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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.
Journal of Bacteriology
|May 29, 2012
Summary
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.
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