Kooperativer Bibliotheksverbund

Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 17 February 2015, Vol.112(7), pp.E766-75
    Description: Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.
    Keywords: RNA-Seq ; Vibrio Cholerae ; Biofilm ; Quorum Sensing ; Srna ; Biofilms ; Sequence Analysis, RNA ; RNA, Viral -- Genetics ; Vibrio Cholerae -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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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: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(5), pp.2124-2129
    Description: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ~64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research. ; Includes references ; p. 2124-2129.
    Keywords: Transcription (Genetics) -- Physiological Aspects ; Cyanobacteria -- Genetic Aspects ; Genetic Regulation -- Research ; Rna Polymerases -- Properties;
    ISSN: 0027-8424
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 23 November 2010, Vol.107(47), pp.20435-40
    Description: The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5' located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5' end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5' domain is sufficient to guide target mRNA degradation and maintain σ(E)-dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3' adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5' domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.
    Keywords: Bacterial Outer Membrane Proteins -- Metabolism ; Gene Expression Regulation, Bacterial -- Genetics ; RNA, Messenger -- Metabolism ; Regulatory Sequences, Ribonucleic Acid -- Genetics ; Regulon -- Genetics ; Salmonella -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 January 2014, Vol.111(4), pp.E501-10
    Description: Phase variation of hypermutable simple sequence repeats (SSRs) is a widespread and stochastic mechanism to generate phenotypic variation within a population and thereby contributes to host adaptation of bacterial pathogens. Although several examples of SSRs that affect transcription or coding potential have been reported, we now show that a SSR also impacts small RNA-mediated posttranscriptional regulation. Based on in vitro and in vivo analyses, we demonstrate that a variable homopolymeric G-repeat in the leader of the TlpB chemotaxis receptor mRNA of the human pathogen Helicobacter pylori is directly targeted by a small RNA (sRNA), RepG (Regulator of polymeric G-repeats). Whereas RepG sRNA is highly conserved, the tlpB G-repeat length varies among diverse H. pylori strains, resulting in strain-specific RepG-mediated tlpB regulation. Based on modification of the G-repeat length within one strain, we demonstrate that the G-repeat length determines posttranscriptional regulation and can mediate both repression and activation of tlpB through RepG. In vitro translation assays show that this regulation occurs at the translational level and that RepG influences tlpB translation dependent on the G-repeat length. In contrast to the digital ON-OFF switches through frame-shift mutations within coding sequences, such modulation of posttranscriptional regulation allows for a gradual control of gene expression. This connection to sRNA-mediated posttranscriptional regulation might also apply to other genes with SSRs, which could be targeting sites of cis- or trans-encoded sRNAs, and thereby could facilitate host adaptation through sRNA-mediated fine-tuning of virulence gene expression.
    Keywords: Homopolymeric Repeat ; Noncoding RNA ; Gene Expression Regulation, Bacterial ; RNA Processing, Post-Transcriptional ; Repetitive Sequences, Nucleic Acid ; Chemotaxis -- Genetics ; Helicobacter Pylori -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 6
    Language: English
    In: Nature, March 11, 2010, Vol.463(7286), p.250(6)
    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 e-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.
    Keywords: Antisense Rna -- Analysis ; Gene Expression -- Analysis ; Transcription (Genetics) -- Analysis ; Helicobacter Pylori -- Genetic Aspects ; Helicobacter Pylori -- Physiological Aspects
    ISSN: 0028-0836
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  • 7
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 01 February 2011, Vol.108(5), pp.2124-9
    Description: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.
    Keywords: Transcription, Genetic ; Synechocystis -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 8
    In: Molecular Microbiology, September 2011, Vol.81(5), pp.1144-1165
    Description: GcvB is one of the most highly conserved Hfq‐associated small RNAs in Gram‐negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB‐mediated regulation in , we combined a genome‐wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild‐type versus mutant sRNA variants revealed that GcvB governs a large post‐transcriptional regulon, impacting ∼1% of all genes via its conserved G/U‐rich domain R1. Complementary predictions of C/A‐rich binding sites in mRNAs and reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base‐pairing. This novel ability of GcvB is focused upon the one target that could feedback‐regulate the glycine‐responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.
    Keywords: Glycine -- Physiological Aspects ; Genetic Research -- Physiological Aspects ; Genomics -- Physiological Aspects ; Messenger Rna -- Physiological Aspects;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 9
    Language: English
    In: Journal of bacteriology, November 2012, Vol.194(21), pp.5864-74
    Description: Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (Δhfq) of S. maltophilia and compared the behaviors of wild-type and Δhfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Δhfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and Δhfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genome-wide map of transcriptional start sites in S. maltophilia.
    Keywords: Host Factor 1 Protein -- Metabolism ; Molecular Chaperones -- Metabolism ; RNA, Bacterial -- Metabolism ; Stenotrophomonas Maltophilia -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 10
    Language: English
    In: Molecular microbiology, June 2011, Vol.80(6), pp.1479-95
    Description: The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a-mediated formation of singlet oxygen ((1)O(2)). Exposure to (1)O(2) induces the alternative sigma factors RpoE and RpoH(II) which then promote transcription of photooxidative stress-related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base-pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to (1)O(2). The higher (1)O(2) sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoH(II)-dependent genes. We used co-immunoprecipitation of FLAG-tagged Hfq combined with RNA-seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, 〉 70% of the Hfq-bound sRNAs are (1)O(2)-affected. Proteomics analysis of the Hfq-deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.
    Keywords: Gene Expression Regulation, Bacterial ; Oxidative Stress ; Protein Binding ; Bacterial Proteins -- Metabolism ; Host Factor 1 Protein -- Metabolism ; Rhodobacter Sphaeroides -- Metabolism
    ISSN: 0950382X
    E-ISSN: 1365-2958
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