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  • Sittka, Alexandra  (9)
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  • 1
    Language: English
    In: Nucleic acids research, 2007, Vol.35(22), pp.7651-64
    Description: In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs.
    Keywords: RNA, Bacterial -- Metabolism ; RNA, Untranslated -- Metabolism ; Ribonucleases -- Physiology ; Salmonella Typhimurium -- Enzymology
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    In: Nature, 2010, Vol.464(7286), p.250
    Description: Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of ~60 small RNAs including the [straight epsilon]-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species. [PUBLICATION ]
    Keywords: 5' Untranslated Regions–Genetics ; Amino Acid Sequence–Genetics ; Base Sequence–Microbiology ; Cells, Cultured–Genetics ; Gene Expression Profiling–Genetics ; Genome, Bacterial–Chemistry ; Helicobacter Infections–Genetics ; Helicobacter Pylori–Metabolism ; Humans–Genetics ; Molecular Sequence Data–Genetics ; Nucleic Acid Conformation–Genetics ; Operon–Genetics ; RNA, Bacterial–Genetics ; RNA, Bacterial–Genetics ; RNA, Bacterial–Genetics ; RNA, Messenger–Genetics ; Sequence Alignment–Genetics ; Transcription, Genetic–Genetics ; Bacteria ; Proteins ; Microbiology ; Gene Expression ; RNA Polymerase ; Cell Division ; E Coli ; 5' Untranslated Regions ; 6s RNA ; RNA, Bacterial ; RNA, Messenger;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 15 May 2012, Vol.109(20), pp.E1277-86
    Description: More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and 〈20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.
    Keywords: Gene Expression Regulation, Bacterial -- Genetics ; RNA, Small Untranslated -- Genetics ; Regulatory Sequences, Ribonucleic Acid -- Genetics ; Salmonella Typhimurium -- Genetics ; Transcription, Genetic -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    In: Molecular Microbiology, January 2007, Vol.63(1), pp.193-217
    Description: The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Q RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, . A deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages . Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non‐coding RNAs, placing Hfq at the centre of post‐transcriptional regulation of virulence gene expression in . In addition, the mutation appears to cause a chronic activation of the RpoE‐mediated envelope stress response which is likely due to a misregulation of membrane protein expression.
    Keywords: Ribonucleic Acid–RNA ; Gene Expression ; Salmonella ; Genotype & Phenotype ; Proteins ; Cell Growth ; Pathology;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 5
    In: Molecular Microbiology, December 2007, Vol.66(5), pp.1174-1191
    Description: The pathogenicity island (SPI‐1) encodes ∼35 proteins involved in assembly of a type III secretion system (T3SS) which endows with the ability to invade eukaryotic cells. We have discovered a novel SPI‐1 gene, , which expresses an abundant small non‐coding RNA (sRNA). The gene, which we identified in a global search for new RNA genes, is activated by the major SPI‐1 transcription factor, HilD, under conditions that favour host cell invasion. The RNA chaperone, Hfq, is essential for the stability of the ∼80 nt InvR RNA. Hfq binds InvR with high affinity , and InvR co‐immunoprecipitates with FLAG epitope‐tagged Hfq in extracts. Surprisingly, deletion/overexpression of revealed no phenotype in SPI‐1 regulation. In contrast, we find that InvR represses the synthesis of the abundant OmpD porin encoded by the core genome. As is conserved in the early branching , we speculate that porin repression by InvR may have aided successful establishment of the SPI‐1 T3SS after horizontal acquisition in the lineage. This study identifies the first regulatory RNA of an enterobacterial pathogenicity island, and new roles for Hfq and HilD in SPI‐1 gene expression.
    Keywords: Ribonucleic Acid–RNA ; Salmonella ; Bacteria ; Bacteriology ; Protein Synthesis ; Genomics;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 6
    Language: English
    In: RNA Biology, 01 July 2009, Vol.6(3), pp.266-275
    Description: The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small noncoding RNAs (sRNAs) in E. coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus janaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA, or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.
    Keywords: Anatomy & Physiology
    ISSN: 1547-6286
    E-ISSN: 1555-8584
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  • 7
    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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  • 8
    Language: English
    In: Molecular microbiology, December 2007, Vol.66(5), pp.1174-91
    Description: The Salmonella pathogenicity island (SPI-1) encodes approximately 35 proteins involved in assembly of a type III secretion system (T3SS) which endows Salmonella with the ability to invade eukaryotic cells. We have discovered a novel SPI-1 gene, invR, which expresses an abundant small non-coding RNA (sRNA). The invR gene, which we identified in a global search for new Salmonella sRNA genes, is activated by the major SPI-1 transcription factor, HilD, under conditions that favour host cell invasion. The RNA chaperone, Hfq, is essential for the in vivo stability of the approximately 80 nt InvR RNA. Hfq binds InvR with high affinity in vitro, and InvR co-immunoprecipitates with FLAG epitope-tagged Hfq in Salmonella extracts. Surprisingly, deletion/overexpression of invR revealed no phenotype in SPI-1 regulation. In contrast, we find that InvR represses the synthesis of the abundant OmpD porin encoded by the Salmonella core genome. As invR is conserved in the early branching Salmonella bongori, we speculate that porin repression by InvR may have aided successful establishment of the SPI-1 T3SS after horizontal acquisition in the Salmonella lineage. This study identifies the first regulatory RNA of an enterobacterial pathogenicity island, and new roles for Hfq and HilD in SPI-1 gene expression.
    Keywords: Gene Expression Regulation, Bacterial ; Porins -- Biosynthesis ; RNA, Untranslated -- Metabolism ; Salmonella -- Genetics
    ISSN: 0950-382X
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