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

Abstract

The Enterococcus faecalis prg and pcf genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis in vitro and blocked pCF10 transfer in vivo. As shown with affinity pulldown assays, PrgJ and the K471E mutant protein interacted with the substrate receptor PcfC and with relaxase PcfG and accessory factor PcfF, which together form the relaxosome at the oriT sequence to initiate plasmid processing. The purified PrgJ and K471E proteins also bound single-and double-stranded DNA substrates without sequence specificity in vitro, and both PrgJ derivatives bound pCF10 in vivo by a mechanism dependent on an intact oriT sequence and cosynthesis of PcfC, PcfF, and PcfG, as shown by a formaldehyde-cross-linking assay. Our findings support a model in which the PcfC receptor coordinates with the PrgJ ATPase to drive early steps of pCF10 processing/transfer: (i) PcfC first binds the pCF10 relaxosome through contacts with PcfF, PcfG, and DNA; (ii) PcfC delivers the plasmid substrate to PrgJ; and (iii) PrgJ catalyzes substrate transfer to the membrane translocase. Substrate engagement with a VirB4-like subunit has not been previously described; consequently, our studies point to a novel function for these signature T4SS ATPases in mediating early steps of type IV secretion.

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

“…Finally, once PcfC binds the pCF10 substrate, the receptor delivers its cargo to the VirB4-like PrgJ ATPase, possibly for further processing prior to translocation (50). Soluble PcfC⌬N103 binds PrgJ in vitro (50), prompting us to test whether AAD PcfC or NBD/ CTD PcfC participates in PrgJ binding.…”

“…Soluble PcfC⌬N103 binds PrgJ in vitro (50), prompting us to test whether AAD PcfC or NBD/ CTD PcfC participates in PrgJ binding. As shown in Fig.…”

“…7D, PrgJHis 6 coeluted with MBP-NBD/CTD PcfC but not MBP alone. In E. coli, PrgJ accumulates as both a full-length protein and a presumptive degradation product (50), and the NBD/CTD PcfC variant bound both forms of this ATPase. In contrast, in the corresponding GST pulldown assays, PrgJ-His 6 did not form a detectable complex with GST-AAD PcfC (Fig.…”

“…Alternatively, the GST tag was cleaved from the AADs by treatment with PreScission enzyme (2 units) at 4°C for 6 h. The protein-Sepharose mixture was centrifuged for 5 min at 0.5 ϫ g, and the supernatant was mixed with 50 l of glutathione-Sepharose beads for 2 h at 4°C to remove residual GST and PreScission enzyme. The purified AAD PcfC or AAD VirD4 fragments were recovered by centrifugation for 5 min at 500 ϫ g. His 6 -tagged forms of PcfF, PcfG, VirD1, VirD2, VirD2⌬C35, and PrgJ were highly enriched by Co 2ϩ affinity chromatography as previously described (30,50). Briefly, 500-ml cultures of BL21(DE3) strains carrying the relevant plasmids were grown at 37°C to an OD 600 of 0.4, induced with IPTG (0.1 mM final concentration), and incubated at room temperature for 3 h. Cells were harvested by centrifugation at 6,000 ϫ g for 10 min, resuspended in 5 ml buffer A, and incubated for 10 min at 37°C.…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…In addition, VirB11 may function with VirB4 to coordinate a structural change in VirB2 pilin protein and mediate VirB2 binding to other T4SS components, which in turn affects pilus polymerization (Kerr and Christie, 2010). Accumulating genetic studies and recent structural studies support a model in which VirB4 dimers or homomultimers contribute both structural information for the assembly of a trans-envelope channel competent for DNA transfer and an ATP-dependent activity for configuring this channel as a dedicated export machine (Fronzes et al, 2009a;Durand et al, 2010Durand et al, , 2011Kerr and Christie, 2010;Li et al, 2012;Pena et al, 2012;Wallden et al, 2012). In addition, the TrIP assay results showed that translocating DNA substrates first interact with the VirD4 receptor, followed by VirB11 ATPase, suggesting VirB11 may function together with VirB4 and deliver the T-DNA substrate to the transfer channel of the T4SS (Cascales and Christie, 2004a;Bhatty et al, 2013;Chandran, 2013;Christie et al, 2014).…”