Table_1_Beyond a Ribosomal RNA Methyltransferase, the Wider Role of MraW in DNA Methylation, Motility and Colonization in Escherichia coli O157:H7.DOCX

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.

MraW是一种16S rRNA甲基转移酶（16S rRNA methyltransferase），参与核糖体解码中心的精细调控。近期研究发现，其可促进金黄色葡萄球菌（Staphylococcus aureus）的毒力形成。本研究针对大肠杆菌O157:H7（Escherichia coli O157:H7）中MraW的功能展开探究，结果显示mraW基因缺失会导致菌株运动性下降、鞭毛生成减少以及DNA甲基化水平降低。全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）结果表明，mraW突变株的全基因组范围内共有336个基因与219个启动子的甲基化水平出现下调，其中涵盖鞭毛相关基因。通过亚硫酸氢盐PCR测序（bisulfite PCR sequencing）验证了鞭毛相关基因的甲基化水平变化。定量反转录PCR（quantitative reverse transcription PCR）结果显示，这些基因的转录水平同样受到影响。进一步通过电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）证实，MraW可直接结合4个鞭毛基因的序列。在这4个鞭毛基因的启动子及编码区的差异甲基化区域（differentially methylated regions, DMRs）中，我们鉴定出一段共有的柔性基序。24个被检测基因中有21个的甲基化水平降低与表达改变呈相关性。体外DNA甲基转移酶活性测定（DNA methyltransferase activity assay）证实了MraW具备DNA甲基化活性，且该活性可被DNA甲基化抑制剂5-氮杂-2′-脱氧胞苷（5-aza-2′-deoxycytidine, 5-aza）抑制。此外，mraW突变株在小鼠体内的定殖能力弱于野生型菌株。我们还发现，mraW突变株中mraZ基因的表达水平上调，证实了mraW对mraZ的拮抗作用。综上，本研究证实MraW是一种DNA甲基化酶，可通过全基因组范围的甲基化调控以及对mraZ的拮抗作用，对大肠杆菌O157:H7产生广泛影响，包括调控其运动性与体内毒力。