Characterization of the cryptic plasmid pTXW from Lactobacillus paracasei TXW

A cryptic plasmid from Lactobacillus paracasei TXW isolated from koumiss, designated as pTXW, was sequenced and characterized. It is 3178 bp in length with a G+C content of 42.9%. The plasmid pTXW was predicted to encode 4 putative ORFs. RepB shared high homology with initiator proteins of plasmids from the rolling circle replication (RCR) pMV158 family. copG was predicted to encode a transcriptional repressor, which was involved in plasmid copy number control together with a putative antisense RNA. Mob putatively acts as a relaxase and belongs to the MOBV relaxase family. Sequence analysis revealed a double strand origin (dso) located upstream the repB gene, which contained the conserved nick sequence of the pMV158 family. A putative single strand origin (sso-like) consisting of successive inverted repeats was also detected. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pTXW replicates via the RCR mechanism. Furthermore, the relative copy number of pTXW was estimated to be 73.5 ± 1.2 copies in each cell by real-time PCR. The new RCR plasmid would be valuable to construct vectors for application in food industry.