| Title: | Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis |
| Author(s): | Kristich CJ; Chandler JR; Dunny GM; |
| Address: | "Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. krist018@umn.edu" |
| DOI: | 10.1016/j.plasmid.2006.08.003 |
| ISSN/ISBN: | 0147-619X (Print) 0147-619X (Linking) |
| 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 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" |
| Keywords: | "*Conjugation, Genetic *DNA, Bacterial Enterococcus faecalis/*genetics Epistasis, Genetic *Genes, Bacterial Genetic Markers Mutation Pheromones/biosynthesis/*genetics Plasmids;" |
| Notes: | "MedlineKristich, Christopher J Chandler, Josephine R Dunny, Gary M eng F32-AI56684/AI/NIAID NIH HHS/ T32 DE007288/DE/NIDCR NIH HHS/ R56 AI058134/AI/NIAID NIH HHS/ AI58134/AI/NIAID NIH HHS/ GM49530/GM/NIGMS NIH HHS/ R01 GM049530/GM/NIGMS NIH HHS/ R01 AI058134/AI/NIAID NIH HHS/ T32DE07288/DE/NIDCR NIH HHS/ F32 AI056684/AI/NIAID NIH HHS/ Research Support, N.I.H., Extramural 2006/09/26 Plasmid. 2007 Mar; 57(2):131-44. doi: 10.1016/j.plasmid.2006.08.003. Epub 2006 Sep 22" |
