Characterization of a Cryptic Rolling-Circle Replication Plasmid pMK8 from Enterococcus durans 1-8.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Cryptic plasmids are extrachromosomal DNA elements found in bacteria that can influence host cell properties.
• Enterococcus durans is a Gram-positive bacterium relevant in various environments, including food and clinical settings.
• Understanding plasmid biology is crucial for deciphering bacterial adaptation and genetic exchange.

Purpose of the Study:

• To sequence and analyze a novel cryptic plasmid, designated pMK8, from Enterococcus durans 1-8.
• To elucidate the replication mechanism and copy number of the pMK8 plasmid.

Main Methods:

• Plasmid DNA sequencing and bioinformatics analysis to identify open reading frames (ORFs) and origins of replication.
• Homology searches to determine the functions of identified ORFs (replication initiation protein RepC, mobilization protein Mob).
• Southern hybridization to detect single-strand DNA (ssDNA) intermediates.
• Real-time PCR to quantify the relative copy number of the plasmid.

Main Results:

• The pMK8 plasmid is 3337 bp with a G+C content of 33.11%.
• Three ORFs were identified, including genes for RepC and Mob proteins.
• Sequence analysis revealed a pT181-family double-strand origin (dso) and an ssoW-family single-strand origin (sso).
• Southern hybridization confirmed ssDNA intermediates, indicating rolling circle replication (RCR).
• Real-time PCR estimated the copy number at approximately 175 ± 14 copies per cell.

Conclusions:

• pMK8 is a novel cryptic plasmid from Enterococcus durans that replicates via the RCR mechanism.
• The plasmid possesses a high copy number, suggesting efficient replication and maintenance.
• The characterization of pMK8 provides insights into plasmid diversity and replication strategies in Enterococcus species.