Stacking of three different restriction and modification systems in Lactococcus lactis by cotransformation

Area of Science:

• Microbiology
• Bacteriology
• Molecular Biology

Background:

• Lactococcus lactis is a key dairy starter culture.
• Bacteriophages (phages) pose a significant threat to industrial L. lactis fermentations.
• Restriction-modification (R/M) systems offer a genetic defense against phage infection.

Purpose of the Study:

• To characterize four novel R/M plasmids for their ability to confer phage resistance in L. lactis.
• To investigate the combined effects of multiple R/M plasmids on phage defense.
• To assess the stability of R/M plasmids in L. lactis.

Main Methods:

• Cotransformation of L. lactis MG1363 with R/M plasmids and a selectable marker plasmid (pVS2).
• Phage plaque assays to determine the efficiency of plating (EOP) on transformant strains.
• Analysis of plasmid stability and compatibility within L. lactis.

Main Results:

• Four distinct R/M plasmids exhibited varying specificities against isometric phage p2 and prolate phage c2.
• Successful cotransformation and maintenance of up to three different R/M plasmids in L. lactis MG1363.
• A dose-dependent increase in phage resistance was observed with increasing numbers of R/M plasmids.
• Instability was noted for certain R/M plasmid combinations, indicating potential replicon incompatibility.

Conclusions:

• Multiple R/M plasmids can be stably combined in L. lactis to enhance phage resistance.
• The combination of R/M plasmids provides a robust strategy for protecting dairy starter cultures against phage predation.
• Further studies are needed to resolve replicon incompatibility issues for optimal industrial application.