Mapping Type IV Secretion Signals on the Primase Encoded by the Broad-Host-Range Plasmid R1162 (RSF1010)

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Type IV secretion is crucial for plasmid conjugal transfer, mobilizing protein relaxases into recipient cells.
• The IncQ plasmid R1162 (RSF1010) utilizes a type IV secretion pathway for DNA transfer.
• MobA is a multifunctional protein of R1162, containing both relaxase and primase domains, with the primase domain also existing as RepB'.

Purpose of the Study:

• To identify and characterize two novel secretion signals within the RepB' primase domain of plasmid R1162.
• To elucidate the role of the accessory protein MobB in mediating the interaction between these signals and the R751 secretion machinery.
• To clarify the location and properties of secretion signals involved in plasmid DNA conjugal transfer.

Main Methods:

• Structural mapping of type IV secretion signals onto the RepB' protein structure.
• Site-directed mutagenesis using arginine-to-alanine substitutions to assess signal function.
• Two-hybrid assays to investigate protein-protein interactions between MobB, TraG, and MobA.
• Conjugal transfer assays to evaluate the functional significance of identified signals and MobB.

Main Results:

• Two distinct type IV secretion signals were identified within RepB': one internal and one C-terminal, both rich in arginine residues.
• Both identified signals are functional in the type IV secretion system of plasmid R751, with the internal signal also supporting conjugal transfer when linked to the relaxase domain.
• The accessory protein MobB is essential for R751-mediated secretion, acting as an adaptor that bridges the RepB' signals to the coupling protein TraG.
• MobB interacts with the membrane-associated domain of TraG and a region of MobA adjacent to the RepB' domain.

Conclusions:

• The RepB' primase domain of plasmid R1162 contains functional signals for type IV secretion and conjugal transfer.
• MobB is a critical adaptor protein for the R751 secretion system, facilitating the recognition of plasmid-encoded signals.
• Understanding these secretion signals and adaptors provides insights into the molecular mechanisms of plasmid DNA mobilization.