The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium Inês de Jesus de Almeida e Silva Insert here an image with rounded corners Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, December, 2012 The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium Inês de Jesus de Almeida e Silva Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Oeiras, December, 2012 Financial Support from Fundação para a Ciência e Tecnologia (FCT) – Ph.D: grant - SFRH / BD / 43211 / 2008. Work performed at: Control of Gene Expression Laboratory Instituto de Tecnologia Química e Biológica Av. da República (EAN) 2781-901 Oeiras – Portugal Tel: +351-21-4469548 Fax: +351-21-4469549 Supervisor : Professora Doutora Cecília Maria Pais de Faria de Andrade Arraiano – Investigadora Coordenadora, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Head of the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) Co-supervisor : Doutora Sandra Cristina de Oliveira Viegas – Investigadora Pós-Doutorada, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Post-doc Fellow in the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) President of the Jury : Doutora Claudina Amélia Marques Rodrigues Pousada – Professora Catedrática Convidada do Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, por delegação; iii Examiners: Professor Doutor Iñigo Lasa Uzcudun – Head of Microbial Biofilm Research Group, Instituto de Agrobiotecnología, Pamplona (Principal Examiner). Professor Doutor Arsénio do Carmo Sales Mendes Fialho – Professor Associado com Agregação, Instituto Superior Técnico, Universidade Técnica de Lisboa (Principal Examiner). Professor Doutor Miguel Agostinho Sousa Pinto Torres Fevereiro – Investigador Principal do Instituto Nacional de Investigação Agrária e Veterinária - INIAV Professora Doutora Isabel Maria Godinho Sá Nogueira – Professora Associada com Agregação, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa To my family, to Dad, Mum, Eduardo and Pedro v "Every experiment proves something. If it doesn’t prove what you wanted it to prove, it proves something else." -Anonymous vii Acknowledgements Acknowledgements I would like to thank to all the people that directly or indirectly supported me during these last four years: - Instituto de Tecnologia Química e Biológica (ITQB) for giving me the opportunity to develop my work during these last years in such a good environment and with such good conditions. I also would like to thank the past and present ITQB directors, namely Professor Doutor José Artur Martinho Simões and Professor Doutor Luís Paulo Rebelo, for all their efforts to maintain ITQB as an excellence institute even in these “crisis” moments. - Fundação para a Ciência e Tecnologia (FCT) for the essential financial support. - My Supervisor Professora Cecília Maria Arraiano for giving me the opportunity to work in her lab, for believe in my capacities, for encouraging me to do the PhD in her lab, for providing excellent work conditions and for all the support during these last four years. Thank you also for giving me the chance to attend to several meetings both in Portugal and abroad where I could present and discuss my work with so many different “scientific people”. - My Supervisor and good friend Sandra Viegas. Thank you for your support, guidance, for all the knowledge that you transmitted me, for your companionship, for always have the right words even in the worst situations. Thank you for being always a wonderful friend. I am sure that my scientific and personal life would not be the same without you. - All my past and present colleagues from CMA Lab… thank you for your support during these important four years of my life. In special, I would like to thank to Sandra, Margarida, Susana, Andreia, Cátia, Rute, Ricardo and Clémentine for your patience, friendship, for all your help and all the good and xi special moments we spent both in and outside the lab. A special thank to my “office mates” and very good friends Sandocha, Margarette and Suzette for your positive vibes and for your support during these last years of my life. Also, a special and grateful thank to Andreia Aires our compass in the lab. Thank you for all your help, support and for being always so helpful and positive! - Franscisco García-del Portillo and Álvaro Ortega from Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain for their collaboration and precious help in the final step of this work. I hope that we can continue our collaboration. - All my friends that during these four years helped me to overcome the difficult moments. Especially: Joana and Ana, my “sisters”, thank you for your support not only during these four years but also along the last ten years. Thank you for your friendship; without you my life would never be the same. Thank you for all the wonderful moments during our weird stay in Göttingen; you were essential to my sanity during those five months. Danke! To my dear friend Catarina thank you for all your support, patience and all the perfect moments we passed. I miss you! - Jónsi, Georg, Kjartan, Orri, Eddie, Jeff, Matt, Stone, Mike, Glen, David, Rita, Chris, Guy, Jonny, Will, Tom, Tim, Richard, Jesse, Gary, Jonny, Nathan, Paul, Tom and many others for all the inspiring moments you provided me with your beautiful songs and wonderful concerts. Takk! xii Acknowledgements - My “new recent family” Susana, José and Manuela Mendes for your support, especially during THE endless moving, and for looking after Pedro when I am not at home. - A special and profound acknowledgement to my grandparents Celina, Avelino and Amália for all your teachings. Even if they had nothing to do with science, they were really important for me in these last years. To my grandfather António thank you for your support, guidance and love. I will always miss you… - To my special friends Sebastião and Belchior for the relaxing moments and for your unconditional love. - To my brother Eduardo and my sister Mónica thank you for all the support and for giving me two of the most precious things of my life. To my nephews António and Mariana thank you for being part of my life! - To my dear mum Luísa and my dear dad José thank you for all the support, unconditional love, friendship… Thank you for telling me always the best words in the right moments. Your advices are precious for me. I have no words to thank you for everything. You are for sure the best parents in the world! This achievement is also yours! - And last but not least… To my boyfriend and best friend Pedro Mendes for being always by my side. Thank you for your support, understanding, strength… thank you for being always so interested in my work even if you don’t understand anything about that. Thank you for being my soulmate, my right- hand man, my gifts’ boy. Thank you for everything! Yoroba ta ta un rosi! xiii Table of contents Table of Contents ABSTRACT ..................................................................................................... XIX RESUMO ...................................................................................................... XXV LIST OF PUBLICATIONS ...................................................................... XXXI DISSERTATION OUTLINE .................................................................. XXXV ABBREVIATIONS........................................................................................ XLI CHAPTER 1 ........................................................................................................ 1 CHAPTER 2 ..................................................................................................... 65 CHAPTER 3 ................................................................................................... 109 CHAPTER 4 ................................................................................................... 151 CHAPTER 5 ................................................................................................... 187 APPENDIX .................................................................................................... 201 xvii Abstract Abstract RNAs are important effectors in the process of gene expression. In bacteria, the levels of the transcripts have to be rapidly adjusted in response to constantly changing environmental demands. The cellular concentration of a given RNA is the result of the balance between its synthesis and degradation. RNA degradation is a complex process encompassing multiple pathways. Ribonucleases are the enzymes that directly process and degrade RNA transcripts, regulating their cellular amounts. The rate at which RNA decay occurs depends on the availability of ribonucleases and their specificities according to the sequence and/or the structural elements of the RNA molecule. Several other factors modulate RNA degradation, namely polyadenylation, which plays a multifunctional role in RNA metabolism. Additionally, small non-coding RNAs are crucial regulators of gene expression, and can directly modulate the stability of their mRNA targets. In many cases this regulation is dependent on Hfq, an RNA binding protein which