Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium

Iyoda, Sunao; Kutsukake, Kazuhiro

doi:10.1007/bf00287103

Cited by 55 publications

(62 citation statements)

References 32 publications

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“…Standard DNA manipulation was performed as described previously (Iyoda & Kutsukake, 1995;Iyoda et al, 2001). PCR amplification was performed on a thermal cycler PE9600 (Perkin Elmer) with TaKaRa EX Taq DNA polymerase (Takara).…”

Section: Methodsmentioning

confidence: 99%

“…Bacteria were grown under the same culture conditions as those for the b-galactosidase assay until they reached an OD 600 of 0?7. The bacteria were removed by centrifugation (10 000 g for 20 min), and the proteins secreted in the culture supernatant were precipitated with 10 % (w/v) TCA and separated on a 12 % (w/v) SDS-polyacrylamide gel, as described previously (Iyoda & Kutsukake, 1995;Iyoda et al, 2001). Western blotting was performed with anti-EspB and anti-Stx2A polyclonal antibodies to probe for each protein in whole-cell lysates derived from equal amounts of bacteria normalized by measurement of OD 600 .…”

Section: Methodsmentioning

confidence: 99%

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Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells

2004

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Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes. In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome. These five perC homologues were characterized, and it was found that three of the homologues (renamed perC homologue pchA, pchB and pchC) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect. Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler. Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the pchABC genes are necessary for full virulence of EHEC O157.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells

2004

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“…Class 2 contains the fliA gene, whose product acts as an alternative sigma factor specific for another subset of the flagellar operons called the class 3 operons (Ohnishi et al, 1990). Class 3 expression is negatively regulated by the flgM gene (Gillen and Hughes, 1991;, which encodes an anti-sigma factor specific for FliA (Ohnishi et al, 1992;Iyoda and Kutsukake, 1995;Chadsey et al, 1998).…”

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confidence: 99%

Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium.

et al. 1999

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The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium. Here we report the molecular structure and in vivo and in vitro expression of the flhD operon. Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site. Primer extension analysis demonstrated six possible transcription start sites for this operon. They include CRP-dependent and CRP-repressible transcription start sites. The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the σ 70 -dependent promoter. The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site. The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site. These two start sites were confirmed by in vitro transcription experiments using σ 70 -RNA polymerase with or without cAMP-CRP.

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“…β-Galactosidase activity was assayed as described previously (Iyoda and Kutsukake, 1995;Iyoda and Watanabe, 2004). E. coli cells grown in LB or DMEM at 37 C with shaking were harvested at an optical density of 0.8 at 600 nm.…”

Section: Methodsmentioning

confidence: 99%

A novel small regulatory RNA enhances cell motility in enterohemorrhagic Escherichia coli

Sudo

Soma

Muto

et al. 2014

J. Gen. Appl. Microbiol.

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Small regulatory RNAs (sRNAs) are conserved among a wide range of bacteria. They modulate the translational efficiency of target mRNAs through base-pairing with the help of RNA chaperone Hfq. The present study identified a novel sRNA, Esr41 (enterohemorrhagic Escherichia coli O157 small RNA #41), from an intergenic region of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai-specific sequence that is not present in the nonpathogenic E. coli K-12. Esr41 was detected as an RNA molecule approximately 70 nucleotides long with a 3 GC-rich palindrome sequence followed by a long poly(U), which is a characteristic of rho-independent terminators and is also a structural feature required for the action of Hfq. EHEC O157 harboring a multicopy plasmid carrying the esr41 gene increased cell motility and the expression of fliC, a gene encoding a major flagellar component. These results indicate that Esr41 stimulates fliC expression in EHEC O157. Furthermore, the increase in cell motility induced by Esr41 was also observed in the E. coli K-12, suggesting that target genes controlled by Esr41 are present in both EHEC O157 and K-12.Key words cell motility; EHEC O157 : H7 Sakai; flagella; small regulatory RNA IntroductionSmall regulatory RNAs (sRNAs) play crucial roles in bacterial stress responses and virulence (Gottesman, 2004;Papenfort and Vogel, 2010;Waters and Storz, 2009). Many sRNAs are induced under specific physiological conditions and control the expression of target genes, primarily at the post-transcriptional levels (Gottesman, 2004;Wassarman, 2002). sRNAs are classified as cis-acting sRNAs or transacting sRNAs. Cis-acting sRNAs are encoded on the strand opposite to the sense strand of the target gene and regulate the target gene expression by perfect base-pairing with its mRNA (Kawano et al., 2005). Trans-acting sRNAs are usually encoded in a different region from that of the target genes and regulate the expression of the target genes by partial base-pairing with their mRNAs. One major class of sRNA binds to RNA chaperone Hfq, which facilitates base-pairing between sRNA and the target mRNA to regulate its translation, mostly negatively but also positively in some cases (Aiba, 2007;Vogel and Luisi, 2011).A comprehensive identification of sRNA has been performed in many bacteria, such as Escherichia coli and None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.

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Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium

Cited by 55 publications

References 32 publications

Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells

Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells

Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium.

A novel small regulatory RNA enhances cell motility in enterohemorrhagic Escherichia coli

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