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Material and Methods Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2011 Investigations of the Quorum sensing circuitry in ”Burkholderia cenocepacia” H111 Inhülsen, Silja Abstract: Burkholderia cenocepacia ist ein Gram-negatives Bakterium, welches ubiquitär in der Umwelt vorkommt. Vor drei Jahrzehnten wurde B. cenocepacia als verursachender Erreger von schwerwiegen- den Lungenentzündungen in Patienten mit zystischer Fibrose (CF) identifiziert. Die Infektionen waren von einer charakteristischen Bakteriämie gekennzeichnet und führten zum frühen Tod der CF-Kranken. Seither wurde B. cenocepacia als ein zunehmend problematisches opportunistisches Pathogen in immun- supprimierten Individuen wahrgenommen, welches die Notwendigkeit für die weitere Erforschung und Aufklärung von Virulenzfaktoren von B. cenocepacia begründet. Es wurde aufgezeigt, dass die Aus- bildung von pathogenen Eigenschaften in B. cenocepacia entscheidend durch „Quorum sensing“ (QS) beeinflusst wird, welches vor allem duch das CepIR-Regulationssystem vermittelt wird, das die Expres- sion bestimmter Gene in Abhängigkeit von der Populationsdichte kontrolliert. In der vorliegenden Arbeit wurde das QS-Regulon von B. cenocepacia H111 entschlüsselt und detalliert analysiert. Die Identifizierung von CepIR-regulierten Funktionen gelang dabei durch die Implementierung einer kombinierten transkrip- tionellen, proteomischen und phänotypischen Herangehensweise. Die Ergebnisse dieser Analyse liefern neue Erkenntnisse über im Vorfeld identifizierte QS-regulierte Phänotypen einschliesslich der Bildung von Biofilmen und der Pathogenität, und offenbaren wichtige Einblicke in den QS- Regulationskreislauf von B. cenocepacia H111. Im zweiten Teil der Arbeit wurden die Produkte von drei QS-aktivierten Gen-Loci, welche in dieser Ausarbeitung identifiziert wurden, funktionell charakterisiert. Diese beinhalteten den „verwaisten“ LuxR-homologen Transkriptionsregulator CepR2, die Lektine BclACB und BCAM1869, welches von einem Gen kodiert wird, das in der intergenetischen Region zwischen den cepR und cepI Genen lokalisiert ist. Die Ergebnisse dieser Arbeit zeigten, dass CepR2 ein AHL-unabhängiger Tran- skriptionsregulator ist. Desweiteren wurde nachgewiesen, dass CepR2 die Expression der Siderophore Pyochelin stimuliert und dabei als Downstream- Regulator im QS-Netzwerk agiert. In der vorliegen- den Arbeit wurde die subzelluläre Lokalisierung des BclB Lektins determiniert und die Bedeutung der BclACB-Proteine bei der strukturellen Entwicklung von Biofilmen in H111 untersucht. Zudem wurde ein neuer Regulator, BCAM1869, identifiziert, welcher die Produktion von AHL-Molekülen in B. cenocepacia H111 hemmt. Hinzukommend scheint BCAM1869 die Aktivität von LuxR-ähnlichen Transkriptionsreg- ulatoren zu beeinträchtigen, einschliesslich des Vibrio fischeri LuxR-Regulators. Die Ergebnisse dieser Arbeit weisen auch darauf hin, dass BCAM1869 die Aktivität von CepR2 in B. cenocepacia H111 posi- tiv beeinflusst. Summary Burkholderia cenocepacia is a Gram-negative bacterium which is ubiquitously found in the environment. Three decades ago, B. cenocepacia was identified as the causative agent of severe pulmonary infections in patients with cystic fibrosis (CF). The disease was accompanied by achar- acteristic bacteraemia that resulted in the early death of CF individuals. Since then, B. cenocepacia has emerged as a problematic opportunistic pathogen in patients with immunodeficiency disorders demon- strating the need for further exploration and elucidation of factors which contribute to B. cenocepacia virulence. It has become clear that the expression of pathogenic traits in B. cenocepacia is at least partly controlled by quorum sensing (QS), a cell density-dependent regulatory process. In the present thesis, the QS regulon of B. cenocepacia H111, which is comprised by the cepIR genes, was mapped and studied in detail. CepIR-regulated functions were identified by a combined transcriptomic, proteomic and phenotypic approach. The results of this analysis provided new insights into the molecular mecha- nisms of previously identified QS-regulated phenotypes, including biofilm formation, and pathogenicity, and revealed novel information of the complex QS circuitry operating in B. cenocepacia H111. In the second part of this thesis, the products of three QS-activated loci, which were identified in our mapping study, were further characterized. These included the orphan LuxR homologous transcriptional regulator CepR2, the BclACB lectins, and BCAM1869, which is encoded by a gene located within the intergenetic region of the cepR and cepI genes. The results of this work identified CepR2 as an AHL-independent transcriptional regulator. Moreover, CepR2 was shown to stimulate expression of the siderophore pyoche- lin, while acting as a downstream regulator of the H111 QS network. In the present thesis, the subcellular localization of lectin BclB was determined, and the importance of the BclACB proteins for the structural development of H111 biofilms was investigated. The results obtained suggest that BCAM1869 fine-tunes QS regulation: BCAM1869 represses the producton of AHL signal molecules in B. cenocepacia H111. Furthermore, BCAM1869 seems to interfere with the activity of LuxR-like transcriptional regulators, in- cluding the Vibrio fischeri LuxR protein. The results of this work also suggest that BCAM1869 positively influences the activity of CepR2 in B. cenocepacia H111. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-59362 Dissertation Published Version Originally published at: Inhülsen, Silja. Investigations of the Quorum sensing circuitry in ”Burkholderia cenocepacia” H111. 2011, University of Zurich, Faculty of Science. 2 Investigations of the Quorum Sensing Circuitry in Burkholderia cenocepacia H111 Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Silja Inhülsen aus Hannover, Deutschland Promotionskomitee Prof. Dr. Leo Eberl (Vorsitz) Prof. Dr. Kathrin Riedel Prof. Dr. Jakob Pernthaler Zürich, 2011 Investigations of the Quorum Sensing Circuitry in Burkholderia cenocepacia H111 Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Silja Inhülsen aus Hannover, Deutschland Promotionskomitee Prof. Dr. Leo Eberl (Vorsitz) Prof. Dr. Kathrin Riedel Prof. Dr. Jakob Pernthaler Zürich, 2011 Table of Contents List of Figures and Tables ..................................................................................................................... i Zusammenfassung ................................................................................................................................ iii Summary ............................................................................................................................................... iv Abbreviations ......................................................................................................................................... v 1 Introduction ............................................................................................................................ 1 1.1 The genus Burkholderia.............................................................................................................. 1 1.1.1 The Burkholderia cepacia complex (Bcc) .......................................................................... 2 1.1.2 Burkholderia cenocepacia: a problematic opportunistic human pathogen ......................... 4 1.2 Cell-to-cell communication via quorum sensing ........................................................................ 5 1.3 Genomic structure and quorum sensing circuitry in B. cenocepacia H111 ................................ 6 1.4 Goals of this work ...................................................................................................................... 9 2 Material and Methods ......................................................................................................... 11 2.1 Material..................................................................................................................................... 11 2.1.1 Bacterial strains and plasmids ........................................................................................... 11 2.1.2 Media, chemicals and other material ................................................................................ 12 2.1.3 Oligonucleotides ............................................................................................................... 15 2.2 Molecular biological methods .................................................................................................. 16 2.2.1 Cultivation of bacterial strains and growth conditions ..................................................... 16 2.2.2 Manipulation of DNA ....................................................................................................... 16 2.2.2.1 DNA preparation and characterization ...................................................................... 16 2.2.2.2 Southern blot hybridization ....................................................................................... 17 2.2.2.3 Precipitation of DNA and RNA and quality analysis by gel electrophoresis............ 18 2.2.2.4 Recombinant
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