TY - THES A1 - Fröhlich, Kathrin T1 - Assigning functions to Hfq-dependent small RNAs in the model pathogen Salmonella Typhimurium T1 - Funktionelle Charakterisierung Hfq-abhängiger kleiner RNAs im Modellpathogen Salmonella Typhimurium N2 - Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators’ individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5’ end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader. N2 - Die bakterielle Genexpression wird unter anderem maßgeblich von nicht-kodierenden RNAs bestimmt. Kleine regulatorische RNAs (sRNAs) sind eine bezüglich Größe und Struktur heterogene Gruppe von Riboregulatoren, die ihre in cis oder in trans-kodierten Zielgene mittels direkter Basenpaarungen kontrollieren. Während der Großteil der sRNAs reprimierend wirkt, konnte für einige RNAs gezeigt werden, dass sie die Expression ihres Zieltranskripts verstärken. Ein wichtiger Kofaktor für die regulatorische Funktion der sRNAs ist das RNA-Chaperon Hfq, welches sowohl die Umfaltung intramolekularer Sekundärstrukturen ermöglicht, als auch die Ausbildung von Basenpaarungen zwischen komplementären RNA-Sequenzen steuert. Zusätzlich schützt Hfq nicht-gepaarte RNAs vor dem Abbau durch Ribonukleasen, und trägt damit zur Stabilität der Moleküle bei. Durch Ko-Immunopräzipitation mit Hfq sowie in weiteren experimentellen als auch bioinformatischen Studien konnten im letzten Jahrzehnt in diversen Enterobakterien, wie z.B. auch Escherichia coli und Salmonellae, mehr als 150 verschiedene sRNAs bestimmt werden. Von so genannten "core sRNAs" (Kern-sRNAs) wird aufgrund ihres hohen Grades an Konservierung in unterschiedlichen Spezies angenommen, dass sie zentrale Funktionen erfüllen. Etwa 25 core sRNAs agieren unter verschiedenen Umweltbedingungen als Regulatoren. Ihre exakte biologische Rolle, sowie ihre Funktionsweise sind jedoch größtenteils noch unbekannt. In der vorliegenden Arbeit wurden die beiden konservierten, Hfq-abhängigen sRNAs, SdsR und RydC, im Modellpathogen Salmonella Typhimurium charakterisiert. SdsR war als eine der abundantesten sRNAs der stationären Phase in E. coli beschrieben worden. Durch Auswertung der Konservierungsmuster der sdsR Promotorsequenz sowie klassische genetische Analyse konnte SdsR als erste sRNA unter direkter Kontrolle des alternativen σ Faktors σS bestimmt werden. In Salmonella führt die Überexpression von SdsR zur Reprimierung des Membranporins OmpD, und die Bindestelle von SdsR auf dem ompD Transkript wurde mittels einer auf 3'-RACE basierenden Methode ermittelt. Obwohl die Expression von ompD auf post-transkriptionaler Ebene von drei weiteren sRNAs kontrolliert wird, konnte eine spezische Regulation des Porins durch SdsR während Aminosäure-Hungerung gezeigt werden. Auch RydC, die zweite in dieser Studie analysierte sRNA, wurde zunächst in E. coli beschrieben und ist aber auch in weiteren γ-Proteobakterien konserviert. Interessanterweise enthält die Sekundärstruktur dieser Hfq-abhängigen sRNA einen Pseudoknoten, während das 5'-Ende ungepaart ist. Die Zielgene von RydC wurden mittels einer Transkriptomanalyse bestimmt, in der die Änderung der Häufigkeitsverteilung aller mRNAs nach kurzzeitiger Überexpression der sRNA auf Microarrays untersucht wurde. RydC bewirkte die spezifische Aktivierung des längeren von insgesamt zwei Versionen der cfa mRNA, die für eine Cyclopropan-fettsäuresynthase kodiert, ein Enzym das zur Modifikation von Phospholipiden dient. Eine Basenpaarung über das freie 5'-Ende der sRNA RydC führt zur Aktivierung der cfa-Expression, und maskiert eine Erkennungssequenz der Endoribonuklease, RNase E, innerhalb des Transkripts. KW - Small RNA KW - Genexpression KW - Hfq KW - Small RNA KW - Hfq KW - Salmonella KW - Salmonella typhimurium Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85488 ER - TY - JOUR A1 - Papenfort, Kai A1 - Vogel, Jörg T1 - Small RNA functions in carbon metabolism and virulence of enteric pathogens JF - Frontiers in Cellular and Infection Microbiology N2 - Enteric pathogens often cycle between virulent and saprophytic lifestyles. To endure these frequent changes in nutrient availability and composition bacteria possess an arsenal of regulatory and metabolic genes allowing rapid adaptation and high flexibility. While numerous proteins have been characterized with regard to metabolic control in pathogenic bacteria, small non-coding RNAs have emerged as additional regulators of metabolism. Recent advances in sequencing technology have vastly increased the number of candidate regulatory RNAs and several of them have been found to act at the interface of bacterial metabolism and virulence factor expression. Importantly, studying these riboregulators has not only provided insight into their metabolic control functions but also revealed new mechanisms of post-transcriptional gene control. This review will focus on the recent advances in this area of host-microbe interaction and discuss how regulatory small RNAs may help coordinate metabolism and virulence of enteric pathogens. KW - sRNA KW - carbon metabolism KW - Hfq KW - CsrA KW - virulence Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197520 SN - 2235-2988 VL - 4 IS - 91 ER - TY - JOUR A1 - Dimastrogiovanni, Daniela A1 - Fröhlich, Kathrin S. A1 - Bandyra, Katarzyna J. A1 - Bruce, Heather A. A1 - Hohensee, Susann A1 - Vogel, Jörg A1 - Luisi, Ben F. T1 - Recognition of the small regulatory RNA RydC by the bacterial Hfq protein JF - eLife N2 - Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target. KW - Hfq KW - small RNA KW - natively unstructured protein KW - protein-RNA recognition KW - gene regulation KW - Escherichia coli-Hfq KW - SM-like protein KW - messenger-RNA KW - chaperone Hfq KW - target recognition KW - noncoding RNAs KW - interaction surfaces KW - crystal-structures KW - soluble-RNAs KW - C-Terminus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114191 SN - 2050-084X VL - 3 IS - e05375 ER - TY - JOUR A1 - Westermann, Alexander J. A1 - Venturini, Elisa A1 - Sellin, Mikael E. A1 - Förstner, Konrad U. A1 - Hardt, Wolf-Dietrich A1 - Vogel, Jörg T1 - The major RNA-binding protein ProQ impacts virulence gene expression in Salmonella enterica serovar Typhimurium JF - mBio N2 - FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. KW - Hfq KW - noncoding RNA KW - ProQ KW - RNA-seq KW - bacterial pathogen KW - posttranscriptional control Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177722 VL - 10 IS - 1 ER -