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

Xuefang Xu,Jianguo Xu,Ying Huang,Dai Wang,Xuelian Luo,Xiaojing Peng,Ruiting Lan,Xiaoyuan Wang,Xuancheng Lu,Changyun Ye,Yuhui Dong,Yuan Zhang,Shuangshuang Lu,Heng Zhang,Changde Wu,Hongqing Zhao,Zhongqiu Teng,Pengya Gao,Ji Pu

doi:10.3389/fmicb.2019.02520

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.

Highlights

Escherichia coli O157:H7 is the most commonly isolated enterohaemorrhagic E. coli (EHEC) and accounts for more than 90% of clinical EHEC cases (Garg et al, 2005; Friedrich et al, 2007; Xu et al, 2012)
The gene encoding MraW was deleted in E. coli O157:H7 strain EDL933 resulting in a mutant strain designated as EDL933 mraW
We investigated the role of MraW in genomewide DNA methylation and its role in virulence in E. coli O157:H7

Summary

Introduction

Escherichia coli O157:H7 is the most commonly isolated enterohaemorrhagic E. coli (EHEC) and accounts for more than 90% of clinical EHEC cases (Garg et al, 2005; Friedrich et al, 2007; Xu et al, 2012) It causes diarrheal diseases and other syndromes such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) with colonization of the intestinal mucosa and subsequent toxin release in the intestinal tract (Friedland et al, 1995). The main virulence factors involved in the intestinal colonization of the host are the type III secretion system (T3SS), curli and flagella (Dobbin et al, 2006; Bretschneider et al, 2007). It was revealed that both rsmI and rsmH (mraW) affected the virulence of Staphylococcus aureus in silk worms by contributing resistance to oxidative stress (Kyuma et al, 2015). It was revealed that both rsmI and rsmH (mraW) affected the virulence of Staphylococcus aureus in silk worms by contributing resistance to oxidative stress (Kyuma et al, 2015). mraW is likely to contribute to the tolerance to aminoglycoside killing as a mraW transposon insertion mutant had a more than 10-fold reduction of gentamicin tolerance in in vitro culture (Zou et al, 2018)

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