & Streiff, 1987; Love et al., 1988; Tiboni et al., 1989), their * Author for correspondence.Tel. (0382) 391578; fax (0382) 528496. Abbreviations : EF, elongation factor ; G APDH, glyceraldehyde- 3-phosphate dehydrogenase; GS, glutamine synthase ; SVP, snake venom phosphodiesterase. The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number x60160. ability to withstand high temperatures appears to be determined only by features of primary structure. Therefore, sequence comparison of homologous proteins from mesophilic and thermophilic organisms may enable sequence traits critical for thermal stabilization to be identified (Fabry et al., 1989; Schultes et al., 1990). In this work on the extremely thermophilic eubac- terium Therrnotogamaritima, attention has been focused on the key enzyme for ammonia assimilation glutamine synthetase (GS). This protein has been extensively investigated at the biochemical and molecular levels in a wide range of phylogenetically diverse bacterial/archaeal and eukaryotic species (Streicher & Tyler, 1980; Bhatnagar et al., 1986; Cullimore & Bennet, 1988). In both Bacteria and Archaea, GS is an oligomeric enzyme, termed GSI, consisting of twelve identical subunits each with a molecular mass of 50-55 kDa. The only known exception is the anaerobic bacterium Bacteroidesfragilis, whose GS is composed of six subunits each of 83 kDa (Hill et al., 1989). The eukaryotic GS, termed GSII, differs from the bacterial and archaeal enzymes in comprising eight subunits, each of 45-48 kDa, whose primary sequence has little similarity to those of the 0001-7137 O 1992 SGM