A novel restriction-modification system is responsible for temperature-dependent phage resistance in Listeria monocytogenes ECII.

Listeria monocytogenes epidemic clone II (ECII) strains are unusual in being completely resistant to phage when grown at low temperatures (≤30°C). In the current study we constructed and characterized a mariner-based mutant (J46C) of the ECII strain H7550-Cd(S) that lacked temperature-dependent resistance to phage. The transposon was localized in LMOh7858_2753 (open reading frame [ORF] 2753), a member of a 12-ORF genomic island unique to ECII strains. ORF 2753 and ORF 2754 exhibited homologies to restriction endonucleases and methyltransferases associated with type II restriction-modification (RM) systems. In silico-based predictions of the recognition site for this putative RM system were supported by resistance of DNA from ECII strains to digestion by BfuI, a type II restriction enzyme specific for GTATCC (N6/5). Similarly to J46C, a mutant harboring an in-frame deletion of ORF 2753 was susceptible to phage regardless of temperature of growth (25°C or 37°C). Genetic complementation restored phage resistance in 25°C-grown cells of ORF 2753 mutants. Reverse transcription (RT) and quantitative real-time PCR data suggested enhanced transcription of ORF 2753 at low temperatures (≤25°C) compared to 37°C. In contrast, available transcriptional data suggested that the putative methyltransferase (ORF 2754) was constitutively expressed at all tested temperatures (4 to 37°C). Thus, temperature-dependent resistance of L. monocytogenes ECII to phage is mediated by temperature-dependent expression of the restriction endonuclease associated with a novel RM system (LmoH7) unique to this epidemic clone.