Beyond a Ribosomal RNA Methyltransferase, the Wider Role of MraW in DNA Methylation, Motility and Colonization in Escherichia coli O157:H7

Abstract

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four 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 activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

Functional Characterization of the Cell Division Gene Cluster of the Wall-less Bacterium Mycoplasma genitalium

“…This residue is involved in translation initiation and its mutation in E. coli leads to an increased doubling time ( Jemiolo et al, 1985 ). Moreover, the loss of MraW function has been directly related to an altered translation fidelity ( Kyuma et al, 2015 ), DNA methylation, and regulation of gene expression ( Xu et al, 2019 ). Our RNA-Seq data from the mraW mutant suggest that MraW does not regulate gene expression in M. genitalium .…”

“…In Staphylococcus aureus , mraW mutants also exhibit an anomalous translation fidelity, along with a reduced growth rate and an increased sensitivity to oxidative stress ( Kyuma et al, 2015 ). More recently, the MraW protein was found to also methylate DNA and alter gene expression in E. coli ( Xu et al, 2019 ). Of note, loss of MraW increases the expression of MraZ, which supports the antagonistic activity between these two proteins described earlier ( Eraso et al, 2014 ; Xu et al, 2019 ).…”

“…More recently, the MraW protein was found to also methylate DNA and alter gene expression in E. coli ( Xu et al, 2019 ). Of note, loss of MraW increases the expression of MraZ, which supports the antagonistic activity between these two proteins described earlier ( Eraso et al, 2014 ; Xu et al, 2019 ).…”

Comparative Analysis of Three Bradyrhizobium diazoefficiens Genomes Show Specific Mutations Acquired during Selection for a Higher Motility Phenotype and Adaption to Laboratory Conditions

“…Although it has been noted that in E. coli , the 16S rRNA modifications are not needed for the functional activity of in vitro -reconstituted ribosomes ( 28 ), several studies indicate that MraW could be important for bacterial fitness, stress tolerance, and virulence and might be essential in some bacteria ( 24 , 29 – 31 ). Additionally, a recent report shows a new function for E. coli MraW as a DNA methylase with a wider role, affecting different flagellar genes and promoters and therefore implicated in motility ( 32 ). The remaining five LP 3008 specific mutations are all missense variants.…”

“…As an example, DNA alteration in AAV28_RS30020-bll6610 could lead to lower motility, indicating a possible indirect role of mraW . Recently, the role of MraW as DNA methylase has been described, with implications for motility ( 32 ), highlighting MraW as one of the most likely candidates to explain the high-motility phenotype of LP 3008. Xu et al ( 32 ) found 336 genes and 219 promoters which showed a decrease of methylation in the mraW mutant, including 4 flagellar gene and promoter sequences.…”