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Development and Characterization of a Self-Bioluminescent Heavy Metal Cyanobacterial Bioreporter Strain

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Development and Characterization of a Self-Bioluminescent Heavy Metal Cyanobacterial Bioreporter Strain Ph.D. Thesis Development and characterization of a self-bioluminescent heavy metal cyanobacterial bioreporter strain Keila Martín Betancor 2017 UAM Ph.D. Thesis DEVELOPMENT AND CHARACTERIZATION OF A SELF-BIOLUMINESCENT HEAVY METAL CYANOBACTERIAL BIOREPORTER STRAIN Keila Martin Betancor Madrid, 14/06/2017 Programa de Doctorado de Microbiología Dirigida por: Dr. Francisca Fernández Piñas Dr. Ismael Rodea Palomares Departamento de Biología Departamento de Biología Facultad de Ciencias Facultad de Ciencias Universidad Autónoma de Madrid Universidad Autónoma de Madrid Memoria presentada para optar al título de Doctor por la Universidad Autónoma de Madrid. La presente memoria ha sido realizada cumpliendo los requisitos para optar a la Mención de Doctorado Internacional FUNDING The thesis was supported by the Spanish government (proyects MICIN CGL2010- 15675 and MINECO CTM2013-45775-C2-2-R) and the Dirección General de Universidades e Investigación de la Comunidad de Madrid, Research Network (Comunidad de Madrid S-2009/AMB/1511) Abbreviations (AA) Allen and Arnon culture medium (ICP/MS) Mass spectrometry (ANOVA) Analyses of variance (IPTG) Isopropyl-β-D-thio-galactoside (ATP) Adenosine 5´-triphosphate (LL.4) Four parameter log-logistic model (Atz) Atrazine (LOD) Limit of detection (BG11) Blue-Green culture medium (LOEC) Lowest observed effect concentration (BIFs) Bioluminescence induction factors (m) Slope of the dose-response curve (BLM) Biotic ligand model (Md) Metal dissolved (CA) Concentration addition (MOA) Mode of Action (CI) Combination Index (MOFf) MOF filtrates (CIw) Weighted Combination Index (MOFs) Metal Organic Frameworks (D) Doses (MOFs) MOF suspensions (DCP) 3,5 diclorophenol (MOPS) 3-(N-morpholino)propanesulfonic (ddH O) Double distilled water 2 acid (Dm) Median doses (MPC) Maximum permissive concentration (DNA) Deoxyribonucleic acid (NMs) Nanomaterials (E) Effect (NOEC) No observed effect concentration (EC) European Community (OD) Optical density (EC ) Effective Concentration x (p) Fractional effects (ED) Effective dose (R) Linear correlation coefficient (EDp) Fractional effective doses (RLU) Relative luminescence units (EDTA) Ethylenediaminetetraacetic acid (SIM) Substituted Imidazolate Material (E ) Maximum level of effect max (TAP-) Tris-minimal phosphate medium (Ep) Fractional effect scale (TAZ) Triazole (EPA) Environmental Protection Agency (TU) Toxic Unit (fa) Fraction affected (UMT) Urogen III methyltransferase (FIAAS) Flow injection atomic absorption (WHAM) Windermere Humic Aqueous (FIM) Free ion model Model (fu) Fraction unaffected (WWTP) Wastewater treatment plant (GFP) Green fluorescent protein (X-Gal) bromo-chloro-indolyl- galactopyranoside (IA) Independent action (ZIF) Zeolite imidazolate frameworks (ICP/AES) Inductively coupled plasma atomic electron spectrometry (β-Gal) β-galactosidase CONTENTS RESUMEN ...................................................................................................................... 1 SUMMARY ................................................................................................................... 11 CHAPTER I: General introduction ............................................................................ 21 1 Heavy metals in the environment ......................................................................... 23 2 Microbial bioreporters .......................................................................................... 26 3 Reporter genes ........................................................................................................ 28 3.1 Colored-protein based bioreporters ........................................................................ 28 3.2 Photoprotein bioreporters ....................................................................................... 28 3.3 Fluorescent bioreporters ......................................................................................... 29 3.4 Bioluminescent bioreporters .................................................................................... 30 4 Microorganisms metal-resistance proteins and gene regulation ....................... 31 4.1 ArsR/SmtB family: .................................................................................................... 34 4.2 MerR family: ............................................................................................................ 35 4.3 CsoR/RcnR family: ................................................................................................... 36 4.4 DtxR, Fur, and NikR family ..................................................................................... 37 5 Heavy metals bioluminescent microorganism-based bioreporters and applications ................................................................................................................... 38 5.1 Bacterial bioreporters: ............................................................................................ 39 5.2 Eukaryotic bioreporters: .......................................................................................... 40 6 Cyanobacteria and their applications in environmental monitoring ................ 44 6.1 Global toxicity cyanobacterial bioreporters ............................................................ 46 6.2 Cyanobacterial bioreporters responsive to heavy metals ........................................ 47 7 Turn-off vs. Turn-on bioreporters dose-response curves .................................. 48 8 Theoretical models of chemical mixture effects .................................................. 50 8.1 Independent Action (IA): .......................................................................................... 51 8.2 Concentration Addition (CA): .................................................................................. 51 9 Current approach to study mixtures in inducible systems ................................ 55 10 Other potential applications of the heavy metals bioreporters ......................... 57 11 Preservation methods ............................................................................................ 59 OBJECTIVES ............................................................................................................... 75 CHAPTER II: Construction of a self-luminescent cyanobacterial bioreporter that detects abroad range of bioavailable heavy metals in aquatic environments ......... 81 Supplementary material .................................................................................................. 95 CHAPTER III: Defining an additivity framework for mixture research in inducible whole-cell biosensors .................................................................................................. 105 Supplementary material ................................................................................................ 121 CHAPTER IV: Co, Zn and Ag-MOFS evaluation as biocidal materials towards photosynthetic organisms .......................................................................................... 155 Supplementary material ................................................................................................ 167 CHAPTER V: High-throughput freeze-dried cyanobacterial bioassay for fresh- waters environmental monitoring ............................................................................. 177 Supplementary material ................................................................................................ 193 GENERAL DISCUSSION ......................................................................................... 201 CONCLUSIONS / CONCLUSIONES ...................................................................... 217 R E S U M E N 1 RESUMEN El control de la calidad del agua y de los suelos requiere el desarrollo de técnicas combinadas para obtener información global sobre la composición química de la muestra y la evaluación de los efectos biológicos. En este sentido, los bioensayos de toxicidad se ha convertido en una herramienta muy útil ya que permiten obtener información sobre los efectos biológicos de un contaminante discriminando la fracción biodisponible y / o la toxicidad de los mismos. Otra aplicación de los bioensayos de toxicidad es el desarrollo y construcción de organismos reporteros o “bioreporters” para aplicaciones ambientales. Un bioreporter es una célula viva, intacta, que ha sido genéticamente modificada para producir una señal medible (gen reportero) en respuesta a un agente químico o físico específico en el medio ambiente. El uso de células enteras tiene una ventaja fundamental por el hecho de que reacciones complejas sólo pueden existir en una célula intacta, metabólicamente activa; De esta manera, pueden evaluarse parámetros globales tales como la biodisponibilidad de los contaminantes o la toxicidad. Los bioreporters constituidos por células enteras pueden ser básicamente de dos tipos: :(turn- off) que se reprimen en presencia de una muestra tóxica (portan fusiones de promotores constitutivos con los genes reporter) y aquellas que se inducen (turn-on) en presencia de un contaminante o un grupo de contaminantes concreto (portan fusiones de promotores inducibles frente a determinados compuestos y los genes reporter). Los metales pesados son uno de los contaminantes que generan mayor preocupación dada su persistencia en el medio ambiente y su alta toxicidad para todos los organismos. Dado que la toxicidad de los metales pesados viene determinada por su biodisponibilidad, la combinación de métodos químicos y bioreportes
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