Conjugal transfer of plasmid DNA from Escherichia coli to enterococci: a method to make insertion mutations

Shuttle vector pAT18 was transferred by conjugation from Escherichia coli S17-1 to Enterococcus faecalis OG1RF and Enterococcus faecium SE34. Transfer was mediated by the transfer functions of plasmid RK2 in E. coli S17-1 and the origin of conjugal transfer (oriT) located on pAT18. The oriT sequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene of E. faecalis OG1RF was cloned into pTEX5235 and transferred by conjugation from E. coli S17-1 to E. faecalis OG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster from E. faecalis, with a transposon insertion in pyrC, was also transferred from E. coli S17-1 to E. faecalis OG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in a pyrC knockout mutant showing an auxotrophic phenotype.