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 degrees 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 degrees C). Genetic complementation restored phage resistance in 25 degrees 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 degrees C) compared to 37 degrees C. In contrast, available transcriptional data suggested that the putative methyltransferase (ORF 2754) was constitutively expressed at all tested temperatures (4 to 37 degrees 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.