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  • 1
    In: Molecular Microbiology, October 2009, Vol.74(1), pp.139-158
    Description: The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome‐wide screens in and . Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial ‘core’ small RNA, yet its function remained unknown. Here, we report that ArcZ acts as a post‐transcriptional regulator in , repressing the mRNAs of the widely distributed (serine uptake) and (oxidative stress) genes, and of STM3216, a horizontally acquired methyl‐accepting chemotaxis protein (MCP). Both and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an deletion strain further argued for the existence of a distinct set of loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady‐state levels of 〉 16% (〉 750) of all mRNAs, and rendered the bacteria non‐motile. Deep sequencing detected a dramatically changed profile of Hfq‐bound sRNAs and mRNAs, suggesting that the unprecedented pleiotropic effects by a single sRNA might in part be caused by altered post‐transcriptional regulation.
    Keywords: Biology;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 2
    In: Molecular Microbiology, May 2008, Vol.68(4), pp.890-906
    Description: Post‐transcriptional repression of porin synthesis has emerged as a major function of Hfq‐dependent, small non‐coding RNAs (sRNAs). Many enterobacteria express OmpX‐like porins, a family of outer membrane proteins whose physiological roles and structural properties have been studied intensively. While regulatory sRNAs have been identified for most major and many minor porins of and , a post‐transcriptional regulator of OmpX levels has never been found. Here, we have taken a ‘reverse target search’ approach by systematic inactivation of sRNA genes, and screening 35 sRNA deletion strains for effects on OmpX synthesis. We have identified the Hfq‐dependent CyaR (formerly RyeE) sRNA as an repressor. Global transcriptomic profiling following induction of CyaR expression suggests that mRNA is the primary target of this sRNA under standard growth conditions. The results of phylogenetic and mutational analyses suggest that a conserved RNA hairpin of CyaR, featuring a C‐rich apical loop, acts to sequester the Shine–Dalgarno sequence of mRNA and to inhibit translational initiation. We have also discovered that expression is tightly controlled by the cyclic AMP receptor protein, CRP. This represents a new link between porin repression and nutrient availability that is likely to be widely conserved among enterobacteria.
    Keywords: Genetic Research -- Genetic Aspects ; Genetic Research -- Physiological Aspects ; Bacterial Genetics -- Genetic Aspects ; Bacterial Genetics -- Physiological Aspects ; Cyclic Adenosine Monophosphate -- Genetic Aspects ; Cyclic Adenosine Monophosphate -- Physiological Aspects ; Salmonella -- Genetic Aspects ; Salmonella -- Physiological Aspects ; Rna -- Genetic Aspects ; Rna -- Physiological Aspects ; Porins -- Genetic Aspects ; Porins -- Physiological Aspects;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 3
    In: Molecular Microbiology, December 2006, Vol.62(6), pp.1674-1688
    Description: The bacterial envelope stress response (ESR) is triggered by the accumulation of misfolded outer membrane proteins (OMPs) upon envelope damage or excessive OMP synthesis, and is mediated by the alternative sigma factor, σ. Activation of the σ pathway causes a rapid downregulation of major mRNAs, which prevents further build‐up of unassembled OMPs and liberates the translocation and folding apparatus under conditions that require envelope remodelling. The factors that facilitate the rapid removal of the unusually stable mRNAs in the ESR were previously unknown. We report that in the ESR relies upon two highly conserved, σ‐controlled small non‐coding RNAs, RybB and MicA. By using a transcriptomic approach and kinetic analyses of target mRNA decay , RybB was identified as the factor that selectively accelerates the decay of multiple major mRNAs upon induction of the ESR, while MicA is proposed to facilitate rapid decay of the single mRNA. In unstressed bacterial cells, the two σ‐dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of σ.
    Keywords: Mrna Turnover ; Envelopes ; Outer Membrane Proteins ; Kinetics ; Non-Coding RNA ; Stress ; Homeostasis ; Sigma Factor ; Translocation ; Salmonella ; RNA ; Genetics & Taxonomy;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 4
    Language: English
    In: PLoS Genetics, 2008, Vol.4(8), p.e1000163
    Description: Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo . The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella . Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria. ; The past decade has seen small regulatory RNA become an important new mediator of bacterial mRNA regulation. This study describes a rapid way to identify novel sRNAs that are expressed, and should prove relevant to a variety of bacteria. We purified the epitope-tagged RNA-binding protein, Hfq, and its bound RNA by immunoprecipitation from the model pathogen, serovar Typhimurium. This new strategy used Next Generation pyrosequencing to identify 727 Hfq-bound mRNAs. The numbers of sRNAs expressed in was doubled to 64; half are associated with Hfq. We defined the exact coordinates of sRNAs, and confirmed that they are expressed at significant levels. We also determined the Hfq regulon in , and reported the role of Hfq in controlling transcription of major pathogenicity islands, horizontally acquired regions, and the flagellar cascade. Hfq is reported to be a global regulator that affects the expression of almost a fifth of all genes. Our new approach will allow sRNAs and mRNAs to be characterized from different genetic backgrounds, or from bacteria grown under particular environmental conditions. It will be valuable to scientists working on genetically tractable bacteria who are interested in the function of RNA-binding proteins and the identification of sRNAs.
    Keywords: Research Article ; Biochemistry -- Bioinformatics ; Genetics And Genomics -- Functional Genomics ; Genetics And Genomics -- Gene Expression ; Microbiology ; Microbiology -- Microbial Evolution And Genomics
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 5
    Language: English
    In: Molecular microbiology, December 2006, Vol.62(6), pp.1674-88
    Description: The bacterial envelope stress response (ESR) is triggered by the accumulation of misfolded outer membrane proteins (OMPs) upon envelope damage or excessive OMP synthesis, and is mediated by the alternative sigma factor, sigmaE. Activation of the GE pathway causes a rapid downregulation of major omp mRNAs, which prevents further build-up of unassembled OMPs and liberates the translocation and folding apparatus under conditions that require envelope remodelling. The factors that facilitate the rapid removal of the unusually stable omp mRNAs in the ESR were previously unknown. We report that in Salmonella the ESR relies upon two highly conserved, sigmaE-controlled small non-coding RNAs, RybB and MicA. By using a transcriptomic approach and kinetic analyses of target mRNA decay in vivo, RybB was identified as the factor that selectively accelerates the decay of multiple major omp mRNAs upon induction of the ESR, while MicA is proposed to facilitate rapid decay of the single ompA mRNA. In unstressed bacterial cells, the two oE-dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of oE.
    Keywords: Bacterial Outer Membrane Proteins -- Metabolism ; RNA, Messenger -- Metabolism ; RNA, Untranslated -- Metabolism ; Salmonella -- Metabolism ; Sigma Factor -- Metabolism
    ISSN: 0950-382X
    E-ISSN: 13652958
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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