DNA Methyltransferase regulates nitric oxide homeostasis and virulence in a chronically adapted Pseudomonas aeruginosa strain

Opportunistic pathogens such as Pseudomonas aeruginosa adapt their genomes rapidly during chronic infections. Understanding their epigenetic regulation may provide biomarkers for diagnosis and reveal novel regulatory mechanisms. We performed single-molecule real-time sequencing (SMRT-seq) to characterize the methylome of a chronically adapted P. aeruginosa clinical strain TBCF10839. Two N6-methyl-adenine (6mA) methylation recognition motifs (RCCANNNNNNNTGAR and TRGANNNNNNTGC) were identified and predicted as new type I methylation sites using REBASE analysis. We confirmed that motif TRGANNNNNNTGCwas methylated by MTase M.PaeTBCFII, according to methylation sensitivity assays in vivo and vitro. Transcriptomic analysis showed that ΔM.PaeTBCFII knockout mutant significantly downregulated nitric oxide reductase (NOR) regulating and coding gene expression such as nosR and norB, which contain methylated motifs in their promoters or coding regions. ΔM.PaeTBCFII exhibited reduced intercellular ...