Beyond a ribosomal RNA methyltransferase, the wider role of MraW in DNA methylation, motility and colonization inEscherichia coliO157:H7

Abstract

ABSTRACTMraW (RsmH) is an AdoMet-dependent 16S rRNA methyltransferase conserved in bacteria and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence ofStaphylococcus aureusin host animals. In this study, we examined the function of MraW inEscherichia coliO157:H7 and found that deletion ofmraWled to decreased motility and flagellar production. Whole-genome bisulfite sequencing showed genome wide decrease of methylation of 336 genes and 219 promoters in themraWmutant. The methylation level of 4 flagellar gene sequences were further confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated the transcription of these genes was also affected. MraW was observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common motif in differentially methylated regions of promoters and coding regions of the 4 flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase (DNMT) activity assayin vitro. ThemraWmutant colonized poorer than wild type in mice. we further found that the expression ofmraZin themraWmutant was increased confirming the antagonistic effect ofmraWonmraZ. In conclusion,mraWwas found to be a DNA methylase and has a wide-ranging effect onE.coliO157:H7 including motility and virulencein vivovia genome wide methylation andmraZantagonism.IMPORTANCEMraW is a well-studied 16S rRNA methyltransferase and was recently found have an impact on bacterial virulence. Here we demonstrated its new function as a DNA methylase and effect on motility, colonization in mice, DNA methylation in genome wide and contribution to virulence. Its direct binding of differentially methylated flagellar-encoding DNA sequences was observed, indicating a correlation between DNA methylation and regulation of flagellar genes. In addition, the expression ofmraZwhich function as an antagonist ofmraWwas increased in themraWmutant.mraWplays an important role in gene regulation likely through DNA methylation. Clearly it plays a role in virulence inE. coliO157:H7. It also opens a new research field for virulence study in bacteria.