Interactions between the F conjugal transfer system and CloDF13::Tna plasmids

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Conjugative plasmid transfer relies on a complex set of genes, including the F transfer (tra) genes.
• Small multicopy plasmids like ColE1 and cloDF13 can utilize F transfer machinery but may have unique requirements.
• The F traD gene and the FinC inhibition system play roles in regulating conjugative transfer.

Purpose of the Study:

• To investigate the specific F transfer genes essential for the conjugation of ColE1 and cloDF13 plasmids.
• To elucidate the role of the F traD gene and the FinC system in the transfer of these small plasmids.
• To characterize mutations affecting FinC-mediated inhibition of plasmid transfer.

Main Methods:

• Conjugation experiments to assess plasmid transfer frequencies.
• Plasmid copy number analysis.
• Phage plaque assays to evaluate F-specific phage propagation.
• Hybridization experiments with lambda tra phage DNA to study gene transcription.
• Isolation and characterization of FinC-insensitive mutants.

Main Results:

• All F transfer genes for stable mating pairs (traN, traG) were essential for ColE1 and cloDF13 transfer, unlike F conjugal DNA metabolism genes (traM, traI, traZ).
• The F traD gene was required for ColE1 transfer but not cloDF13; R100-1 efficiently transferred cloDF13 but not ColE1.
• A cloDF13 copy number mutant inhibited F transfer and f2 phage plaque formation, suggesting FinC targets traD function rather than synthesis.
• A FinC-insensitive mutant with an up-promoter mutation increased traJ and traD expression, counteracting FinC inhibition.

Conclusions:

• Plasmid transfer by ColE1 and cloDF13 utilizes a subset of F transfer genes, with distinct requirements for traD.
• The FinC inhibition system targets the function of the traD protein, not its synthesis.
• Mutations enhancing traD expression can overcome FinC-mediated inhibition, providing insights into transfer regulation.