Open AccessA new theta‐type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis
Author(s) -
Frère Jacques,
Benachour Abdellah,
Giard JeanChristophe,
Laplace JeanMarie,
Flahaut Sigrid,
Auffray Yanick
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb12935.x
Subject(s) - replicon , plasmid , enterococcus faecalis , mutant , gene , biology , homologous recombination , microbiology and biotechnology , shuttle vector , genetics , recombinant dna , vector (molecular biology) , escherichia coli
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 .