Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp mesenteroides FR52, and its use as a food grade vector

A 1.8-kb cryptic plasmid pFR18 was isolated from Leuconostoc mesenteroides ssp, mesenteroides FR52 and characterized. The identification of single-str anded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replica tion of pFR18 is directed by a rolling-circle mechanism (RCR), Sequence ana lysis revealed a single open reading frame (rep18) encoding a putative 335- amino acid protein homologous to the pT181 replicase. Furthermore, a putati ve double strand origin similar to that of the pT181 plasmid family was ide ntified, A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential reg ion of the plasmid, The resulting construction was able to transform Lactob acillus sake and various species of Leuconostoc. It was stable in L. mesent eroides, however, the segregational stability of a pFR18 derivative contain ing large Escherichia coil DNA fragments was affected. Nevertheless, the ne w RCR plasmid pFR18 may be useful for the construction of food grade vector s. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.