Development of versatile shuttle vectors for Deinococcus grandis

Abstract

To develop new shuttle vectors for Deinococcus species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2467-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 repABC-type plasmids of a-proteobacteria . On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of D. radiodurans SARK and Thermus species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in E. coli with a chloramphenicol resistance determinant, were shown to autonomously replicate in D. grandis ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in D. grandis. On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in D. grandis. An expression plasmid that possesses the D. radiodurans minimal groE promoter was constructed, and a firefly luciferase gene was successfully expressed in D. grandis. The D. grandis host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms.