Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids

Area of Science:

• Molecular Biology
• Plasmid Biology
• Genetics

Background:

• Plasmids are extrachromosomal DNA elements crucial for bacterial adaptation and evolution.
• IncFI group plasmids are a significant class of bacterial plasmids with diverse applications.
• Understanding plasmid replication mechanisms is key to controlling their behavior in host cells.

Purpose of the Study:

• To isolate and characterize a novel autonomous replicon, RepFIB, from the P307 plasmid.
• To elucidate the functional role of DNA repeats within the RepFIB replicon.
• To investigate the determinants of plasmid copy number and incompatibility.

Main Methods:

• Isolation and sequencing of the RepFIB replicon.
• Maxicell analysis for protein identification.
• Construction and replication proficiency testing of RepFIB deletion mutants in a polA1 strain.
• Incompatibility assays.

Main Results:

• RepFIB shares homology with IncFI group replicons and contains eleven 21-bp repeats flanking an open reading frame encoding a ~40 kDa protein.
• A minimal replication region of 1.6 kb was defined, encompassing repeats flanking the open reading frame.
• Deletion of downstream repeats increased plasmid copy number, indicating their role in copy number control.
• All three sets of repeats mediated incompatibility with a resident RepFIB plasmid.

Conclusions:

• The RepFIB replicon is a functional unit within IncFI plasmids, characterized by specific repeat sequences.
• DNA repeats within RepFIB play a critical role in regulating plasmid copy number.
• These repeats are also responsible for expressing plasmid incompatibility, a key factor in plasmid maintenance.