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Appl Environ Microbiol

Title:		Development of a method for markerless genetic exchange in Enterococcus faecalis and its use in construction of a srtA mutant
Author(s):		Kristich CJ; Manias DA; Dunny GM;
Address:		"420 Delaware St. S.E., MMC196, Minneapolis, MN 55455, USA"
Journal Title:		Appl Environ Microbiol
Year:		2005
Volume:		71
Issue:		10
Page Number:		5837 - 5849
DOI:		10.1128/AEM.71.10.5837-5849.2005
ISSN/ISBN:		0099-2240 (Print) 1098-5336 (Electronic) 0099-2240 (Linking)
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"
Keywords:		"Aminoacyltransferases/*genetics Bacterial Proteins/*genetics Conjugation, Genetic Cysteine Endopeptidases Drug Resistance, Bacterial Enterococcus faecalis/enzymology/genetics/growth & development Fluorouracil/pharmacology Gene Deletion Gene Expression Reg;"
Notes:		"MedlineKristich, Christopher J Manias, Dawn A Dunny, Gary M eng F32-AI56684/AI/NIAID NIH HHS/ T32 HD007381/HD/NICHD NIH HHS/ HL51987/HL/NHLBI NIH HHS/ R01 HL051987/HL/NHLBI NIH HHS/ HD07381-12/HD/NICHD NIH HHS/ F32 AI056684/AI/NIAID NIH HHS/ Evaluation Study Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S. 2005/10/06 Appl Environ Microbiol. 2005 Oct; 71(10):5837-49. doi: 10.1128/AEM.71.10.5837-5849.2005"