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A new theta-type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis

Frère, Jacques ; Benachour, Abdellah ; Giard, Jean-Christophe ; Laplace, Jean-Marie ; Flahaut, Sigrid ; Auffray, Yanick

In: FEMS Microbiology Letters, 1998, vol. 161, no. 1, p. 107-114

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    Titel
    • A new theta-type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis
    Autor
    • Frère, Jacques. Laboratoire de Microbiologie de l'Environnement, IRBA, Université de Caen, F-14032 Caen cedex, France
    • Benachour, Abdellah. Institut de Biochimie, rue du Musée, 5, Université de Fribourg, CH-1700 Fribourg, Suisse
    • Giard, Jean-Christophe. Department of Microbiology and Immunology, University of South Alabama, MSB 2096, Mobile, AL, USA
    • Laplace, Jean-Marie. Laboratoire de Microbiologie de l'Environnement, IRBA, Université de Caen, F-14032 Caen cedex, France
    • Flahaut, Sigrid. Laboratoire de Biologie Cellulaire et Moléculaire, Université du Littoral - Côte d'Opale, BP 299, F-62228 Calais cedex, France
    • Auffray, Yanick. Laboratoire de Microbiologie de l'Environnement, IRBA, Université de Caen, F-14032 Caen cedex, France
    Dokumententyp
    • Postprint
    OAI-PMH-ID
    • oai:doc.rero.ch:295262
    Summary
    We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42°C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30°C to 42°C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis