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

Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, 6431 Fannin, Houston, Texas 77030 USA; Department of Microbiology, University of Minnesota Medical School, 1460 Mayo Bldg., MMC 196, 420 Delaware St. SE, Minneapolis, MN 55445; Department of Biology and Biochemistry, University of Houston, Houston, TX 77204

^* To whom correspondence should be addressed. Email: Peter.J.Christie{at}uth.tmc.edu.

	Abstract

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of Gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCN103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCN103 bound pCF10 but not pcfG or oriT mutant plasmids as shown by Transfer DNA Immunoprecipitation (TrIP), indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCN103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.