Functional characterization of bacterial sRNAs involved in stress responses and quorum sensing of bacterial pathogens

Pathogenic bacteria such as Vibrio cholerae, the causative agent of cholera disease, thrive in host and non-host environments. This lifestyle requires constant monitoring of environmental signals to mediate adaptation through changes in gene expression. Furthermore, V. cholerae employs quorum sensing systems to synchronize group behaviors, such as biofilm formation and virulence gene expression. Both processes frequently involve regulation of gene expression by small regulatory RNAs (sRNAs). Bacterial sRNAs base-pair to trans encoded target mRNAs to alter their stability or translation. Recent studies in V. cholerae identified 107 putative sRNAs with yet undescribed functions. The present work aimed to characterize candidate sRNAs with respect to their involvement in stress responses and quorum sensing. Specifically, a bioinformatical approach aiming to identify binding sites of the envelope stress related, alternative sigma factor σE, identified the MicV sRNA as a member of the σE regulon. The σE stress response of V. cholerae involves regulation by another sRNA, VrrA. Expression of both sRNAs is activated under high cell density conditions or by treatment with membrane damaging agents. Global transcriptome analyses revealed that both sRNAs act together to control outer membrane protein expression, and to maintain envelope homeostasis under membrane damaging conditions. We pinpoint collective functions of both sRNAs to the presence of a conserved base-pairing domain in both sRNAs. Finally, laboratory selection experiments employing a library of synthetic sRNAs revealed that regulation of a single porin is sufficient to mediate stress relief. The third sRNA studied in this thesis, VadR, was identified using a genetic screen to assess sRNAs affecting cell curvature of V. cholerae. Analyses of the vadR promoter revealed that vadR expression is activated in response to cell wall damage by the VxrAB two component system. Transcriptome analyses revealed that VadR regulates a large gene cluster responsible for synthesis of the biofilm matrix, and biofilm formation was inhibited in cells overexpressing vadR. Additionally, we found that VadR regulates cell curvature by inhibiting translation of crvA, encoding the major curvature determinant of V. cholerae. Finally, we pinpoint regulation of crvA by VadR to be critical to mediate resistance to cell wall damage. Expression of the fourth sRNA analyzed in this study, VqmR, is controlled by quorum sensing system, involving the autoinducer DPO. Analyses aiming to identify additional mRNA targets for the VqmR sRNA found five additional targets, including the low cell density master regulator aphA. VqmR base-pairs to aphA using an unusual site located in the rho-independent terminator of the sRNA. Regulation of aphA by VqmR resulted in reduced virulence gene expression. Finally, the present work found that the interplay of all three quorum sensing systems is required to achieve a full quorum sensing response.Bakterielle Pathogene wie Vibrio cholerae, der Erreger der Cholera, finden sich in der Umwelt und in Wirtsorganismen. Dieser Lebenszyklus setzt ein konstantes Überwachen von Umweltsignalen voraus, um sich durch entsprechende Genexpression anzupassen. Zudem verwendet V. cholerae bakterielle Kommunikationssysteme um Gruppenverhalten, wie das Bilden von Biofilmen und die Expression von Virulenzgenen, in der Population zu synchronisieren. Diese beiden Prozesse beinhalten häufig die Regulation der Genexpression durch kleine regulatorische RNAs (sRNAs). Bakterielle sRNAs interagieren über Basen-paarungen mit trans-kodierten mRNAs, um deren Stabilität oder Translation zu regulieren. Jüngste Studien haben 107 potenzielle sRNAs, mit bisher ungeklärten Funktionen, in V. cholerae identifiziert. Ziel der vorliegenden Arbeit war es diese sRNAs, unter Berücksichtigung ihrer Beteiligung an Stressantworten und bakterieller Kommunikation, zu charakterisieren. Im Einzelnen wurde ein bioinformatischer Ansatz verwendet, um die Bindestellen des Membranstress-Sigmafaktors σE zu finden und dabei wurde die sRNA MicV als Teil des σE-Regulons identifiziert. Die σE Stressantwort in V. cholerae beinhaltet Regulation durch eine weitere sRNA, VrrA. Die Expression beider sRNAs wird bei hohen Zelldichten, oder durch Behandlung mit membranschädigenden Substanzen, aktiviert. Transkriptomanalysen zeigten das beide sRNAs gemeinsam die Expression von Proteinen der äußeren Membran regulieren, um die Membranhomeostase unter Membranstressbedingungen zu gewährleisten. Die überlappenden Funktionen beider sRNAs begründen sich durch das Vorhandensein einer konservierten Domäne zur Basenpaarung. Abschließend zeigten wir durch Selektionsexperimente mit einer Sammlung von synthetischen sRNAs, dass die Regulation eines einzigen Porins ausreicht, um Membranstress zu lindern. Die dritte sRNA in dieser Studie, VadR, wurde durch einen genetischen Screen für sRNAs die die Zellkrümmung von V. cholerae beeinflussen gefunden. Analysen des vadR Promotors zeigten das die Expression von vadR bei Zellwandstress durch das VxrAB Zwei-komponentensystem aktiviert wird. Transkriptomanalysen zeigten das VadR ein großes Gencluster, verantwortlich für die Synthese der Biofilm Matrix, reguliert. Demzufolge war die Bildung von Biofilmen in Zellen die vadR überexprimieren inhibiert. Zusätzlich regulierte VadR durch Inhibieren der Translation der crvA mRNA, welche für eine wichtige Zellkrümmungs-determinante kodiert, die Zellkrümmung. Letztendlich zeigten wir das die Regulation von crvA durch VadR entscheidend für die Resistenz gegen Zellwandstress ist. Die Expression der vierten sRNA in dieser Studie, VqmR, wird durch ein bakterielles Kommunikationssystem und den Autoinducer DPO reguliert. Durch Analysen, die darauf abzielten neue Ziel-mRNAs der VqmR sRNA zu identifizieren, konnten fünf weitere Ziel-mRNAs identifiziert werden, welche unter anderem, für den Hauptregulator bei niedriger Zelldichte, AphA, kodieren. Die Basenpaarung zwischen VqmR und aphA ist ungewöhnlich, und benötigte eine Region im Rho-unabhängigen Terminator der sRNA. Die Regulation von aphA durch VqmR resultierte in einer Reduktion der Virulenzgenexpression. Schlussendlich konnte die vorliegende Arbeit zudem zeigen das drei bakterielle Kommunikationssysteme zusammenwirken, um ihre volle Wirkung zu entfalten

RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae

Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae

Vibrio cholerae biofilm dispersal regulator causes cell release from matrix through type IV pilus retraction

The extracellular matrix is a defining feature of bacterial biofilms and provides structural stability to the community by binding cells to the surface and to each other. Transitions between bacterial biofilm initiation, growth, and dispersion require different regulatory programs, all of which result in modifications to the extracellular matrix composition, abundance, or functionality. However, the mechanisms by which individual cells in biofilms disengage from the matrix to enable their departure during biofilm dispersal are unclear. Here, we investigated active biofilm dispersal of Vibrio cholerae during nutrient starvation, resulting in the discovery of the conserved Vibrio biofilm dispersal regulator VbdR. We show that VbdR triggers biofilm dispersal by controlling cellular release from the biofilm matrix, which is achieved by inducing the retraction of the mannose-sensitive hemagglutinin (MSHA) type IV pili and the expression of a matrix protease IvaP. We further show that MSHA pili have numerous binding partners in the matrix and that the joint effect of MSHA pilus retraction and IvaP activity is necessary and sufficient for causing biofilm dispersal. These results highlight the crucial role of type IV pilus dynamics during biofilm dispersal and provide a new target for controlling V. cholerae biofilm abundance through the induction and manipulation of biofilm dispersal

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus

The alphaproteobacteriumCaulobacter crescentusthrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level inC. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of thehfqgene is associated with a severe loss of fitness inC. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq inC. crescentusand demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of theC. crescentuscell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotrophCaulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy inC. crescentushas been lacking. Here, we identified sRNAs and mRNAs associated with Hfqin vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters inC. crescentus