Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

Christopher J. Kristich, Josephine R. Chandler, Gary M Dunny

Research output: Contribution to journalArticlepeer-review

110 Scopus citations

Abstract

Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

Original languageEnglish (US)
Pages (from-to)131-144
Number of pages14
JournalPlasmid
Volume57
Issue number2
DOIs
StatePublished - Mar 2007

Bibliographical note

Funding Information:
This work was supported by Grants AI58134 and GM49530 from the NIH to G.M.D. C.J.K. was supported by NRSA fellowship F32-AI56684 from the NIH. J.R.C. is a predoctoral trainee supported by Grant T32DE07288 from the NIH. We thank Tim Leonard for photography and technical advice, and Barry Wanner for bringing the work of Kast and colleagues on E. coli pheS to our attention.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

  • Counterselection
  • Enterococcus faecalis
  • Epistasis
  • Markerless exchange
  • Pheromone biosynthesis
  • p-chloro-phenylalanine
  • pheS

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