PCR-mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon Sulfolobus shibatae: identification of putative amino-acid signatures for extremophilic adaptation

Abstract

Highly degenerate oligodeoxyribonucleotides (oligos) were used to PCR amplify the most conserved region of the glutamate dehydrogenase (GDH)-encoding gene from the extreme thermophilic archaeon, Sulfolobus shibatae. The amplified fragment was cloned and sequenced, and then used as a homologous probe to clone a genomic restriction fragment containing the near-complete gdhA gene. The deduced amino acid (aa) sequence shows a very high degree of similarity with the aa sequence previously determined by direct sequencing of the purified enzyme from Sulfolobus solfataricus [Maras et al., Eur. J. Biochem. 203 (1992) 81-87]. The introduction of this new sequence into our GDH phylogenetic trees [Benachenhou-Lahfa et al., J. Mol. Evol. 35 (1993) 335-346] showed that it is a new member of hexameric GDH family II, and did not modify the topology of the trees. Comparison of the primary structures of extremophilic GDH enzymes (halophilic, thermophilic and hyperthermophilic) with those of their non-halophilic and mesophilic counterparts in this family II led us to identify a few aa changes which seem to be specific either to hyperthermophilic or halophilic adaptation.