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-stranded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replication of pFR18 is directed by a rolling-circle mechanism (RCR). Sequence analysis revealed a single open reading frame (rep18) encoding a putative 335-amino acid protein homologous to the pT181 replicase. Furthermore, a putative double strand origin similar to that of the pT181 plasmid family was identified. A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential region of the plasmid. The resulting construction was able to transform Lactobacillus sake and various species of Leuconostoc. It was stable in L. mesenteroides, however, the segregational stability of a pFR18 derivative containing large Escherichia coli DNA fragments was affected. Nevertheless, the new RCR plasmid pFR18 may be useful for the construction of food grade vectors.
Biet
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Institut de Biologie Moléculaire et d'Ingénierie Génétique, CNRS-ESA 6031, Université de Poitiers, 40 avenue du recteur Pineau, 86022, Poitiers, Cedex, France. franck.biet@pasteur-lille.fr
Cenatiempo
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Fremaux
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000032:analysis; D019649:Food Industry; D007934:Leuconostoc / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids
D004277:DNA, Single-Stranded