Identification of the minimal replicon of the streptomycete plasmid pIJ101.

The minimal replicon of pIJ101 was defined to a 2.0-kb BalI-SacII fragment containing the rep ORF and a non-coding region of DNA, but not orf56 (pQR435). This non-coding region of DNA was found to be crucial for plasmid replication in Streptomyces lividans and may contain either the origin of replication or the rep promoter (or both). A 1.4-kb NotI-SacII fragment containing only the rep ORF produced a 54-kDa protein in vitro when expressed from the lacZ promoter. pQR435 was structurally unstable in S. lividans and accumulated single-stranded (ss)DNA. The insertion of the site for second-strand initiation (ssi) into pQR435 did not prevent the plasmid from rearranging or from accumulating ssDNA.