Fig. 2

Southern blotting confirms the ModM3 methylation target sequence 5′-AC^m6ATC-3′. a Schematic representation of the restriction-inhibition assay used to confirm the ModM3 methylation site. The location of the Southern blot probe, and the BtsCI and ModM3 recognition sites are shown. The central BtsCI site overlaps the ModM3 recognition sequence and is sensitive to overlapping N6-methyladenine methylation. b Restriction-inhibition assay of modM3 ON, modM3 OFF and ∆modM3 genomic DNA using the methyl-sensitive restriction endonuclease BtsCI. The restriction endonuclease HindIII is not sensitive to methylation and is included as a control for digestion. c Southern blot of BtsCI digested genomic DNA isolated from modM3 ON, modM3 OFF, and ∆modM3 strains. Methylated DNA in the ModM3 ON strain is protected from BtsCI digestion resulting in a 1.5 kb band. All BtsCI sites are cleaved in the modM3 OFF and ∆modM3 strains as ModM3 methylation is absent, resulting in a 0.5 kb band. d qRT-PCR indicating the relative abundance of methylated, undigested genomic DNA in modM3 ON and modM3 OFF relative to ∆modM3 following digestion with BtsCI (Ct values of 18.32, 21.97, 24.77, respectively, normalised to copB reference)