Dataset Information

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

ABSTRACT: The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

SUBMITTER: Tran CN

PROVIDER: S-EPMC3185424 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

Tran Chi Nhan CN Giangrossi Mara M Prosseda Gianni G Brandi Anna A Di Martino Maria Letizia ML Colonna Bianca B Falconi Maurizio M

Nucleic acids research 20110630 18

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind ...[more]

PMID: 21724612

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Project description:VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF-RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to "fish" out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5'-UUUUaGYcUuUau-3') that is the RNA-converted consensus sequence previously proposed for the VirF-DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF-RNA interaction, in particular when exposed in a stem-loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA-RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem-loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA-RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.

Project description:The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.

Dataset Information

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

Publications

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

Similar Datasets

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