Faculty Collaboration Database

Development of a method for markerless genetic exchange in Enterococcus faecalis and its use in construction of a srtA mutant. Appl Environ Microbiol 2005 Oct;71(10):5837-49

Date

10/06/2005

Pubmed ID

16204495

Pubmed Central ID

PMC1265997

DOI

10.1128/AEM.71.10.5837-5849.2005

Scopus ID

2-s2.0-26844580763 (requires institutional sign-in at Scopus site)   54 Citations

Abstract

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, genetic analysis of these organisms has thus far been limited in scope due to the lack of advanced genetic tools. To broaden the repertoire of genetic tools available for manipulation of E.faecalis, we investigated the use of phosphoribosyl transferases as elements of a counterselection strategy. We report here the development of a counterselectable markerless genetic exchange system based on the upp-encoded uracil phosphoribosyl transferase of E. faecalis. Whereas wild-type E. faecalis is sensitive to growth inhibition by the toxic base analog 5-fluorouracil (5-FU), a mutant bearing an in-frame deletion of upp is resistant to 5-FU. When a cloned version of upp was ectopically introduced into the deletion mutant, sensitivity to 5-FU growth inhibition was restored, thereby providing the basis for a two-step integration and excision strategy for the transfer of mutant alleles to the enterococcal chromosome by recombination. This method was validated by the construction of a DeltasrtA mutant of E. faecalis and by the exchange of the surface protein Asc10, encoded on the pheromone-responsive conjugative plasmid pCF10, with a previously isolated mutant allele. Analysis of the DeltasrtA mutant indicated that SrtA anchors Asc10 to the enterococcal cell wall, facilitating the pheromone-induced aggregation of E. faecalis cells required for high-frequency conjugative plasmid transfer in liquid matings. The system of markerless exchange reported here will facilitate detailed genetic analysis of these important pathogens.

Author List

Kristich CJ, Manias DA, Dunny GM

Author

Christopher J. Kristich PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Aminoacyltransferases
Bacterial Proteins
Conjugation, Genetic
Cysteine Endopeptidases
Drug Resistance, Bacterial
Enterococcus faecalis
Fluorouracil
Gene Deletion
Gene Expression Regulation, Bacterial
Genetic Markers
Genetic Techniques
Membrane Proteins
Mutation
Pentosyltransferases
Plasmids
Recombination, Genetic