title: Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.
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 (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, 6431 Fannin, Houston, TX 77030, USA.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhang
authors.forename: Dawn
authors.initials: D
authors.lastname: Manias
authors.forename: Hye-Jeong
authors.initials: HJ
authors.lastname: Yeo
authors.forename: Gary M
authors.initials: GM
authors.lastname: Dunny
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Christie
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001692:Biological Transport; D002352:Carrier Proteins; D004269:DNA, Bacterial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010675:Pheromones / Q000502:physiology; D010957:Plasmids / Q000235:genetics*; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D010675:Pheromones; D037521:Virulence Factors; D000251:Adenosine Triphosphatases