Figure 2.

Graphical representation of the silenced JHP0414/JHP0415 Type I MTase of J99-R3 (A), the putative phase-variable Type III MTases (B), BcgI-like R-M systems (C) and Type ISP MTases (D). (A) The gene sequences of HsdM (JHP0415) and HsdS (JHP0414) are displayed as gray bars and the protein sequences as yellow bars. SMRT sequencing of the J99-R3 genome did not reveal methylation by the Type I MTase complex (dashed lines), while expression of the enzyme complex on a plasmid in E. coli 2796 and subsequent SMRT analyses demonstrate methylation of the recognition site AAAN₆TGG. (B–D) The gene sequences (gray arrows) contain one or two putative or authentic frameshifts that would prevent translation of full-length proteins (colored bars). Each frameshift was repaired through site-directed mutagenesis (addition and deletion of an indicated number of nucleotides, +15 Nt—TAAGGTTAATATATG) and correction was verified by targeted Sanger sequencing. The activity of the MTase proteins was pretested with a methylation activity assay. If an MTase showed activity in the assay, the gDNA of the corresponding E. coli host strain ER2796 was subjected to SMRT sequencing and analyzed for methylation. ‘mut’ marks alleles with frameshift corrections, ‘trunc’ labels not fully translated alleles, ‘n.d.’ points out that no SMRT sequencing was performed and dashed lines indicate that no methylation could be detected through SMRT sequencing. Filled inverted triangles highlight the position of the frameshift mutations. JHP—ORFs of J99, HP—ORFs of 26695. Homologous systems are displayed in the same color.