Cloning and Characterization of the Replicon of the Nocardia italica Plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.