Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

Abstract

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 poorlytransformed bacteria.

Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding

“…The tra region of pCF10 carries three functionally distinct subsets of genes encoding (i) the Dtr (DNA transfer and replication) functions required for processing of the plasmid for transfer, (ii) the Mpf (mating-pair formation) proteins composing the mating or translocation channel, and (iii) 3 cell-wall-anchored proteins implicated in formation of the donor-recipient cell junction (2,8,9,12,21,37,43). The two Dtr proteins, the relaxase PcfG and the accessory factor PcfF, assemble at the plasmid's origin of transfer (oriT) sequence as the relaxosome to cleave the DNA strand destined for transfer (here termed the T strand) (8,43,44).…”

“…The two Dtr proteins, the relaxase PcfG and the accessory factor PcfF, assemble at the plasmid's origin of transfer (oriT) sequence as the relaxosome to cleave the DNA strand destined for transfer (here termed the T strand) (8,43,44). PcfF initiates processing by binding the double-stranded (ds) oriT sequence, and oriT-bound PcfF then recruits the PcfG relaxase for strand-specific cleavage at the nic site.…”

Enterococcus faecalisPcfC, a Spatially Localized Substrate Receptor for Type IV Secretion of the pCF10 Transfer Intermediate

“…This 67.6-kb plasmid is a member of the family of sex pheromone plasmids found to date only in the enterococci but with the capacity to mobilize transfer of oriT-containing plasmids to Lactococcus lactis and Streptococcus agalactiae (64). pCF10 carries the tetracycline resistance conjugative transposon Tn925 (22) and codes for pheromone sensing and response functions, conjugation functions, and a surface adhesin termed aggregation substance (AS) which is important both for conjugation and virulence (38,68,69).…”

“…One or more processing factors termed the DNA transfer and replication (Dtr) proteins initiate transfer by assembling as a relaxosome at the origin of transfer sequence (oriT) to catalyze cleavage of the DNA strand (T strand) destined for transfer (33). For pCF10, the PcfG relaxase and the accessory factor PcfF function together to cleave DNA at the nic site within an IncP-type oriT sequence (18,64). Next, by a mechanism that is poorly understood, the relaxosome or the processed transfer intermediate interacts with a substrate receptor, also termed the type IV coupling protein (T4CP) (3).…”

“…The DNA processing reactions of several gram-positive conjugative plasmids have been characterized in detail (14,15,18,46,63,64). By contrast, structure-function studies of the cognate mating pair formation and substrate transfer mechanisms are still in their infancy, although an interaction network for putative Mpf subunits of the S. agalactiae pIP501-encoded T4S system recently was described (1).…”