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Material and Methods Genome-wide RNA-seq analysis of quorum sensing-dependent regulons in the plant-associated Burkholderia glumae strain PG1 Dissertation with the aim of achieving a doctoral degree At the Faculty of Mathematics, Informatics and Natural Sciences Department of Biology of the Universität of Hamburg Submitted by Rong Gao From Jiangsu, China 2015 in Hamburg Day of oral defense: 15.06.2015 The following evaluators recommend the admission of the dissertation: Professor Dr. Wolfgang Streit Professor Dr. Wilhelm Schäfer Abstract Burkholderia glumae PG1 is a soil-associated motile plant pathogenic bacterium possessing a cell density-dependent regulatory mechanism called quorum sensing (QS). Its genome encodes three distinct putative autoinducer-1 synthase genes, here designated bgaI1-3, which are responsible for synthesizing the N-acyl-homoserine lactones (AHL). In this study, all three bgaI genes were characterized as the functional AHL synthase genes using AHL bioreporters A. tumefaciens NTL4 and C. violaceum CV026. To elucidate functions of these QS systems, B. glumae PG1 ΔbgaI1-3 mutants were generated through allelic exchange. The deletion of each bgaI gene resulted in the strong reduction in motility, extracellular lipolytic activity, plant maceration and rice pathogenicity and colony morphology variation. To further identify the QS-regulated genes, the genome-wide transcriptome analysis of three ΔbgaI mutants vs. the parental strain was performed in the transition from exponential to stationary growth phase. In comparison with the parental strain, 481 of these genes were regulated in mutant ΔbgaI1, 213 in ΔbgaI2, and 367 in ΔbgaI3. In total, a set of 745 QS-regulated genes was identified. Among them, the majority of the QS-regulated genes linked to metabolic activities and the most pronounced regulation was observed for the genes involved in rhamnolipid, Flp pili, lipase, flagella, type VI secretion system and genes linked to a CRIPSR-Cas system. Notably, a cross-species analysis of QS-regulated genes revealed that B. glumae PG1 has more similarities with B. thailandensis than with the other two Bptm members, B. pseudomallei and B. mallei in lifestyle. Acknowledgement First and foremost I would like to thank my supervisor Prof. Dr. Wolfgang Streit who gave me this opportunity as a PhD student in his lab and provided me the challenging and interesting research projects and invaluable guidance for my research over the past five years and I am also grateful for his help for proofreading my thesis. I would like to thank Prof. Dr. Wilhelm Schäfer for agreeing to be my second superviser and Jun. Prof. Dr. Mirjam Perner for being a language proofreader of my thesis. I would like to thank Sabine Zumbraegel for supervising me laboratory techniques and Dr. Claudia Katharina Hornung for sharing her microbiological knowledge with me, Nicolas Rychlik for providing the vector pNPTS138-R6KT, Dr. Jennifer Chow for providing the control strain of the lipase test and Angela Jordan for her input in the TLC assay, Elke Woelken and PD Dr. Eva Spieck for their help in the TEM expriments, Marion Klötzl for the assistance with growing the rice plant; Dr. Andreas Knapp for the bioinformatics analysis of CRISPR and T6SS. I would like to thank Dr. Yuchen Han and Jiajia Tang for proofreading my thesis and their scientific suggestions. I would like to thank all people who contributed to the transcriptome publication based on this work: Dr. Dagmar Krysciak for proofreading my publication, Christian Utpatel for producing my transcriptome circular maps, Dr. Christel Schmeisser for her scientic suggestions of mutation technique, Prof. Rolf Daniel and Dr. Sonja Voget for their RNA-seq service from the Göttingen Genomics Laboratory of Georg-August-Universität Göttingen, and Dr. Andreas Knapp and Prof. Karl-Erich Jaeger for their assistance with the qPCR experiments from the institute of molecular enzymtechnology of Heinrich Heine University Düsseldorf, Prof. Dr. Wolfgang Streit. I would like to thank all my colleagues: Andreas, Angela, Arek, Andrea, Boris, Birhanu, Christel, Christian, Christiane, Dagmar, Dominik, Ebrahim, Gabi, Gesche, Han, Hanae, Henning, Ines, Janine, Jessica, Jenny C., Jenny H., Joana, Julia, Jeremy, Katja, Maria, Mariita, Martina, Mirjam, Moritz, Nele, Nico R., Nico A., Norwin, Patrick, Philipp, Regina, Sabine K., Sabine Z., Sascha, Steffi, Simon, Simone, Tanja, Ulrich, Uschi and Wenke. Thanks for your help and supports. I would like to thank my country and Chinese Scholarship Council for subsidizing me to study in Germany. Special thanks to my parents and my wife for their unselfish supports and love. At last, many many many thanks for the people who helped me and supported me!!! The results of this thesis were published as follows: Gao R, Krysciak D, Utpatel C, Knapp A, Schmeisser C, Daniel R, Voget S, Jaeger KE, Streit WR. 2015. Genome-wide RNA sequencing analysis of quorum sensing-controlled regulons in the plant-associated Burkholderia glumae strain PG1. Appl Environ Microbiol., accepted. Knapp A, Voget S, Gao R, Zaburannyi N, Poehlein A, Krysciak D, Breuer M, Hauer B, Streit WR, Müller R, Daniel R, Jaeger KE. 2015. Biotechnological potential of the lipase producing strain Burkholderia glumae PG1. Appl Microbiol Biotechnol., Submitted. Table of contents VII Table of Contents I. Introduction ......................................................................................................................................... 1 1. Quorum sensing ................................................................................................................................ 1 1.1. Classification of QS systems ........................................................................................................ 1 1.1.1. AHL-based QS systems in Gram-negative bacteria .............................................................. 2 1.1.2 Oligopeptide-mediated QS systems in Gram-positive bacteria.............................................. 4 1.1.3. Autoinducer-2-based QS systems in Gram-negative and Gram-positive bacteria................ 7 1.1.4. Additional QS systems ........................................................................................................... 8 1.2. N-acyl-homoserine lactone (AHL) ................................................................................................ 9 1.2.1. Structure of AHL .................................................................................................................... 9 1.2.2. Synthesis process of AHL.................................................................................................... 10 2. The genus Burkholderia and its QS systems ............................................................................... 12 2.1 The genus Burkholderia .............................................................................................................. 12 2.1.1. Taxonomy of the genus Burkholderia .................................................................................. 13 2.1.2 Bukholderia glumae .............................................................................................................. 15 2.2. QS in the genus Burkholderia .................................................................................................... 16 2.2.1. AHL-based QS in Burkholderia ........................................................................................... 16 2.2.2 QS in B. glumae ................................................................................................................... 18 3. Intentions of this research ............................................................................................................. 19 II. Material and Methods ...................................................................................................................... 21 1. Bacteria strains, constructs, vectors and primers ...................................................................... 21 2. Culture media, supplements and solutions .................................................................................. 24 2.1. Culture media ............................................................................................................................. 24 2.1.1. Lysogeny broth (LB) medium .............................................................................................. 24 2.1.2. AT medium ........................................................................................................................... 24 2.2. Supplements ............................................................................................................................... 25 3. Cultivation of bacteria ..................................................................................................................... 25 3.1. Cultivation of B.glumae strains ................................................................................................... 25 3.2. Cultivation of Escherichia coli strains ......................................................................................... 26 3.3. Cultivation of Agrobacterium tumefaciens NTL4 ........................................................................ 26 3.4. Cultivation of Chromobacteium violaceum CV026 ..................................................................... 26 3.5. Maintenance of bacteria strains ................................................................................................. 26 Table of contents VIII 3.6.
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