Conjugation is a major contributor to lateral gene transfer in bacteria, and pheromone-inducible conjugation systems in Enterococcus faecalis play an important role in the dissemination of antibiotic resistance and virulence in enterococci and related bacteria. We have genetically dissected the determinants of DNA processing of the enterococcal conjugative plasmid pCF10. Insertional inactivation of a predicted relaxase gene pcfG, via insertion of a splicing-deficient group II intron, severely reduced pCF10 transfer. Restoration of intron splicing ability by genetic complementation restored conjugation. The pCF10 origin of transfer (oriT) was localized to a 40-nucleotide sequence within a non-coding region with sequence similarity to origins of transfer of several other plasmids in gram positive bacteria. Deletion of the oriT reduced pCF10 transfer by more than five orders of magnitude without affecting pCF10-dependent mobilization of co-resident oriT-containing plasmids. Although the host range for pCF10 replication is limited to enterococci, we found that the pCF10 conjugation system promotes mobilization of oriT-containing plasmids to multiple bacterial genera. Therefore, this transfer system may have applications for gene delivery to a variety of poorly-transformed bacteria.
Bibliographical noteFunding Information:
The authors thank Larry McKay of the University of Minnesota for the generous gift of L. lactis LM2301RF, Craig Rubens of the University of Washington for the generous gift of S. agalactiae COH3r, and Chris Kristich from our laboratory for supplying strains and integrative plasmids prior to publication. This work was supported by PHS Grant 1RO1-GM49530 from the NIH to G.M.D. J.H.S. was supported by NIH training Grants T32AI07421, T32GM08347, and MSTP grant T32GM08244. E.M.B. was supported by NIH training Grant T32AI07421.
- Origin of transfer