Recent Submissions

  • Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.

    Westbrook, Alexandra; Tang, Xun; Marshall, Ryan; Maxwell, Colin S; Chappell, James; Agrawal, Deepak K; Dunlop, Mary J; Noireaux, Vincent; Beisel, Chase L; Lucks, Julius; Franco, Elisa; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley-Blackwell, 2019-01-13)
    To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modelling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity. This article is protected by copyright. All rights reserved.
  • The Major RNA-Binding Protein ProQ Impacts Virulence Gene Expression in Salmonella enterica Serovar Typhimurium.

    Westermann, Alexander J; Venturini, Elisa; Sellin, Mikael E; Förstner, Konrad U; Hardt, Wolf-Dietrich; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Amercan Society of Microbiology, 2019-01-02)
    FinO domain proteins such as ProQ of the model pathogen
  • Functional analysis of Salmonella Typhi adaptation to survival in water.

    Kingsley, Robert A; Langridge, Gemma; Smith, Sarah E; Makendi, Carine; Fookes, Maria; Wileman, Tom M; El Ghany, Moataz Abd; Keith Turner, A; Dyson, Zoe A; Sridhar, Sushmita; Pickard, Derek; Kay, Sally; Feasey, Nicholas; Wong, Vanessa; Barquist, Lars; Dougan, Gordon; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley-Blackwell, 2018-11-18)
    Contaminated water is a major risk factor associated with the transmission of Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of human typhoid. However, little is known about how this pathogen adapts to living in the aqueous environment. We used transcriptome analysis (RNA-seq) and transposon mutagenesis (TraDIS) to characterize these adaptive changes and identify multiple genes that contribute to survival. Over half of the genes in the S. Typhi genome altered expression level within the first 24 h following transfer from broth culture to water, although relatively few did so in the first 30 min. Genes linked to central metabolism, stress associated with arrested proton motive force and respiratory chain factors changed expression levels. Additionally, motility and chemotaxis genes increased expression, consistent with a scavenging lifestyle. The viaB-associated gene tviC encoding a glcNAc epimerase that is required for Vi polysaccharide biosynthesis was, along with several other genes, shown to contribute to survival in water. Thus, we define regulatory adaptation operating in S. Typhi that facilitates survival in water.
  • The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.

    Liao, Chunyu; Slotkowski, Rebecca A; Achmedov, Tatjana; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-10-12)
    The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.
  • Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

    Tawk, Caroline; Nigro, Giulia; Rodrigues Lopes, Ines; Aguilar, Carmen; Lisowski, Clivia; Mano, Miguel; Sansonetti, Philippe; Vogel, Jörg; Eulalio, Ana; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-11-02)
    While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.
  • A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

    Nolan, Laura M; Whitchurch, Cynthia B; Barquist, Lars; Katrib, Marilyn; Boinett, Christine J; Mayho, Matthew; Goulding, David; Charles, Ian G; Filloux, Alain; Parkhill, Julian; Cain, Amy K; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Microbiology Society, 2018-11-01)
    Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
  • Genome organization and DNA accessibility control antigenic variation in trypanosomes.

    Müller, Laura S M; Cosentino, Raúl O; Förstner, Konrad U; Guizetti, Julien; Wedel, Carolin; Kaplan, Noam; Janzen, Christian J; Arampatzi, Panagiota; Vogel, Jörg; Steinbiss, Sascha; Otto, Thomas D; Saliba, Antoine-Emmanuel; Sebra, Robert P; Siegel, T Nicolai; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
    Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
  • Long Noncoding RNA SSR42 Controls Staphylococcus aureus Alpha-Toxin Transcription in Response to Environmental Stimuli.

    Horn, Jessica; Klepsch, Maximilian; Manger, Michelle; Wolz, Christiane; Rudel, Thomas; Fraunholz, Martin; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-11-15)
    Staphylococcus aureus is a human pathogen causing a variety of diseases by versatile expression of a large set of virulence factors that most prominently features the cytotoxic and hemolytic pore-forming alpha-toxin. Expression of alpha-toxin is regulated by an intricate network of transcription factors. These include two-component systems sensing quorum and environmental signals as well as regulators reacting to the nutritional status of the pathogen. We previously identified the repressor of surface proteins (Rsp) as a virulence regulator. Acute cytotoxicity and hemolysis are strongly decreased in rsp mutants, which are characterized by decreased transcription of toxin genes as well as loss of transcription of a 1,232- nucleotide (nt)-long noncoding RNA (ncRNA), SSR42. Here, we show that SSR42 is the effector of Rsp in transcription regulation of the alpha-toxin gene, hla. SSR42 transcription is enhanced after exposure of S. aureus to subinhibitory concentrations of oxacillin which thus leads to an SSR42-dependent increase in hemolysis. Aside from Rsp, SSR42 transcription is under the control of additional global regulators, such as CodY, AgrA, CcpE, and B, but is positioned upstream of the two-component system SaeRS in the regulatory cascade leading to alpha-toxin production. Thus, alpha-toxin expression depends on two long ncRNAs, SSR42 and RNAIII, which control production of the cytolytic toxin on the transcriptional and translational levels, respectively, with SSR42 as an important regulator of SaeRS-dependent S. aureus toxin production in response to environmental and metabolic signals. IMPORTANCE Staphylococcus aureus is a major cause of life-threatening infections. The bacterium expresses alpha-toxin, a hemolysin and cytotoxin responsible for many of the pathologies of S. aureus. Alpha-toxin production is enhanced by subinhibitory concentrations of antibiotics. Here, we show that this process is dependent on the long noncoding RNA, SSR42. Further, SSR42 itself is regulated by several global regulators, thereby integrating environmental and nutritional signals that modulate hemolysis of the pathogen.
  • Nuclear lncRNA stabilization in the host response to bacterial infection.

    Munschauer, Mathias; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-07-02)
    Long non-coding RNAs (lncRNAs) play important roles in many cellular pathways, but their contribution to the defense of eukaryotic cells against pathogens remains poorly understood. A new study from Imamura et al in The EMBO Journal reports that Salmonella infection in human cells impacts nuclear RNA decay, which in turn drives the accumulation of otherwise unstable nuclear lncRNAs, some of which may have protective effects against this common bacterial pathogen. These unexpected findings demand more efforts to fully decrypt the molecular functions of lncRNAs in innate and adaptive immunity.
  • HHV-6 encoded small non-coding RNAs define an intermediate and early stage in viral reactivation.

    Prusty, Bhupesh K; Gulve, Nitish; Chowdhury, Suvagata Roy; Schuster, Michael; Strempel, Sebastian; Descamps, Vincent; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
    Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions.
  • RNA Structure-A Neglected Puppet Master for the Evolution of Virus and Host Immunity.

    Smyth, Redmond P; Negroni, Matteo; Lever, Andrew M; Mak, Johnson; Kenyon, Julia C; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
    The central dogma of molecular biology describes the flow of genetic information from DNA to protein via an RNA intermediate. For many years, RNA has been considered simply as a messenger relaying information between DNA and proteins. Recent advances in next generation sequencing technology, bioinformatics, and non-coding RNA biology have highlighted the many important roles of RNA in virtually every biological process. Our understanding of RNA biology has been further enriched by a number of significant advances in probing RNA structures. It is now appreciated that many cellular and viral biological processes are highly dependent on specific RNA structures and/or sequences, and such reliance will undoubtedly impact on the evolution of both hosts and viruses. As a contribution to this special issue on host immunity and virus evolution, it is timely to consider how RNA sequences and structures could directly influence the co-evolution between hosts and viruses. In this manuscript, we begin by stating some of the basic principles of RNA structures, followed by describing some of the critical RNA structures in both viruses and hosts. More importantly, we highlight a number of available new tools to predict and to evaluate novel RNA structures, pointing out some of the limitations readers should be aware of in their own analyses.
  • New RNA-seq approaches for the study of bacterial pathogens.

    Saliba, Antoine-Emmanuel; C Santos, Sara; Vogel, Jörg; Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2017-01-01)
    Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself. Dual RNA-seq analyzing pathogen and host simultaneously has revealed roles of noncoding RNAs during infection and enabled the correlation of bacterial gene activity with specific host responses. Single-cell RNA-seq studies have addressed how heterogeneity among individual host cells may determine infection outcomes.
  • Molecular mechanism of mRNA repression in by a ProQ-dependent small RNA.

    Smirnov, Alexandre; Wang, Chuan; Drewry, Lisa L; Vogel, Jörg; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2017-04-13)
    Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 30 end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-a. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-a. Similarities and differences between ProQ- and Hfqmediated regulation will be discussed.
  • ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes.

    Yu, Sung-Huan; Vogel, Jörg; Förstner, Konrad U; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-09-01)
    To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at
  • Small synthetic molecule-stabilized RNA pseudoknot as an activator for -1 ribosomal frameshifting.

    Matsumoto, Saki; Caliskan, Neva; Rodnina, Marina V; Murata, Asako; Nakatani, Kazuhiko; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-08-02)
    Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.
  • The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

    Heidrich, Nadja; Hagmann, Antony; Bauriedl, Saskia; Vogel, Jörg; Schoen, Christoph; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-07-30)
    Neisseria meningitidis, a commensal β-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.
  • Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

    Cain, Amy K; Boinett, Christine J; Barquist, Lars; Dordel, Janina; Fookes, Maria; Mayho, Matthew; Ellington, Matthew J; Goulding, David; Pickard, Derek; Wick, Ryan R; Holt, Kathryn E; Parkhill, Julian; Thomson, Nicholas R; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-06-29)
    Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col
  • CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level.

    El Mouali, Youssef; Gaviria-Cantin, Tania; Sánchez-Romero, María Antonia; Gibert, Marta; Westermann, Alexander J; Vogel, Jörg; Balsalobre, Carlos; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
    Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3'UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3'UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3'UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.
  • Machine learning identifies signatures of host adaptation in the bacterial pathogen Salmonella enterica.

    Wheeler, Nicole E; Gardner, Paul P; Barquist, Lars; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
    Emerging pathogens are a major threat to public health, however understanding how pathogens adapt to new niches remains a challenge. New methods are urgently required to provide functional insights into pathogens from the massive genomic data sets now being generated from routine pathogen surveillance for epidemiological purposes. Here, we measure the burden of atypical mutations in protein coding genes across independently evolved Salmonella enterica lineages, and use these as input to train a random forest classifier to identify strains associated with extraintestinal disease. Members of the species fall along a continuum, from pathovars which cause gastrointestinal infection and low mortality, associated with a broad host-range, to those that cause invasive infection and high mortality, associated with a narrowed host range. Our random forest classifier learned to perfectly discriminate long-established gastrointestinal and invasive serovars of Salmonella. Additionally, it was able to discriminate recently emerged Salmonella Enteritidis and Typhimurium lineages associated with invasive disease in immunocompromised populations in sub-Saharan Africa, and within-host adaptation to invasive infection. We dissect the architecture of the model to identify the genes that were most informative of phenotype, revealing a common theme of degradation of metabolic pathways in extraintestinal lineages. This approach accurately identifies patterns of gene degradation and diversifying selection specific to invasive serovars that have been captured by more labour-intensive investigations, but can be readily scaled to larger analyses.
  • Bacterial RNA Biology on a Genome Scale.

    Hör, Jens; Gorski, Stanislaw A; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-16)
    Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over.

View more