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Small RNA-Controlled Gene Regulatory Networks in Pseudomonas Putida Downloaded from orbit.dtu.dk on: Sep 23, 2021 Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida Bojanovic, Klara Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Bojanovic, K. (2016). Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida. Novo Nordisk Foundation Center for Biosustainability. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida PhD Thesis Klara Bojanovič Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark August 2016 Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida PhD thesis written by Klara Bojanovič Supervisor Katherine S. Long © PhD Thesis 2016 Klara Bojanovič Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Kemitorvet 220, 2800 Kgs. Lyngby Denmark When you tread your way, Always go to the end. In spring, to a flower so sweet, In summer, to a shower of wheat, In autumn, to pantries that glow, In winter, to the lady of snow, In life, to the truth that is thine, Until color leaks into your cheeks. And if you don’t climb the first time, To the top and reap the best crop, Try it once more And over and over again. Ko hodiš, pojdi zmeraj do konca. Spomladi do rožne cvetice, poleti do zrele pšenice, jeseni do polne police, pozimi do snežne kraljice, v knjigi do zadnje vrstice, v življenju do prave resnice, v sebi do rdečice čez eno in drugo lice. A če ne prideš ne prvič, ne drugič do krova in pravega kova poskusi: vnovič in zopet in znova.” – Tone Pavček (Slovenian poet) Preface This thesis is written as a partial fulfillment of the requirements to obtain a PhD degree at the Technical University of Denmark. The work presented in this thesis was carried out from September 2013 to August 2016 at the Novo Nordisk Center for Biosustainability, Technical University of Denmark in Hørsholm. The work was supervised by Associate Professor Katherine S. Long. Funding was provided by the Novo Nordisk Foundation and an ITN grant from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7-People-2012-ITN), under grant agreement No. 317058, Bactory. The thesis was evaluated by Rebecca M. Lennen, Senior researcher at DTU (Denmark); Birgitte Hahr Kallipolilis, Associate Professor at Syddansk Universitet (Denmark); and Professor Claudio Valverde from Universidad Nacional de Quilmes (Argentina). Klara Bojanovič Lyngby, August 2016 i Acknowledgements During my PhD journey I encountered a whole rainbow of feelings and emotions and nothing could be done without the help and support of many people. I would like to thank my supervisor Katherine S. Long for her time, interest and encouragement throughout this project and the willingness to wait for me for some months when the rest of the group has already started. A thank you goes to Søren Molin and Mette Munk for all Bactory-related activities and opportunities, I am happy to have been a part of it. I would also like to thank Alex T. Nielsen for always having a positive attitude at CfB and for the scientific advices given at any time. I also had the opportunity to work in Novozymes and I am grateful to Anne Breüner and Allan Kent Nielsen for having me there and for their guidance and enthusiasm. Isotta you have been a special character in this chapter. When we were lost in the data and the terminal commands, feeling ‘different’ at the Czech course, under the rain in Mexico with your head broken, looking puzzled at the results or at our cultures growing ‘weird’ – I am so happy we were always shoulder to shoulder. Thank you for always being there and letting me bother you. The rest of my group – thank you Mikkel for keeping up with me and patiently answering countless questions about R, terminal commands, Danish translations, etc., as well as Xioachen for going for dinners and for sometimes pissing me off so I felt like you were my brother (meant in the most positive way) . Mafalda, thank you for always being there on the long working days, prepared to have fun at any moment, and for having such a strong personality. Patricia, Sofie and Henrique being part of the Bactory Summer School organizers was a special experience trying our skills outside of science and I am ii happy we did it so well together. ‘Best-office-ever’– Isotta, Mafalda, Patricia, Sofie, Xiaochen, Mikkel, Dario, and Mr. Kim thank you everyone! It was a true highway to laughter, culture-mixing, trouble- sharing, and colorful emotions. Being part of that office was one of the best things in these three years – I’ll keep all the chocolate treats, BBQs, Friday music afternoons, and the cemetery in my memory. Also I am grateful to be one of the Bactory students (those not yet mentioned – Ivan, Songyuan, Stephanie, Christina, Eric, and Sonia) because it was very nice to be a part of a group, sharing the problems and the happiness – together everything was much easier. I am grateful to many people at CfB who shared their knowledge with me: Martin Holm Rau, Anna Koza, Tune Wulff, Stefan Kol, Thomas Beuchert Kallehauge, Rebecca Lennen, and Basti Bergdahl. Special thanks to many others who have made a nice working environment at CfB: Maja, Ida, Rosa, Virginia, João, Nuša, Elleke, Daniel, GG, Fen, Virginia, Christian, Nabin… My PhD journey has been with many ups and downs and Joanna thank you for worrying about me when I was working late and keeping my head in the every-day things far from science. Marina, I am so happy I have met you on the plane even before I moved to Cph and all the girl’s time we had. Marina, Barbara, Benjamin, Rok, and Eva thank you for keeping up my Slovenian spirit in Scandinavia. The best thing that happened to me on this journey is you, Henrique. Thank you for being supportive, loving, open-minded, adventurous, and strong-willed. With you everything is better and easier. I am grateful to have amazing friends and family whose support and presence is the foundation of me being myself. Ana Ž., Maruša, Ana H., Neja, Lea, Ana Š., Didi, Neža, Mojca… Thank you for the all the fun, craziness, Skype iii conversations, and keeping me down to Earth. Thank you to my family, especially to mom, dad, Katja, and Miha – without a solid base no one can be great. Thank you for the support, without which I would never be where I am. Also thank you for the packages of Milka and Slovenian specialties that kept me going. I would also not be where I am without Jure Piškur, who set me to the path I have taken. Thank you for showing me hospitality, life abroad, and how a scientist can be crazy and fun and at the same time immensely smart. Thank you for taking me into your group where I met people that will forever be close to my heart – Dinesh, Nerve, Khadija, Citla, Edi, Andrea… JP, rest in peace. And to everyone I met in Cph – I hope one day our paths cross again! iv Abstract Bacteria commonly encounter stressful conditions during growth in their natural environments and in industrial biotechnology applications such as the biobased production of chemicals. As the coordinated regulation of gene expression is necessary to adapt to changing environments, bacteria have evolved numerous mechanisms to control gene expression in response to specific environmental signals. In addition to two-component systems, small regulatory RNAs (sRNAs) have emerged as major regulators of gene expression. The majority of sRNAs bind to mRNA and regulate their expression. They often have multiple targets and are incorporated into large regulatory networks and the RNA chaperone Hfq in many cases facilitates interactions between sRNAs and their targets. Some sRNAs also act by binding to protein targets and sequestering their function. In this PhD thesis we investigated the transcriptional response of Pseudomonas putida KT2440 in different conditions via identification of differentially expressed mRNAs and sRNAs. P. putida is a soil bacterium with a versatile metabolism and innate stress endurance traits, which makes it suitable as future cell factory for the production of valuable compounds. Detailed insights into the mechanisms through which P. putida responds to different stress conditions and increased understanding of bacterial adaptation in natural and industrial settings were gained. Additionally, we identified genome-wide transcription start sites, and many regulatory RNA elements such as sRNAs and riboswitches. Further, the sRNAome during the growth of bacteria was investigated and compared to the strain without Hfq protein. Hfq has a big impact on v sRNAs and gene expression in P. putida, hence many Hfq-associated sRNAs and mRNAs were found. Together, the results reported here significantly increase the knowledge of adaptation mechanisms in P. putida, as well as its transcriptome and regulatory networks.
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