Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis
作者： Christopher J. Kristich , Josephine R. Chandler , Gary M. Dunny
DOI: 10.1016/J.PLASMID.2006.08.003
关键词:
摘要: Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract. E. also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite clinical significance enterococci, genetic analysis has been restricted by limitations inherent in tools. To facilitate manipulation faecalis, we developed conjugative delivery system for high-frequency introduction cloned DNA into target strains and host-genotype-independent counterselectable marker use markerless exchange. We used these tools construct collection mutant carrying defined mutations several genes, including ccfA, eep, gelE, sprE, alternative sigma factor (sigH). Furthermore, combined various permutations create double mutants, triple quadruple enabled tests epistasis be conducted on pheromone biosynthesis pathway. Analysis cCF10 production mutants revealed both ccfA2 delta eep10 are epistatic gelE/sprE. our knowledge, this represents first example applied chromosomally encoded biosynthetic pathway enterococci. Thus, advanced reported here enable efficient sophisticated important pathogens.
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