Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations |
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| Authors: | Fang Teng Barbara E. Murray George M. Weinstock |
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| Affiliation: | aDepartment of Microbiology and Molecular Genetics, Division of Infectious Diseases, University of Texas Medical School, Houston, Texas, 77030;bDivision of Infectious Diseases, Department of Medicine, University of Texas Medical School, Houston, Texas, 77030;dDivision of Infectious Diseases, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas, 77030;cDivision of Infectious Diseases, Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston, Texas, 77030 |
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| Abstract: | Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence 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 ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. 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 fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. 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 apyrCknockout mutant showing an auxotrophic phenotype. |
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