A new theta-type thermosensitive replicon from Lactococcus lactis as an integration vector for Enterococcus faecalis.
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 degrees 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 degrees C to 42 degrees 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.
Frère
J
J
Laboratoire de Microbiologie de l'Environnement, IRBA, Université de Caen, France. frere@ibba.unicaen.fr
Benachour
A
A
Giard
J C
JC
Laplace
J M
JM
Flahaut
S
S
Auffray
Y
Y
D000595:Amino Acid Sequence; D013293:Enterococcus faecalis / Q000235:genetics*; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D012093:Replicon