AbstractUropathogenicEscherichia coli(UPEC) persister bacteria play crucial roles in clinical treatment failure and relapse. DNA methylation is known to regulate gene expression in bacteria, but its role in persister formation has not been investigated. Here, we created adenine methylation deletion mutant (Δdam) and cytosine methylation mutant (Δdcm) from UPEC strain UTI89 and found that theΔdammutant but notΔdcmmutant had significant defect in persister formation during exposure to various antibiotics (gentamicin, fluroquinolones and cephalosporin) and stresses (acid pH and hyperosmosis), and that complementation of thedammutant restored its persister defect phenotype. PacBio sequencing of epigenetic genomewide methylation signature and RNA sequencing of theΔdammutant were performed to define, for the first time, the role of adenine methylation in persister formation. Methylome data analysis showed that 99.73% of m6A modifications on GATC were demethylated in theΔdammutant, and demethylation nucleotide site related genes suggested an overwhelming effect on transcription and metabolic processes. Transcriptome analysis of theΔdammutant in comparison to wild type showed that flagella biosynthesis, galactitol transport/utilization, and signaling related genes were upregulated while pilus, fimbriae, virulence, glycerol, nitrogen metabolism pathways and transcriptional regulators were downregulated. The comparative COG analysis of methylome and transcriptome enriched pathways identified translation, ribosomal structure and biogenesis, and cell motility were upregulated, whereas DNA repair, secondary metabolite biosynthesis and diverse transport systems, some of which are known to be involved in persister formation, were downregulated in theΔdammutant. These findings provide new insights about the molecular basis of how DNA adenine methylation may be involved in persister formation and offer novel therapeutic targets for combating persister bacteria.