Characterization of pR18, a novel rolling-circle replication plasmid from Lactobacillus plantarum

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Lactobacillus plantarum PA18, isolated from Pandanus amaryllifolius, harbors the pR18 plasmid.
• Plasmids are crucial for bacterial adaptation and evolution, often carrying antibiotic resistance genes.

Purpose of the Study:

• To characterize the pR18 plasmid, including its replication mechanism, genetic content, and evolutionary implications.
• To investigate the potential for horizontal gene transfer of antibiotic resistance determinants.

Main Methods:

• Nucleotide sequence analysis of the pR18 plasmid.
• Southern hybridization and mung bean nuclease treatment to confirm replication mechanism.
• Phylogenetic tree analysis of plasmid-encoded proteins.
• Quantitative PCR to determine plasmid copy number.

Main Results:

• The pR18 plasmid is a 3211bp circular molecule containing two open reading frames (ORFs).
• ORF2 encodes a replication protein similar to the Rep proteins of the RCR pC194/pUB110 family.
• ORF1 encodes linA, an enzyme inactivating lincomycin, confirming antibiotic resistance.
• Replication occurs via the rolling circle (RCR) mechanism.
• Phylogenetic analysis suggests horizontal transfer of antibiotic resistance determinants between diverse bacteria.
• The relative copy number of pR18 is approximately 39 copies per chromosome equivalent.

Conclusions:

• The pR18 plasmid utilizes the RCR replication mechanism and confers lincomycin resistance.
• Horizontal transfer of antibiotic resistance genes, potentially without mobilization genes, occurs broadly across bacterial species.
• Studying such transfers is vital for understanding Lactobacillus plasmid origins and evolution.