Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. Plasmid 2007 Mar;57(2):131-44
Date
09/26/2006Pubmed ID
16996131Pubmed Central ID
PMC1852458DOI
10.1016/j.plasmid.2006.08.003Scopus ID
2-s2.0-33846993832 (requires institutional sign-in at Scopus site) 144 CitationsAbstract
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 delta eep10 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.
Author List
Kristich CJ, Chandler JR, Dunny GMAuthor
Christopher J. Kristich PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Conjugation, GeneticDNA, Bacterial
Enterococcus faecalis
Epistasis, Genetic
Genes, Bacterial
Genetic Markers
Mutation
Pheromones
Plasmids
