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Doctoral Thesis 2016 UNDERSTANDING the AROMATIC HYDROCARBON DEGRADATION POTENTIAL of PSEUDOMONAS STUTZERI

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Doctoral Thesis 2016 UNDERSTANDING the AROMATIC HYDROCARBON DEGRADATION POTENTIAL of PSEUDOMONAS STUTZERI Doctoral Thesis 2016 UNDERSTANDING THE AROMATIC HYDROCARBON DEGRADATION POTENTIAL OF PSEUDOMONAS STUTZERI: A PROTEO-GENOMIC APPROACH Isabel Brunet Galmés Doctoral Thesis 2016 Doctoral Program of “Microbiologia Ambiental i Biotecnologia” UNDERSTANDING THE AROMATIC HYDROCARBON DEGRADATION POTENTIAL OF PSEUDOMONAS STUTZERI: A PROTEO-GENOMIC APPROACH Isabel Brunet Galmés Thesis Supervisor: Dr. Rafael Bosch Thesis Supervisor: Dra. Balbina Nogales Doctor by the Universitat de les Illes Balears A mumpare i a mumare Agraïments Gràcies Rafel i Balbina per dirigir aquesta tesis, que és tant meva com vostre. A en Rafel per l’oportunitat que em vares donar, ja fa 7 anys, d’entrar al laboratori. Així com també per engrescar-me dins el món de la ciència, i ensenyar-me a treballar tant dins com fora del laboratori. I a na Balbina, pels mil consells que m’ha donat aquests anys, per ensenyar-me a ser més meticulosa amb el que faig i per ajudar-me amb tot el que ha pogut. A en Jordi i n’Elena, per acollir-me dins aquest grup de recerca, pels vostres consells i noves idees per continuar aquesta feina. Voldria agrair també a en Toni Bennasar i en Sebastià les crítiques constructives que m’heu anat fent al llarg d’aquests anys, des del projecte final de màster fins ara. Gràcies també a tots els companys de laboratori, a més de companys sou uns grans amics. Sempre estaré agraïda a na Marga, en Toni Busquets i n’Arantxa, pels grans consells que m’heu donat, tant dins com fora del laboratori, i per estar sempre disposats a donar-me una mà. A na Joana, que tant em va ajudar durant els meus primers anys i a en Sebas, que sempre alegrava el laboratori. A na Cris, na Magda i na Claudia, per escoltar-me sempre que perdia la paciència amb els experiments. A na Maria i en David, per ajudar-me en els mals moments, i acompanyar-me en els bons. Sense oblidar a les darreres incorporacions, en Dani i na Cati, vos desig el millor amb les vostres tesis. A na Lady, en Farid i en Mohamed, pels punts de vista diferents que m’heu aportat. Gràcies Angel, Xisco i Guillem, per mantenir sempre l’ordre en el laboratori, i fer-ho sempre amb un somriure. També agrair a na Trinidad per no perdre els nervis amb les meves seqüències, que han estat moltes. Gràcies també al grup de bioquímica, sense la seva ajuda no hauria acabat mai els gels de proteïnes! A en Joseph, per ensenyar-me a fer feina dins un laboratori i per tots els seus consells que encara ara em dóna. També per oferir-me un lloc en el seu grup durant la meva estància. Igualment agraeixo a la seva família, na Mar, na Lluc i na Neus, per donar-me tan bona benvinguda a Anglaterra. My sincere thanks to Sue and Alex for their invaluable help during the proteomic studies at the University of Warwick, I surely appreciate your help. I’m also grateful to Despina and Amandeep, both of you made my stay in Coventry really pleasant, and I hope I will see you soon. Gràcies a la meva família, tots vosaltres heu fet possible acabar aquesta etapa. Especialment agraeixo a mumpare i a mumare el seu suport, sense vosaltres mai hauria arribat a entrar al laboratori, i menys encara hauria acabat aquesta tesis. Agraeixo molt quant em vàreu animar a ser alumne col·laboradora ara fa 8 anys, enlloc de cercar una feina d’estiu. Igual d’important ha estat en Pere, especialment aquests darrers anys. El millor company que podria tenir, gràcies per fer agradables els moments més durs i pel coratge que sempre m’has donat. A na Magdalena, per tenir sempre unes paraules d’ànim i donar-me coratge per seguir endavant. A na Catalina i en Rafel, per acollir-me com ho heu fet, pels vostres ànims i per fer-me sempre costat. I a en Rafel, com a cunyat, per escoltar amb tant interès els meus dilemes de feina, i com a fisio perquè sense ell, potser aquesta tesis s’hauria acabat, però la meva salut se n’hauria ressentit molt! A tots els meus amics, i especialment a ses nines, perquè encara que no sempre vos he pogut dedicar tot el temps que voldria, sempre sou allà. Sense oblidar el BioTim, les nostres xerrades no tenen preu, qualque dia arreglarem el món! Finalment, m’agradaria donar les gràcies a les diferents entitats que han fet possible realitzar aquest treball. Primer de tot, a la Conselleria d’Educació, Cultura i Universitats per concedir-me una beca de formació de personal investigador 2011-2015, amb cofinançament del Fons Social Europeu. El suport financer es va obtenir del Ministeri d’Economia i Competitivitat a través del projecte Consolider CSD2009-00006. El model d’estudi AN10 serveix com a marc comparatiu pel projecte CTM2011-24886, amb cofinançament FEDER. Thanks also to the European Molecular Biology Organization, for making possible my work at the University of Warwick with the Short-Term Fellowship, which is part of this thesis. A tots, mil gràcies! Table of Contents ABSTRACT .................................................................................................................................... 1 INTRODUCTION............................................................................................................................ 7 1. Degradation of aromatic compounds .............................................................................. 9 2. What defines a Pseudomonas stutzeri? ......................................................................... 11 2.1. Taxonomy ................................................................................................................ 11 2.2. Habitat .................................................................................................................... 12 2.3. Denitrification ......................................................................................................... 13 2.4. Nitrogen fixation ..................................................................................................... 13 2.5. Aromatic hydrocarbon degradation ........................................................................ 15 3. Genomic studies ............................................................................................................. 17 4. Proteo-genomic studies ................................................................................................. 19 OBJECTIVES ................................................................................................................................ 23 MATERIALS AND METHODS ...................................................................................................... 27 1. Genomic analysis ........................................................................................................... 29 2. Culture conditions .......................................................................................................... 32 3. Selection of a benzoate-degrader derivative of strain P. stutzeri AN10 ........................ 33 4. Proteomic studies .......................................................................................................... 34 4.1. Naphthalene experiment ........................................................................................ 34 4.2 Experiment with strain BZ4D ................................................................................... 36 5. Gene cloning and complementation .............................................................................. 37 RESULTS AND DISCUSSION ....................................................................................................... 41 CHAPTER 1: Genomic comparison of Pseudomonas stutzeri ............................................... 43 1. General characterization of P. stutzeri genomes ........................................................... 45 2. Characteristic CDSs of P. stutzeri ................................................................................... 46 3. Phylogenomic analysis of P. stutzeri .............................................................................. 51 4. Pan- and core- proteome of P. stutzeri .......................................................................... 55 5. Identification of putative transposases in P. stutzeri genomes ..................................... 57 CHAPTER 2: Aromatic hydrocarbon degradation potential of Pseudomonas stutzeri........ 61 1. Analysis of aromatic hydrocarbon degradation gene clusters ....................................... 63 1.1. 4-Hydroxyphenylpyruvate degradation gene cluster ............................................. 65 1.2. 4-Hydroxybenzoate degradation gene cluster ........................................................ 68 1.3. Benzoate degradation gene cluster ....................................................................... 71 1.4. Homoprotocatechuate degradation gene cluster ................................................... 75 I 1.5. Carbazole degradation gene cluster ....................................................................... 77 1.6. Phenol degradation gene cluster ............................................................................ 81 1.7. Naphthalene degradation gene cluster .................................................................. 83 2. Dehydroabietic acid degradation gene cluster .............................................................. 91 3. Growth of P. stutzeri with aromatic hydrocarbons ....................................................... 93 CHAPTER 3: Proteomic study of naphthalene degradation by Pseudomonas stutzeri .... 101 1. P.stutzeri proteins detected by proteomics ................................................................. 103
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