Two OB-fold proteins from a Gram-positive conjugative element engage in relaxosome assembly and DNA processing

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Integrative and conjugative elements (ICEs) are mobile genetic elements crucial for bacterial evolution, often spreading adaptive genes like antibiotic resistance.
• ICEs utilize bacterial conjugation for propagation, a process initiated by relaxase enzymes processing DNA at the origin of transfer.
• The ICESt3/Tn916/ICEBs1 ICE superfamily in Gram-positive bacteria employs unique MOBT relaxases, distinct from those typically found in other bacteria.

Purpose of the Study:

• To elucidate the roles of OrfL and OrfM, two OB-fold proteins encoded by ICESt3, in the conjugative transfer process.
• To characterize the structural and functional interactions of OrfL and OrfM within the ICESt3 relaxosome.
• To investigate the broader implications of OB-fold proteins in relaxosome assembly and function.

Main Methods:

• Bacterial conjugation assays were performed to assess the necessity of OrfL and OrfM for ICESt3 transfer.
• Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the 3D structure of OrfM.
• Bacterial Adenylate Cyclase Two-Hybrid (BACTH) assays were employed to investigate protein-protein interactions.

Main Results:

• OrfL and OrfM form a functional complex and act as essential auxiliary proteins for ICESt3 conjugative transfer.
• The NMR structure of OrfM revealed an OB-fold protein with intrinsically flexible regions.
• OrfL demonstrated interactions with both the ICESt3 relaxase (RelSt3) and the host PcrA helicase, suggesting a networking role.

Conclusions:

• OB-fold proteins, OrfL and OrfM, are critical components of the ICESt3 relaxosome, essential for its conjugative transfer.
• The findings highlight a novel mechanism of relaxosome assembly involving OB-fold proteins, contrasting with the more common ribbon-helix-helix proteins found in other bacterial systems.
• OrfL's interaction network underscores the integration of ICEs with host cellular machinery during transfer.