Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenic Escherichia coli

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life, extending also to bacterial virulence. Both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. However, the potential regulatory role of DNA methylation mediated by archetypal Type I systems in Escherichia coli has never been studied. We demonstrated that removal of DNA methylated mediated by three different Escherichia coli Type I RMSs in three distinct E. coli strains had no detectable effect on gene expression or growth in a screen of 1190 conditions. Additionally, deletion of the Type I RMS EcoUTI in UTI89, a prototypical cystitis strain of E. coli, which led to loss of methylation at >750 sites across the genome, had no detectable effect on virulence in a murine model of ascending urinary tract infection (UTI). Finally, introduction of two heterologous Type I RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of "regulation avoidance" for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome. Our results provide a clear and (currently) rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli, further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer. Author summaryDNA methylation is perhaps the most common epigenetic modification, and it is commonly associated with gene regulation (in nearly all organisms) and virulence (particularly well studied in bacteria). Regarding bacterial virulence, the current DNA methylation literature has focused primarily on orphan methyltransferases or phasevariable restriction modification systems (RMSs). Interestingly, no reports have studied the potential regulatory role of the first RMS discovered, the Type I RMS EcoKI. We used transcriptomics, Phenotype Microarrays, and a murine model of urinary tract infection to screen for functional consequences due to Type I methylation in three unrelated strains of E. coli. Remarkably, we found zero evidence for any epigenetic regulation mediated by these Type I RMSs. Thus, these Type I RMSs appear to function exclusively in host defense against incoming DNA (the canonical function of RMSs), while the methylation status of many hundreds of the corresponding recognition sites has no detectable impact on gene expression or any phenotypes. This led us to the concept of "regulation avoidance" by such DNA methyltransferases, which contrasts with the current literature on bacterial epigenetics. Our study hints at the existence of an entire class of regulation avoidant systems, which provides new perspectives on methylation-mediated gene regulation and bacterial genome evolution.