Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium

A hyperthermophilic and amylolytic prokaryote, designated Rt3, was isolated from a thermal spring near Rotorua, New Zealand. The 16S rRNA gene of Rt3 was cloned and sequenced with the aim of determining its phylogenetic affiliations. The phylogenetic analysis of this sequence, which included a selection of archaebacterial and eubacterial 16S rRNA sequences, indicates that Rt3 most likely belongs to the archaebacterial order Thermococcales. An amylase gene (amyA) from Rt3, encoding a highly thermostable amylase activity, was cloned and its DNA sequence determined. Transcriptional signals typical of archaebacteria were evident in this sequence. The sequence is homologous to a broad range of enzymes from the AMY superfamily and contains a typical N-terminal signal peptide. Phylogenetic analysis and comparison of structural features with other AMY superfamily enzymes reveals that, firstly, the closest homologues of the Rt3 amylase are members of the Bacillus and Plant alpha-amylase groups; and secondly, that the Rt3 amylase is closely related to only one other currently known archaebacterial enzyme, i.e. an (AMY superfamily) alpha-amylase from Natronococcus.