Cianobacterias

PLASMID

ARTICLE NO.

39, 182–186 (1998) PL981336

Conjugal Transfer of Plasmid DNA from Escherichia coli to Enterococci: A Method to Make Insertion Mutations
Fang Teng,* Barbara E. Murray,*,†,‡ and George M. Weinstock*,‡,§,1
*Department of Microbiology and Molecular Genetics, Division of Infectious Diseases, †Department of Medicine, §Department of Biochemistry and Molecular Biology, and‡Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston, Texas 77030 Received July 24, 1997; revised January 6, 1998 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 originof 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 fromE. 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 recombinedinto the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. 1998 Academic Press This resulted in a pyrC knockout mutant showing an auxotrophic phenotype.

Enterococci are one of the major causes of hospital infections. In the past decade, their development of resistance to many antibiotics has made their infections even harder to treat. It is important toidentify virulence factors of enterococci and making knockout mutants is essential for this research. Electroporation has been widely applied to introduce DNA into enterococci and to knock out genes (Solioz, 1990; Cruz-Rodz, 1990; Friesenegger, 1991; Waser, 1992; Shepard, 1995). While some strains are transformable by electroporation, others are not or the efficiency is very low. Alternative waysto make knockout mutants of enterococci are thus of interest. In previous studies, shuttle vectors carrying an origin of conjugal transfer (oriT) have been mobilized from Escherichia coli to gram-posiTo whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Texas Medical School, 6431 Fannin Street, Houston, Texas 77030. Fax: (713) 500-0652.E-mail: georgew@utmmg.med.uth.tmc.edu. 0147-619X/98 $25.00
Copyright 1998 by Academic Press All rights of reproduction in any form reserved.
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tive hosts, including the Enterococcus faecalis strain BM4110 (Trieu-Cuot, 1991). The mobilization of these vectors was due to transfer functions provided by an IncP plasmid in the donor E. coli cells. In this report, we demonstrate that this conjugationsystem can be used to make knockout and other insertion mutants of enterococci. Vectors were constructed for this conjugation system and applied to make a gene disruption mutation in the autolysin gene of E. faecalis OG1RF. It was also shown that a large cosmid could be transferred into OG1RF by conjugation to make a knockout mutation in the pyrC gene.
MATERIALS AND METHODS

Bacterial Strains andMedia The bacterial strains and plasmids used in this study are listed in Table 1. Brain heart infusion (BHI) broth and agar (Difco Laboratories) were used for bacterial growth. For filter conjugation, glycine (1–2.5%) was added in

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INSERTION MUTANTS TABLE 1 Strains and Plasmids Strain or plasmid Escherichia coli strains S17-1 Enterococcus strains OG1RF SE34 Plasmids pAT18 pTEX5235...