Barriers to genetic manipulation of Enterococci: Current Approaches and Future Directions

Abstract Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens. The molecular biology of Enterococcus faecalis and Enterococcus faecium has lagged behind other Gram-positive pathogens due to physical and enzymatic barriers restricting DNA transfer, with this review summarising the barriers and highlighting how improving transformation will accelerate our understanding of these important pathogens.