Cloning and characterization of the replicon of the Nocardia italica plasmid, pN1100

A 19-kb, plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading fram e (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three con sensus sequences and a P-loop-like motif in the Rep protein of plas mid pSG 5, 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 th e Streptomyces lividans protoplast. This result strongly suggests that pNI1 00 is a conjugative plasmid and probably replicates by a rolling-circle rep lication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was c onstructed and found to be a useful cloning shuttle vector. (C) 2000 Academ ic Press.