Characterization of a novel rolling-circle replication plasmid pYSI8 from Lactobacillus sakei YSI8

A plasmid from Lactobacillus sakei YSI8, designated as pYSI8, was sequenced and characterized. It consisted of a 4973 bp circular molecule with a G + C content of 35.6%. The plasmid pYSI8 was predicted to contain five putative ORFs, in which ORF1 shared 79% and 76% identity with Rep proteins of pLH2 and pLC2, members of rolling-circle replication (RCR) pMV158 family. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and mung bean nuclease treatment confirmed that pYSI8 replicated via the RCR mechanism. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA (dsDNA). Furthermore, the copy number of pYSI8 was estimated to be 41.9 ± 0.5 in each cell by real-time polymerase chain reaction.