Assembling Flagella in Salmonella Mutant Strains Producing a Type III Export Apparatus without FliO

Barker, Clive S.; Meshcheryakova, Irina V.; Inoue, Tomoharu; Samatey, Fadel A.

doi:10.1128/jb.02184-14

Cited by 17 publications

(19 citation statements)

References 38 publications

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“…A potential binding motif and direct binding were found between MraW protein and flagellar related nucleotide sequences including fliJ, fliR, fhiA , and fliK , indicating a direct physical interaction between MraW and these DNA sequences. The flagellum is composed of three parts: the basal body, the hook, and the filament (32, 33). FliR is one of the six integral membrane proteins of the export apparatus.…”

Section: Discusssionmentioning

confidence: 99%

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

Xu¹,

Huang²,

Zhang

et al. 2018

Preprint

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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), ...

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Section: Discusssionmentioning

confidence: 99%

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

Xu¹,

Huang²,

Zhang

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…Immunoblotting was performed similarly to a previously described method (Barker et al, 2014). For detection of FlhA proteins in whole cell lysates, fresh colonies of Salmonella containing the desired plasmid were inoculated into 6 ml LB with antibiotic and grown with shaking at 37˚C until an OD 600 nm 0.8 and induced for 2 h with the addition of 1 mM IPTG.…”

Section: Immunoblot Analysesmentioning

confidence: 99%

Function of the conserved FHIPEP domain of the flagellar type III export apparatus, protein FlhA

Barker

Inoue

Meshcheryakova

et al. 2016

Molecular Microbiology

Self Cite

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The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.

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“…2 A recent study reported the formation of a single polar flagellum formed by flagellins from two different gene clusters in a Sphingomonas strain [21]. 3 The sixth protein of the flagellar export apparatus, FliO, which has no homolog in the injectisome, has recently been found to regulate FliP during assembly of the flagellum [34].…”

Section: Structural and Functional Homologies Among The T3ss And Othementioning

confidence: 99%

Type III secretion systems: the bacterial flagellum and the injectisome

Diepold

Armitage

2015

Phil. Trans. R. Soc. B

184

175

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

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Assembling Flagella in Salmonella Mutant Strains Producing a Type III Export Apparatus without FliO

Cited by 17 publications

References 38 publications

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

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

Function of the conserved FHIPEP domain of the flagellar type III export apparatus, protein FlhA

Type III secretion systems: the bacterial flagellum and the injectisome

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