Abstract
The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homo-dimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.
Highlights
Enterococcus faecalis can transfer pheromone-inducible plasmids in a highly efficient manner upon sensing the peptide pheromones produced by recipient cells
The encoded T4SSs of these pheromone regulated mobile genetic elements (MGEs) are tightly regulated at the transcriptional level by sensing of peptide pheromones originating from recipient cells (Dunny, 2013; Dunny and Berntsson, 2016)
The molecular mass of wild type PcfF from the size exclusion chromatography (SEC) peak was determined by gas-phase electrophoretic mobility macromolecule analysis (GEMMA, termed Macroion mobility spectrometer) to be ∼32 kDa, very close an apparent dimer (Figure 1A; Kaufman et al, 1996; Bacher et al, 2001)
Summary
Enterococcus faecalis can transfer pheromone-inducible plasmids in a highly efficient manner upon sensing the peptide pheromones produced by recipient cells. The broad medical importance of this large family of pheromone-inducible plasmids is underscored by the fact that they serve as reservoirs for genes encoding many different virulence factors, adhesins and antibiotic resistance They can mobilize other MGEs to both enterococcal and nonenterococcal recipients (Antiporta and Dunny, 2002; Staddon et al, 2006). Some G− accessory factors have been well studied, including the TraM and TraY proteins of the F plasmid, MbeC from the ColE1 plasmid, TrwA of the R388 plasmid and NikA from the R64 plasmid (Luo et al, 1994; Moncalian and de la Cruz, 2004; Yoshida et al, 2008; Varsaki et al, 2009; Wong et al, 2011) These proteins have all been shown to contain a Ribbon-Helix-Helix (RHH) domain responsible for binding to DNA. They suggest the importance of other intrinsic, e.g., DNA bending, and extrinsic factors for relaxosome assembly in vivo
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