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

Abstract

MraW (RsmH) is an AdoMet-dependent 16S rRNA methyltransferase 45 conserved in bacteria and plays a role in the fine-tuning of the ribosomal decoding 46 center. It was recently found to contribute to the virulence of Staphylococcus aureus in 47 host animals. In this study, we examined the function of MraW in Escherichia coli 48 O157:H7 and found that deletion of mraW led to decreased motility and flagellar 49 production. Whole-genome bisulfite sequencing showed genome wide decrease of 50 methylation of 336 genes and 219 promoters in the mraW mutant. The methylation level 51 of 4 flagellar gene sequences were further confirmed by bisulfite PCR sequencing. 52Quantitative reverse transcription PCR results indicated the transcription of these genes 53 was also affected. MraW was observed to directly bind to the four flagellar gene 54 sequences by electrophoretic mobility shift assay (EMSA). A common motif in 55 differentially methylated regions of promoters and coding regions of the 4 flagellar 56 genes was identified. Reduced methylation was correlated with altered expression of 57 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA 58 methyltransferase (DNMT) activity assay in vitro. The mraW mutant colonized poorer 59 than wild type in mice. we further found that the expression of mraZ in the mraW 60 mutant was increased confirming the antagonistic effect of mraW on mraZ. In 61 conclusion, mraW was found to be a DNA methylase and has a wide-ranging effect on 62 E. coli O157:H7 including motility and virulence in vivo via genome wide methylation 63 and mraZ antagonism. 64 65 66 4 IMPORTANCE MraW is a well-studied 16S rRNA methyltransferase and was 67 recently found have an impact on bacterial virulence. Here we demonstrated its new 68 function as a DNA methylase and effect on motility, colonization in mice, DNA 69 methylation in genome wide and contribution to virulence. Its direct binding of 70 differentially methylated flagellar-encoding DNA sequences was observed, indicating 71 a correlation between DNA methylation and regulation of flagellar genes. In addition, 72 the expression of mraZ which function as an antagonist of mraW was increased in the 73 mraW mutant. mraW plays an important role in gene regulation likely through DNA 74 methylation. Clearly it plays a role in virulence in E. coli O157:H7. It also opens a new 75 research field for virulence study in bacteria. 76 77 78 KEYWORDS E. coli O157:H7, MraW, DNA methylation, motility, intestine 79 colonization 80 81 82 83 84 85 86 87 88 5 Escherichia coli O157:H7 is the most commonly isolated enterohaemorrhagic E. coli 89 (EHEC) and accounts for more than 90% of clinical EHEC cases (1-3). It causes 90 diarrheal diseases and other syndromes such as hemorrhagic colitis (HC) and hemolytic 91 uremic syndrome (HUS) with colonization of the intestinal mucosa and subsequent 92 toxin release in the intestinal tract (4). The main virulence factors involved in intestinal 93 colonization of the host are the type III secretion system (T3SS), ...

A Single Origin of Life and Death on Earth

“…The exact mechanisms by which SA decreases anaerobic growth, diversity and motility of microbial communities are unclear. In conjunction with other observations [1][2][3][4][5][6][7][8][9] , very simple experimental findings presented here suggest that SA might inhibit microbial biosynthesis of THF under anaerobic conditions. An as-yet-undefined, universally conserved anaerobic catalyst might be the target of SA in plants.…”

“…These very simple findings suggest that (1) SA decreases E. coli motility under anaerobic but not aerobic conditions, and (2) exogenous methionine counteracts this effect. Taken together with other observations [1][2][3][4][5][6][7][8][9] , these basic experimental findings support the idea that SA might decrease anaerobic motility of E. coli by inhibiting anaerobic biosynthesis of THF leading to methionine and SAM deficiency. SA might inhibit an as-yet-undefined, universally conserved anaerobic catalyst common to all domains of life.…”

“…To begin to explore this possibility, the motility of E. coli was examined with a light microscope under anaerobic (including hypoxic) and aerobic conditions after incubation with SA and/or methionine. Methionine, derived from THF, is converted to SAM 6 , required for most biological methylation processes including E. coli motility 9 . Wildtype E. coli was grown for 4.5 hours at 37°C in 50 mL Luria-Bertani medium either alone (control), supplemented with 2.3 mM SA, or supplemented with 2.3 mM SA plus 0.5 mM methionine.…”