Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea.

Abstract

The pNB101 is the first plasmid to be isolated from an haloalkaliphilic archaea. With insertion of the ColE1 replicon of Escherichia coli, as well as two antibiotic resistance genes at its unique Hin dIII site, a novel shuttle vector between haloarchaea and E. coli was developed. This vector, named pNB102, was successfully transformed into two non-alkaliphilic haloarchaea, Halobacterium salinarum SNOB and Haloarcula hispanica ATCC33960. The presence and stability of pNB102 in the transformants were confirmed by PCR identification, Southern blotting and restriction endonuclease digestion. Results also indicated that the presence of restriction-modification (R-M) systems in some Halobacterium species prevented this transformation. It is the first report that the replicon of pNB101 has such a wide host range, and has taken the first step for construction of the vector/host system in haloalkaliphilic archaea.