The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria

• The replication region of a plasmid from O. oeni is used to generate a cloning vector.
• pRS7Rep is a vector that replicates in Escherichia coli and in lactic acid bacteria.
• pRS7Rep contains single restriction sites: BamHI, XbaI, SalI, HincII, SphI and PstI.
• pRS7Rep is maintained at acceptable rate without selective pressure in L. plantarum.
• Optimisation of the technical conditions of electrotransformation was performed.

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 107), Lactobacillus plantarum (5.7 × 105), Lactobacillus casei (2.3 × 105), Leuconostoc citreum (2.7 × 105), and Enterococcus faecalis (2.4 × 105). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.