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  • Article  (11)
  • Vogel, Jorg  (11)
  • PMC (PubMed Central)  (11)
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  • Article  (11)
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  • 1
    Language: English
    In: PLoS Biology, 2008, Vol.6(3), p.e64
    Description: Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3′ end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms. ; Hierarchical action of regulators is a fundamental principle in gene expression control, and is well understood in protein-based signaling pathways. We have discovered that small noncoding RNAs (sRNAs), a new class of gene expression regulators, can also act hierarchically and form a regulatory cascade. Two highly similar sRNAs function after transcription to activate the mRNA, which codes for an essential function in amino-sugar metabolism. It is somewhat unusual for two sRNAs to act upon the same target mRNA, and despite their seeming homology, these two sRNAs (GlmY and GlmZ) employ different molecular mechanisms and function hierarchically to activate expression: GlmZ directly activates translation via disruption of an mRNA structure that inhibits translation, whereas GlmY controls the processing of GlmZ to prevent the inactivation of this direct activator. We also found that GlmY is itself controlled by an RNA processing event (3′ end polyadenylation), which typically destabilizes bacterial RNA. Our data unequivocally demonstrate that is exceptionally dependent on RNA-based mechanisms for its genetic control. Given the large number of noncoding RNAs of unknown function, we believe that similar regulatory RNA cascades may operate in other organisms. ; A regulatory RNA cascade that posttranscriptionally activates the mRNA is identified, with two highly similar small noncoding RNAs acting hierarchically in a manner thus far known only in protein-based regulatory circuits.
    Keywords: Research Article ; Biochemistry ; Genetics And Genomics ; Molecular Biology
    ISSN: 1544-9173
    E-ISSN: 1545-7885
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  • 2
    In: Nature, 2011, Vol.471(7340), p.602
    Description: CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans -encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
    Keywords: Article;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    Language: English
    In: PLoS ONE, 2011, Vol.6(3), p.e17296
    Description: P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella -induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri , arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Microbiology ; Molecular Biology ; Cell Biology
    E-ISSN: 1932-6203
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  • 4
    In: PLoS ONE, 2015, Vol.10(11)
    Description: Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizosphere-mimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis- encoded antisense RNAs, as well as trans- encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus . Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.
    Keywords: Research Article
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: Nucleic acids research, March 2012, Vol.40(5), pp.2020-31
    Description: The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.
    Keywords: RNA, Small Untranslated -- Metabolism ; Virulence Factors -- Genetics ; Xanthomonas Campestris -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 6
    In: PLoS Pathogens, 2017, Vol.13(2)
    Description: The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.
    Keywords: Review ; Biology And Life Sciences ; Research And Analysis Methods ; Medicine And Health Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Medicine And Health Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Research And Analysis Methods
    ISSN: 1553-7366
    E-ISSN: 1553-7374
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  • 7
    In: PLoS Genetics, 2016, Vol.12(4)
    Description: While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans , the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK . Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein. Author Summary As the function of conserved core-genome-encoded small RNAs (sRNA) reflects the basic lifestyle of bacteria, the function of non-conserved island-encoded sRNAs remains enigmatic. The island-encoded sRNA IsrK belongs to Gifsy-1 prophage of Salmonella . Here, we report a complex mechanism in which the IsrK RNA functions as both sRNA and mRNA to control the production of the toxic AntQ protein. The isrK promoter directs the synthesis of two distinct RNA species: a full-length translationally inactive target mRNA and the correctly terminated, shorter IsrK sRNA. IsrK sRNA binds the full-length inactive mRNA producing an antiterminator protein, AntQ, which interferes with transcription termination. Expression of antQ results in bacterial growth arrest and ultimately cell death. Fluorescence microscopy of E . coli and Salmonella expressing antQ revealed condensed chromatin morphology as observed upon exposure to DNA-damaging agents. We propose that expression of the phage antiterminator protein results in conflicts between transcription and replication machineries and thus facilitates DNA damage. In summary, the RNA regulator IsrK presents a new regulatory principle in which a horizontally acquired sRNA controls genome integrity.
    Keywords: Research Article ; Biology And Life Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Research And Analysis Methods ; Biology And Life Sciences ; Biology And Life Sciences ; Research And Analysis Methods ; Research And Analysis Methods ; Biology And Life Sciences
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 8
    Language: English
    In: PLoS Pathogens, 2016, Vol. 12(6)
    Description: Salmonella Typhimurium (S. Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S. Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf(-/-) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S. Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.
    Keywords: Medical And Health Sciences ; Clinical Medicine ; Infectious Medicine ; Medicin Och Hälsovetenskap ; Klinisk Medicin ; Infektionsmedicin ; Medical And Health Sciences ; Basic Medicine ; Microbiology In The Medical Area ; Medicin Och Hälsovetenskap ; Medicinska Och Farmaceutiska Grundvetenskaper ; Mikrobiologi Inom Det Medicinska Området
    ISSN: 1553-7366
    E-ISSN: 15537374
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  • 9
    Language: English
    In: PLoS Computational Biology, 2009, Vol.5(9), p.e1000502
    Description: With few exceptions, current methods for short read mapping make use of simple seed heuristics to speed up the search. Most of the underlying matching models neglect the necessity to allow not only mismatches, but also insertions and deletions. Current evaluations indicate, however, that very different error models apply to the novel high-throughput sequencing methods. While the most frequent error-type in Illumina reads are mismatches, reads produced by 454's GS FLX predominantly contain insertions and deletions (indels). Even though 454 sequencers are able to produce longer reads, the method is frequently applied to small RNA (miRNA and siRNA) sequencing. Fast and accurate matching in particular of short reads with diverse errors is therefore a pressing practical problem. We introduce a matching model for short reads that can, besides mismatches, also cope with indels. It addresses different error models. For example, it can handle the problem of leading and trailing contaminations caused by primers and poly-A tails in transcriptomics or the length-dependent increase of error rates. In these contexts, it thus simplifies the tedious and error-prone trimming step. For efficient searches, our method utilizes index structures in the form of enhanced suffix arrays. In a comparison with current methods for short read mapping, the presented approach shows significantly increased performance not only for 454 reads, but also for Illumina reads. Our approach is implemented in the software segemehl available at http://www.bioinf.uni-leipzig.de/Software/segemehl/ . ; The successful mapping of high-throughput sequencing (HTS) reads to reference genomes largely depends on the accuracy of both the sequencing technologies and reference genomes. Current mapping algorithms focus on mapping with mismatches but largely neglect insertions and deletions—regardless of whether they are caused by sequencing errors or genomic variation. Furthermore, trailing contaminations by primers and declining read qualities can be cumbersome for programs that allow a maximum number of mismatches. We have developed and implemented a new approach for short read mapping that, in a first step, computes exact matches of the read and the reference genome. The exact matches are then modified by a limited number of mismatches, insertions and deletions. From the set of exact and inexact matches, we select those with minimum score-based E-values. This gives a set of regions in the reference genome which is aligned to the read using Myers bitvector algorithm . Our method utilizes enhanced suffix arrays to quickly find the exact and inexact matches. It maps more reads and achieves higher recall rates than previous methods. This consistently holds for reads produced by 454 as well as Illumina sequencing technologies.
    Keywords: Research Article ; Computational Biology ; Computational Biology -- Genomics ; Genetics And Genomics -- Bioinformatics
    ISSN: 1553-734X
    E-ISSN: 1553-7358
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  • 10
    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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