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  • 1
    Language: English
    In: PLoS Genetics, 2012, Vol.8(6), p.e1002782
    Description: RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus . However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo . Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. ; Control of mRNA stability is crucial for bacteria to survive and rapidly adapt to environmental changes and stress conditions. The molecular players and the degradation pathways involved in these adaptive processes are poorly understood in . The universally conserved double-strand-specific endoribonuclease III (RNase III) in is known to repress the synthesis of several virulence factors and was recently implicated in genome-wide mRNA processing mediated by antisense transcripts. We present here the first global map of direct RNase III targets in . Deep sequencing was used to identify RNAs associated with epitope-tagged wild-type RNase III and two catalytically impaired but binding-competent mutant proteins . Experimental validation revealed an unexpected variety of structured RNA transcripts as novel RNase III substrates. In addition to rRNA operon maturation, autoregulation, degradation of structured RNAs, and antisense regulation, we propose novel mechanisms by which RNase III increases mRNA translation. Overall, this study shows that RNase III has a broad function in gene regulation of . We can now address more specifically the roles of this universally conserved enzyme in gene regulation in response to stress and during host infection.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 2
    Language: English
    In: 2013, Vol.9(5), p.e1003495
    Description: Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter , which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level. ; Many species have evolved into diverse strains with phenotypic and genotypic variations that facilitate adaptation to different ecological niches and, in the case of pathogens, to different hosts. Whereas comparison of genome sequences reveals differences and similarities among strains, the consequences of genomic variations can be tracked by studying the functional output from the genome. RNA sequencing has been revolutionizing transcriptome analyses of both pro- and eukaryotes. However, the bioinformatics-based analysis is still lagging behind, and transcriptome features are often manually annotated, which is laborious and time-consuming. This is even more compounded for the analyses of multiple strains. Here we compared the primary transcriptomes of four isolates of , the leading cause of bacterial gastroenteritis in humans, and provide genome-wide transcriptional start site (TSS) maps using a novel automated annotation method. Our comparative RNA–seq showed that most TSS are conserved in multiple strains, but we also observed SNP–dependent promoter usage. Furthermore, we identified a novel minimal RNA–based CRISPR immune system as well as strain-specific small RNA repertoires. Our automated, comparative TSS annotation will facilitate and improve transcriptome annotation for a wider range of organisms and provides insights into the contribution of transcriptome differences to phenotypic variation among closely related species.
    Keywords: Research Article ; Biology
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 3
    Language: English
    In: 2015, Vol.11(3), p.e1005087
    Description: One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y . pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans -acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated ‘acetate switch’, which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles. ; Many bacterial pathogens cycle between environmental sources and mammalian hosts. Adaptation to the different natural habitats and host niches is achieved through complex regulatory networks which adjust synthesis of the large repertoire of crucial virulence factors and fitness determinants. To uncover underlying control circuits, we determined the first in-depth single-nucleotide resolution transcriptome of . This revealed important novel genetic information, such as global locations of transcriptional start sites, non-coding RNAs, potential riboswitches and provided a set of virulence-relevant expression profiles, which constitute a valuable tool for the research community. The analysis further uncovered a temperature-induced global reprogramming of central metabolic functions, likely to support intestinal colonization of the pathogen. This is accompanied by a major reorganization of the CRP regulon, which involves a multitude of regulatory RNAs. The primary consequence is a fine-tuned, coordinated control of metabolism and virulence through a plethora of environmentally controlled regulatory RNAs allowing rapid adaptation and high flexibility during life-style changes.
    Keywords: Research Article
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 4
    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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  • 5
    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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  • 6
    Language: English
    In: PLoS ONE, 2011, Vol.6(5), p.e19361
    Description: Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA . However, over 16% of meningococcal carriage isolates lack ctrA , rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC , is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC -based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C t ) value of 35, with 100% average reaction efficiency and an average R 2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC -positive, with a range of average C t values from 13.0 to 29.5. The mean sodC C t value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA -negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.
    Keywords: Research Article ; Biology ; Medicine ; Genetics And Genomics ; Infectious Diseases ; Microbiology ; Biotechnology ; Biochemistry
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: PLoS Biology, 2004, Vol.2(12), p.e423
    Description: Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34 + cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors. ; A primate model of gene transfer into hematopoietic stem cells demonstrated MLV integration around transcription start sites whereas SIV integrated into gene-dense regions, indicating distinct safety implications for each.
    Keywords: Research Article ; Genetics And Genomics ; Virology ; Viruses ; Primates
    ISSN: 1544-9173
    E-ISSN: 1545-7885
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