Mobile restriction-modification systems are key pillars of the antiphage architecture of Lactococcus

The genomes of 43 distinct strains of dairy lactococci were reconstructed by a combination of long- and short-read sequencing technologies, resolving the plasmid complement and methylome of these strains in full. The genomes comprised 43 chromosomes of approximately 2.5 Mb each and 269 plasmids ranging in size from 2 – 211 kb (at an average occurrence of 6 per strain). A total of 936 antiphage genes present in all strains were catalogued and a cross-correlation of identified genes with co-occurrent mobile elements was performed, which determined that over 50 % of these genes can be considered mobile. Detailed analysis established that restriction-modification (R-M) systems form a significant portion of this mobile phage defensome. We therefore undertook to assign each detected methylated motif (as associated with Type I, II and III R-M systems) to the specific gene(s) on the chromosome or plasmidome encoding the methylating enzyme through both homology searching and molecular cloning. The cumulative antiphage activity of selected systems and the ability of truncated R-M genes to contribute to methylation were demonstrated. This study reveals the dairy lactococcal plasmidome to be a rich reservoir of Type I R-M specificity subunit encoding genes, and the implications for complete bacterial genome sequencing, methylation analysis, dairy strain selection, phage defence, plasmid prevalence, plasmid mobility, and the wider antiphage landscape are discussed.