DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Plasmids are crucial genetic elements in bacteria, influencing adaptation and traits.
• Lactococcus lactis is a key bacterium in dairy fermentation, with plasmids often carrying important functionalities.
• Understanding plasmid biology is essential for genetic engineering and industrial applications.

Purpose of the Study:

• To determine the complete nucleotide sequence of the plasmid pAR141 from Lactococcus lactis subsp. lactis M14.
• To identify genetic elements responsible for plasmid replication and control.
• To investigate the replication mechanism of pAR141.

Main Methods:

• Plasmid isolation from Lactococcus lactis subsp. lactis M14.
• Complete nucleotide sequencing of the plasmid pAR141.
• Bioinformatic analysis of the plasmid sequence.
• S1 nuclease and Southern hybridization analyses to confirm replication mode.

Main Results:

• The complete nucleotide sequence of pAR141 (1,594 base pairs) was determined.
• pAR141 contains elements for replication and control, including the copG and repB genes in a single operon.
• A countertranscribed RNA (ctRNA) coding region was identified between copG and repB.
• The presence of double-strand origin (dso) and single-strand origin (sso) indicated rolling circle replication (RCR).
• Experimental analyses confirmed the RCR replication mode.

Conclusions:

• The plasmid pAR141 does not confer any known industrial advantages.
• pAR141 utilizes a rolling circle replication mechanism, characteristic of RCR plasmids.
• The identified genetic elements provide insights into plasmid maintenance and stability in Lactococcus lactis.