UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS Departamento de Bioquímica y Biología Molecular TESIS DOCTORAL Domesticating predatory bacteria for biotechnological tools Bacterias depredadoras para ser aplicadas en procesos biotecnológicos MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Cristina Herencias Rodríguez Directora Mª Auxiliadora Prieto Jiménez Madrid Ed. electrónica 2019 © Cristina Herencias Rodríguez, 2019 UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas Departamento de Bioquímica y Biología Molecular DOMESTICATING PREDATORY BACTERIA FOR BIOTECHNOLOGICAL TOOLS BACTERIAS DEPREDADORAS PARA SER APLICADAS EN PROCESOS BIOTECNOLÓGICOS Tesis doctoral Cristina Herencias Rodríguez Directora Mª Auxiliadora Prieto Jiménez Madrid, 2019 1 UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas Departamento de Bioquímica y Biología Molecular DOMESTICATING PREDATORY BACTERIA FOR BIOTECHNOLOGICAL TOOLS BACTERIAS DEPREDADORAS PARA SER APLICADAS EN PROCESOS BIOTECNOLÓGICOS Tesis doctoral Cristina Herencias Rodríguez Directora María Auxiliadora Prieto Jiménez Consejo Superior de Investigaciones Científicas – CSIC Centro de Investigaciones Biológicas - CIB Madrid, 2019 3 And those who were seen dancing were thought to be insane by those who couldn’t hear the music. Aquellos que eran vistos bailando, eran considerados locos por quienes no podían escuchar la música. F. Nietzsche 5 ACKNOWLEDGEMENTS First and foremost, I would like to express my wide and sincere gratitude to my research supervisor, Dr. Mª Auxiliadora Prieto Jiménez, for giving me the opportunity to research and providing invaluable guidance throughout this thesis. Her dynamism, vision, and motivation have deeply inspired me during these years. I would like to acknowledge Dr. Aljoscha Wahl for accepting me into his research group in TUDelft and leading me to work on the fascinating field of metabolism. I would also like to thank Prof. José Luis García, Dr. Eduardo Díaz, Dr. Pedro García, and Prof. Ernesto García for their constructive advice during these years. Thanks to Virginia Martínez and Juan Nogales for introducing me to the interesting fields of predatory bacteria and metabolism. I would also like to thank to Rosa del Campo for her enthusiasm with which she understands the microbiology and for her fruitful advices. I am indebted to the institutions that have financially supported this work: Ministerio de Economía y Competitividad and EMBO which supported over FPI fellowship the accomplishment of this Ph.D. Thesis at CIB and the short stay at Delft University of Technology. I appreciate the work of CIB facilities such as microscopy, photography and technical support team, as well as ICTP facilities (SEM). I would like to give special thanks to Natalia, Vir, Sergio, Gonn, Fran and Roberto, without whom it would not be possible. To Elena and Edu for their inconditional support whatever happens. Wilson, thanks for listening to my music and making me dance. Lastly, I would like to thank my family for all the support during my whole life. Special thanks to Jose, who design the beautiful cover of this book. To my mum and my grandfather who always encourage me to get my dreams. 7 INDEX I. ABBREVIATIONS .................................................................................................................. 13 II. SUMMARY ........................................................................................................................... 15 III. RESUMEN ............................................................................................................................. 19 IV. GENERAL INTRODUCTION ................................................................................................... 23 1. PREDATION: A GENERAL OVERVIEW ............................................................................... 23 2. BACTERIAL PREDATION ................................................................................................... 24 3. BDELLOVIBRIO AND LIKE ORGANISMS ............................................................................ 27 4. BDELLOVIBRIO BACTERIOVORUS HD100 ......................................................................... 28 4.1 Prey range and predatory efficiency ....................................................................... 30 4.2 Epibiotic predation by B. bacteriovorus .................................................................. 31 4.3 Resistance to B. bacteriovorus ................................................................................ 32 5. HOST-INDEPENDENT B. BACTERIOVORUS ....................................................................... 32 6. BDELLOVIBRIO AS A TOOL FOR BIOTECHNOLOGICAL APPLICATIONS ............................. 34 6.1 B. bacteriovorus as a source of biocatalysts............................................................ 34 6.2 Bdellovibrio strains as a living antibiotics ................................................................ 34 6.3 Bdellovibrio strains as a biocontrol agents .............................................................. 35 6.4 Genetic tools for B. bacteriovorus ........................................................................... 36 V. MOTIVATIONS AND AIMS .................................................................................................... 39 VI. COMMON MATERIALS AND METHODS ............................................................................... 41 1. GROWTH CONDITIONS OF B. BACTERIOVORUS HD100 .................................................. 41 2. B. BACTERIOVORUS HD100 AND PREY VIABILITY CALCULATION .................................... 41 3. BIOMASS CALCULATION .................................................................................................. 42 4. PHASE CONTRAST AND FLUORESCENCE MICROSCOPIC TECHNIQUES ........................... 42 5. SCANNING ELECTRON AND TRANSMISSION ELECTRON MICROSCOPIC TECHNIQUES ... 43 6. STATISTICAL ANALYSES .................................................................................................... 43 RESULTS ....................................................................................................................................... 45 VII. COMPREHENSIVE ANALYSIS OF THE PREDATORY BACTERIA BDELLOVIBRIO BACTERIOVORUS THROUGH A GENOME SCALE METABOLIC MODEL ........................................ 47 1. INTRODUCTION ............................................................................................................... 49 2. MATERIALS AND METHODS ............................................................................................ 51 2.1 General concepts in the metabolic reconstruction ................................................. 51 2.2 Genome-scale metabolic network reconstruction: iCH457 .................................... 54 2.3 Biomass Function .................................................................................................... 54 9 2.4 Reaction essentiality analysis .................................................................................. 56 2.5 Generation of condition-specific models: iCHAP and iCHGP .................................. 56 2.6 Software .................................................................................................................. 57 3. RESULTS ........................................................................................................................... 59 3.1 Characteristics of B. bacteriovorus metabolic reconstruction ................................ 59 3.2 Model-driven assessment of auxotrophies and biomass building block transport systems highlight the predatory lifestyle of B. bacteriovorus ................................. 61 3.3 iCH457 exhibits high accuracy by predicting physiological states of B. bacteriovorus under different nutrients scenarios ........................................................................ 62 3.4 Reaction essentiality towards understanding the predator´s lifestyle ................... 64 3.5 Analysis of the predator´s lifestyle using condition-specific models: Attack Phase (iCHAP) and Growth Phase (iCHGP) models ............................................................ 66 VIII. DEVELOPMENT OF AN ACTIVE AND FUNCTIONAL AXENIC CULTURE OF B. BACTERIOVORUS HD100 ............................................................................................................. 71 1. INTRODUCTION ............................................................................................................... 73 2. MATERIALS AND METHODS ............................................................................................ 75 2.1 Strain media and axenic growth conditions ............................................................ 75 2.2 Quantification of the genome number of B. bacteriovorus .................................... 75 2.3 Amplification of DNA and hit locus sequencing ...................................................... 76 2.4 Analysis of extracellular metabolites by GC-TOF-MS .............................................. 77 2.5 Intracellular ATP measurements of B. bacteriovorus cells growing in rich medium………………………………………………………………………………………………………………79 2.6 Consumption, production rate and growth rate calculations in BdQ10 cells ......... 79 3. RESULTS ........................................................................................................................... 81 3.1 Feasibility of developing axenic cultures of B. bacteriovorus HD100: Quantification