UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR Regulation of gene expression by the cAMP-Crp system in the soil bacterium Pseudomonas putida TESIS DOCTORAL Memoria presentada para optar al grado de Doctor en Ciencias Alejandro Arce Rodríguez DIRECTORES DE TESIS: Víctor de Lorenzo Prieto Belén Calles Arenales CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (CSIC) CENTRO NACIONAL DE BIOTECNOLOGÍA Madrid, 2012 En memoria de mi abuela Alicia Corrales Campos (1938-2012), quien un día se durmió en su particular “País de las maravillas” para no volver jamás. QEPD A Dios y a mi familia. Soy quien soy gracias a ustedes! In memory of my grandmother Alicia Corrales Campos (1938-2012), who one day fell asleep in her particular “Wonderland” to never come back. RIP To God and my family. Acknowledgements This work would not have been possible without the support of many people who I would like to thank with a few words. To Victor de Lorenzo, to whom I am especially grateful for the opportunity to join his group, for his teaching, his guidance and specially for supporting and encouraging me always, in the bad and good moments. Thank you very much F !! Also to Beléeeeeeen Calles for accepting to be the co-director of this Thesis, and of course for her teaching, her patience, her support and her friendship. Thank you both for showing me how to be a better scientist! Many thanks to Fernando Rojo, for accepting to be my tutor. I would like to thank Tino Krell and Raúl Platero, for their contribution with the ITC experiments and for the useful discussions about the thermodynamic properties of Crp. To Teresa Suárez at CIB, for helping me with the D. discoideum chemotaxis experiments and to Max Chavarría, for his collaboration with the quantification of cAMP. I am also indebted to Antoine Danchin, for useful and inspiring discussions about cAMP regulation and to H. Aiba and D. Ladant, for valuable materials. I want to acknowledge also to Silvia Juárez and Sergio Ciordia at the proteomics facility, and to Juan Carlos Alonso at the 214 lab of CNB for their help with Crp purification and protein techniques. Also, to Juan Carlos Oliveros at the genomic facility for his contribution with the analysis of the RNA-seq results Of course, I would like to thank the people of the VdL lab: To Sofía, for her technical support, but especially for her invaluable friendship and for encourage me always to keep going! To Gonzalo for his help with IVT and EMSA assays, and for always laugh with my bad jokes! To Esteban, Rafael, Pablo, Juhyun (a.k.a Chu!) and Danilo for their collaboration, critical discussions and their willingness to teach me new techniques. To Inés, for the assistance with the administrative tasks. To Ilaria, for her friendship and her contagious laugh, and to Alberto also for his friendliness. But especially I want to thank you guys for making me feel always like I am at home, outside or inside the lab! I could not forget the former members of the lab, especially Carlos Álvarez, with whom I am grateful for the preparation of the RNA polymerase of P. putida. Also, I am thankful to Aitor, José Ignacio, David, Olga, Esther and Paola for their help with my first steps in the laboratory. Much of this work was certainly achieved thanks to you! There are special persons in both shores of the Atlantic Ocean to whom I am indebted for their invaluable friendship and for pushing me up since I started the Thesis some years ago. So, Cicelio, María José, Mauricio, Luigi, Ana E, Bocha, Isella, Pau, Pri, Adolfo, Guega, Héctor, Caro, José Raúl, Lorena (from Costa Rica) and Isa (and her family), María, Leti, Jacobo, Juan Carlos, Stephanie, Noelia, Mar (from Europe) part of this work is also because of you! Finally, there are some persons that have always played a main role in the theatre of life and to whom I am immensely grateful. They are my parents Ricardo and Xinia, and my brothers Alex Ricardo, Andreina and Fabián Alonso. Thank you for the support, for the advices, for the prayers, for the words of encouragement, for the lots of love and for making me feel that 9000 km could be reduced to a few steps. And of course, I am immensely grateful to Andrea González, my dear girl who has attended me in the last part of the Thesis. Thanks for your care, your patience and for light up my world with your smile. How I wish you were here! Love you guys. ¡Pura vida a todos! Aleyo GENERAL INDEX Index of Figures ......................................................................................................... iv Index of Tables .......................................................................................................... vi Abbreviations ............................................................................................................ vii Abstract ...................................................................................................................... xi Resumen .................................................................................................................. xiii I. Introduction .......................................................................................................... 1 1 Global regulation of gene expression ........................................................................ 3 2 The archetypal cAMP-Crp system of E. coli ............................................................. 5 2.1 The cAMP-receptor protein (Crp) ..................................................................... 5 2.2 Carbon Catabolite Repression mediated by cAMP-Crp in E. coli ....................... 8 2.3 Transcription activation at simple Crp-dependent promoters .......................... 11 3 Adenylate cyclase (CyaA) ........................................................................................ 14 4 cAMP phosphodiesterase (cAMP PDE) .................................................................. 15 5 Virulence factor regulator (Vfr) of Pseudomonas aeruginosa ..................................... 16 6 cAMP-Crp system in Pseudomonas putida ............................................................... 17 7 The issue at stake .................................................................................................... 19 II. Objectives ........................................................................................................... 21 III. Materials and Methods ...................................................................................... 25 1 Culture conditions and media ................................................................................ 27 2 Bacterial strains ...................................................................................................... 27 3 Plasmids ................................................................................................................. 29 4 Recombinant DNA techniques .............................................................................. 31 5 Protein techniques .................................................................................................. 34 6 Construction of ∆crp and ∆cyaA strains of P. putida ............................................... 35 7 crp and cyaA complementation assays ..................................................................... 35 7.1 Construction of single ∆crp and double ∆crp ∆cyaA strains of E. coli ............... 35 7.2 Design of expression vectors for crp and cyaA genes of E. coli and P. putida ..... 36 8 Dictyostelium discoideum cAMP chemotaxis biosensor ............................................ 37 9 Quantification of cAMP produced by E. coli and P. putida .................................... 38 i 10 Protein purification .............................................................................................. 39 10.1 Production and purification of MBP-CrpP. putida fusion protein ....................... 39 10.2 Cleavage of MBP-Crp and purification of CrpP. putida ...................................... 40 10.3 Production and purification of apo-CrpP. putida ................................................ 40 11 Analytical ultracentrifugation of CrpP. putida ............................................................ 41 12 Partial proteolysis of CrpP. putida .............................................................................. 42 13 Isothermal titration calorimetry (ITC) ................................................................. 42 14 Electrophoretic mobility shift assays (EMSA) ....................................................... 43 15 In vitro transcription assays (IVT) ........................................................................ 44 16 β-galactosidase assays ............................................................................................ 45 16.1 Construction of plasmids encoding lacZ transcriptional and translational fusions ...................................................................................................................... 45 16.2 Measurements of the β-galactosidase activity ................................................. 45 17 RNA-seq transcriptome analysis of crp and cyaA mutants in P. putida .................. 46 17.1 Culture conditions, RNA isolation and rRNA removal .................................. 46 17.2 RNA-Seq using the Illumina Genome Analyzer ............................................. 47 17.3 Bioinformatic analysis .................................................................................... 48 17.4 Estimation of Differential