Detection and characterization of a theta-replicating plasmid pLP60 from PC518 by inverse PCR.

Plasmid DNA of PC518 was isolated by an improved method which contained a washing step for removing lysozyme. Three plasmid DNA libraries were constructed. A pair of outward primers was designed at both ends of the novel plasmid fragment obtained from plasmid DNA libraries, and the remainder of the circle plasmid was amplified by inverse PCR (iPCR). The whole sequence of plasmid was analyzed by the basic local alignment search tool, Tandem Repeats Finder, DNAMAN V6.0, DNASTAR and MEGA X software. The copy number was measured using quantitative real-time PCR. Plasmid extract showed 7 bands on agarose gel, indicating that PC518 contains multiple plasmids. The complete sequence of plasmid pLP60 was obtained by plasmid DNA libraries and iPCR. pLP60 is 6006 bp in length with a G + C content of 41.19 %, which encodes 8 open reading frames (ORFs). The site like theta-type could be located upstream of , which contains a short tandem repeats (sTR) and a long tandem repeats (lTR). RepB of pLP60 only had low similarity with Rep protein of known theta-type plasmids, but phylogenetic tree analysis showed that plasmids whose Rep proteins are similar to pLP60 have lTR at , and the conservativeness of lTR is consistent with similarity of Rep proteins, suggesting that RepB of pLP60 is a theta-replicating protein. So pLP60 was classified as class A of theta replication. The copy number of pLP60 was measured as 5 copies per cell by qPCR.