Mutational analyses revealed the 21.5 kb plasmid-encoded lactose fermentation and proteolytic activity properties in Lactococcus lactis ssp. lactis MN24. Reductions in maximum specific growth rate and population density of the 21.5 kb plasmid-cured mutant of MN24 confirmed the data obtained by mutation tests. Plasmid curing, polymerase chain reaction and DNA sequence analyses data showed that the lacticin 481 operon was located on 22.4 kb plasmid. The phage resistance system in strain MN24 was identified as an adsorption inhibition type and chromosomally encoded via phage-host interaction tests and mutation analyses.