Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: determination of the minimal replicon and functionality identification of the putative sso.

Abstract In order to determine the minimal replicon and the single strand origin ( sso ) of the plasmid pM4, different fragments of pM4 were amplified by polymerase chain reaction (PCR) and cloned into pBEm, a replication probe vector for Lactobacillus . The deletion analysis results showed that the minimal replicon of pM4 could be determined within a 1280 bp fragment consisting of double strand origin ( dso ) and rep gene encoding replication protein. Based on plasmid segregation stability assay and its ability to convert single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) by Southern hybridization, an sso of replication was located at nucleotides −118–92 in the plasmid pM4, about 300 bp upstream of dso . In addition, the host range assay indicated that plasmid pM4 could replicate in L. casei 05–21, L. rhamnosus AS 1.2466 T and L. plantarum 05–19 of all the tested Lactobacillus strains. Analysis of the pM4 replicon will allow its use in constructing a food-grade vector for application in food industry.