Development of versatile shuttle vectors for

Sato, Katsuya; Tu, Z.*; Oba, Hirofumi; Narumi, Issei

To develop new shuttle vectors for species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2,467-bp plasmid possesses two open reading frames, one encoding 88 amino acid residues (Orf1) and the other encoding 501 amino acid residues (Orf2). The predicted amino acid sequence encoded by Orf1 exhibits similarity to the N-terminal regions of replication proteins encoded by -type plasmids of -proteobacteria. On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of SARK and species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in with a chloramphenicol resistance determinant, were shown to autonomously replicate in ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in . On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in . An expression plasmid that possesses the minimal promoter was constructed, and a firefly luciferase gene was successfully expressed in . The host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms.