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Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
Published on: January 4, 2017
Enterococcus faecalis PrgJ, a VirB4-like ATPase, mediates pCF10 conjugative transfer through substrate binding
Feng Li1, Cristina Alvarez-Martinez, Yuqing Chen
1Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas, USA.
Insights
The PrgJ ATPase, part of the type IV secretion system in Enterococcus faecalis, binds and hydrolyzes ATP to facilitate plasmid transfer. This study reveals a novel role for VirB4-like ATPases in initiating secretion processes.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Enterococcus faecalis plasmid pCF10 utilizes a type IV secretion system (T4SS) for conjugative transfer.
- The prg and pcf genes encode this T4SS, with PrgJ identified as a VirB4 family ATPase crucial for function.
Purpose of the Study:
- To investigate the biochemical activities and in vivo function of the PrgJ ATPase in the context of pCF10 plasmid transfer.
- To elucidate the role of PrgJ in the early steps of type IV secretion and plasmid processing.
Main Methods:
- Purification and characterization of PrgJ protein, including ATP binding and hydrolysis assays.
- Site-directed mutagenesis of the putative NTP binding site (K471E) in PrgJ.
- Affinity pulldown assays to assess protein-protein interactions with pCF10 conjugation factors (PcfC, PcfG, PcfF).
- In vitro DNA binding assays using single- and double-stranded DNA substrates.
- In vivo formaldehyde-cross-linking assays to study PrgJ-DNA interactions and dependence on other factors.
Main Results:
- Purified PrgJ dimers exhibit ATP binding and hydrolysis activities.
- A K471E mutation in PrgJ diminished ATP binding and abolished hydrolysis in vitro, while blocking pCF10 transfer in vivo.
- PrgJ and its K471E mutant interacted with PcfC, PcfG, and PcfF, components of the pCF10 relaxosome.
- Both PrgJ forms bound DNA non-specifically in vitro and bound pCF10 in vivo dependent on oriT and relaxosome components.
- A model is proposed where PcfC coordinates with PrgJ to drive early pCF10 processing and transfer steps.
Conclusions:
- PrgJ, a VirB4-like ATPase, plays a critical role in the initiation of pCF10 conjugative transfer.
- PrgJ's ATPase activity is essential for pCF10 transfer, and it interacts with key components of the relaxosome.
- This study identifies a novel function for VirB4-like ATPases in mediating substrate engagement and transfer initiation in type IV secretion systems.
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.
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