
<p><strong>Doctoral Thesis </strong><br><strong>2016 </strong></p><p><strong>UNDERSTANDING THE AROMATIC HYDROCARBON </strong><br><strong>DEGRADATION POTENTIAL OF PSEUDOMONAS </strong><br><strong>STUTZERI: A PROTEO-GENOMIC APPROACH </strong></p><p><strong>Isabel Brunet Galmés </strong><br><strong>Doctoral Thesis </strong><br><strong>2016 </strong></p><p><strong>Doctoral Program of “Microbiologia Ambiental i </strong><br><strong>Biotecnologia” </strong></p><p><strong>UNDERSTANDING THE AROMATIC HYDROCARBON </strong><br><strong>DEGRADATION POTENTIAL OF PSEUDOMONAS </strong><br><strong>STUTZERI: A PROTEO-GENOMIC APPROACH </strong></p><p><strong>Isabel Brunet Galmés </strong><br><strong>Thesis Supervisor: Dr. Rafael Bosch Thesis Supervisor: Dra. Balbina Nogales </strong></p><p><strong>Doctor by the Universitat de les Illes Balears </strong></p><p>A mumpare i a mumare </p><p><strong>Agraïments </strong></p><p>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. </p><p>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 </p><p>constructives que m’heu anat fent al llarg d’aquests anys, des del projecte final de màster fins </p><p>ara. Gràcies també a tots els companys de laboratori, a més de companys sou uns grans amics. </p><p>Sempre estaré agraïda a na Marga, en Toni Busquets i n’Arantxa, pels grans consells que m’heu </p><p>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! </p><p>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. </p><p>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. </p><p>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 </p><p>ser alumne col·laboradora ara fa 8 anys, enlloc de cercar una feina d’estiu. Igual d’important ha </p><p>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 </p><p>a fisio perquè sense ell, potser aquesta tesis s’hauria acabat, però la meva salut se n’hauria </p><p>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! </p><p>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 </p><p>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. </p><p>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. </p><p>A tots, mil gràcies! </p><p><strong>Table of Contents </strong></p><p><a href="#19_0"><strong>ABSTRACT </strong></a><a href="#19_0">.................................................................................................................................... 1 </a><a href="#25_0"><strong>INTRODUCTION</strong></a><a href="#25_0">............................................................................................................................ 7 </a><br><a href="#27_0">1. Degradation of aromatic compounds ..............................................................................</a><a href="#27_0"> </a><a href="#27_0">9 </a><a href="#29_0">2. What defines a </a><a href="#29_0"><em>Pseudomonas stutzeri</em></a><a href="#29_0">? .........................................................................</a><a href="#29_0"> </a><a href="#29_0">11 </a><br><a href="#29_1">2.1. Taxonom</a><a href="#29_1">y</a><a href="#29_1">.</a><a href="#29_1">...............................................................................................................</a><a href="#29_1"> </a><a href="#29_1">11 </a><a href="#30_0">2.2. Habitat ....................................................................................................................</a><a href="#30_0"> </a><a href="#30_0">12 </a><a href="#0_0">2.3. Denitrification .........................................................................................................</a><a href="#0_0"> </a><a href="#0_0">13 </a><a href="#0_1">2.4. Nitrogen fixation .....................................................................................................</a><a href="#0_1"> </a><a href="#0_1">13 </a><a href="#0_2">2.5. Aromatic hydrocarbon degradation........................................................................</a><a href="#0_2"> </a><a href="#0_2">15 </a><br><a href="#0_3">3. Genomic studie</a><a href="#0_3">s</a><a href="#0_3">.</a><a href="#0_3">............................................................................................................</a><a href="#0_3"> </a><a href="#0_3">17 </a><a href="#0_4">4. Proteo-genomic studies .................................................................................................</a><a href="#0_4"> </a><a href="#0_4">19 </a><br><a href="#0_5"><strong>OBJECTIVES </strong></a><a href="#0_5">................................................................................................................................ 23 </a><a href="#0_6"><strong>MATERIALS AND METHODS </strong></a><a href="#0_6">...................................................................................................... 27 </a><br><a href="#0_0">1. Genomic analysis ...........................................................................................................</a><a href="#0_0"> </a><a href="#0_0">29 </a><a href="#0_7">2. Culture condition</a><a href="#0_7">s</a><a href="#0_7">.</a><a href="#0_7">.........................................................................................................</a><a href="#0_7"> </a><a href="#0_7">32 </a><a href="#0_8">3. Selection of a benzoate-degrader derivative of strain </a><a href="#0_8"><em>P. stutzeri </em></a><a href="#0_8">AN1</a><a href="#0_8">0</a><a href="#0_8">.</a><a href="#0_8">....................... 33 </a><a href="#0_9">4. Proteomic studies ..........................................................................................................</a><a href="#0_9"> </a><a href="#0_9">34 </a><br><a href="#0_10">4.1. Naphthalene experiment ........................................................................................</a><a href="#0_10"> </a><a href="#0_10">34 </a><a href="#0_0">4.2 Experiment with strain BZ4D ...................................................................................</a><a href="#0_0"> </a><a href="#0_0">36 </a><br><a href="#0_11">5. Gene cloning and complementation..............................................................................</a><a href="#0_11"> </a><a href="#0_11">37 </a><br><a href="#0_6"><strong>RESULTS AND DISCUSSION </strong></a><a href="#0_6">....................................................................................................... 41 </a></p><p><a href="#0_12"><strong>CHAPTER 1: Genomic comparison of </strong></a><a href="#0_12"><strong>Pseudomonas stutzer</strong></a><a href="#0_12"><strong>i</strong></a><a href="#0_12">............................................... 43 </a></p><p><a href="#0_0">1. General characterization of </a><a href="#0_0"><em>P. stutzeri </em></a><a href="#0_0">genomes ...........................................................</a><a href="#0_0"> </a><a href="#0_0">45 </a><a href="#0_13">2. Characteristic CDSs of </a><a href="#0_13"><em>P. stutzeri </em></a><a href="#0_13">................................................................................... 46 </a><a href="#0_14">3. Phylogenomic analysis of </a><a href="#0_14"><em>P. stutzeri </em></a><a href="#0_14">.............................................................................. 51 </a><a href="#0_15">4. Pan- and core- proteome of </a><a href="#0_15"><em>P. stutzer</em></a><a href="#0_15"><em>i</em></a><a href="#0_15">.......................................................................... 55 </a><a href="#0_16">5. Identification of putative transposases in </a><a href="#0_16"><em>P. stutzeri </em></a><a href="#0_16">genomes .....................................</a><a href="#0_16"> </a><a href="#0_16">57 </a></p><p><a href="#0_17"><strong>CHAPTER 2: Aromatic hydrocarbon degradation potential of </strong></a><a href="#0_17"><strong>Pseudomonas stutzeri</strong></a><a href="#0_17">........ 61 </a></p><p><a href="#0_0">1. Analysis of aromatic hydrocarbon degradation gene clusters.......................................</a><a href="#0_0"> </a><a href="#0_0">63 </a><br><a href="#0_18">1.1. 4-Hydroxyphenylpyruvate degradation gene cluster .............................................</a><a href="#0_18"> </a><a href="#0_18">65 </a><a href="#0_19">1.2. 4-Hydroxybenzoate degradation gene cluster........................................................</a><a href="#0_19"> </a><a href="#0_19">68 </a><a href="#0_20">1.3. Benzoate degradation gene cluster</a><a href="#0_20"> </a><a href="#0_20">....................................................................... 71 </a><a href="#0_21">1.4. Homoprotocatechuate degradation gene cluster...................................................</a><a href="#0_21"> </a><a href="#0_21">75 </a></p><p>I<br><a href="#0_22">1.5. Carbazole degradation gene cluster .......................................................................</a><a href="#0_22"> </a><a href="#0_22">77 </a><a href="#0_0">1.6. Phenol degradation gene cluster ............................................................................</a><a href="#0_0"> </a><a href="#0_0">81 </a><a href="#0_23">1.7. Naphthalene degradation gene cluster ..................................................................</a><a href="#0_23"> </a><a href="#0_23">83 </a><br><a href="#0_24">2. Dehydroabietic acid degradation gene cluster ..............................................................</a><a href="#0_24"> </a><a href="#0_24">91 </a><a href="#0_25">3. Growth of </a><a href="#0_25"><em>P. stutzeri </em></a><a href="#0_25">with aromatic hydrocarbons .......................................................</a><a href="#0_25"> </a><a href="#0_25">93 </a></p><p><a href="#0_17"><strong>CHAPTER 3: Proteomic study of naphthalene degradation by </strong></a><a href="#0_17"><strong>Pseudomonas stutzeri </strong></a><a href="#0_17">.... 101 </a></p><p><a href="#0_0">1. </a><a href="#0_0"><em>P.stutzeri </em></a><a href="#0_0">proteins detected by proteomics.................................................................</a><a href="#0_0"> </a><a href="#0_0">103 </a><a href="#0_26">2. General proteome compariso</a><a href="#0_26">n</a><a href="#0_26">.</a><a href="#0_26">...................................................................................</a><a href="#0_26"> </a><a href="#0_26">103 </a><a href="#0_27">3. Comparison of proteomes from naphthalene and succinate cultures</a><a href="#0_27"> </a><a href="#0_27">....................... 107 </a><br><a href="#0_28">3.1. Proteomic changes in naphthalene degradation proteins ...................................</a><a href="#0_28"> </a><a href="#0_28">107 </a><a href="#0_29">3.2. Putative transporters involved in naphthalene degradation ...............................</a><a href="#0_29"> </a><a href="#0_29">110 </a><br><a href="#0_30">4. Comparison of proteomes from salicylate and succinate cultures .............................</a><a href="#0_30"> </a><a href="#0_30">117 </a></p><p><a href="#0_17"><strong>CHAPTER 4: Benzoate degradation potential of </strong></a><a href="#0_17"><strong>Pseudomonas stutzeri </strong></a><a href="#0_17"><strong>AN10 </strong></a><a href="#0_17">................. 121 </a></p><p><a href="#0_0">1. </a><a href="#0_0"><em>P. stutzeri </em></a><a href="#0_0">AN10 potential to metabolize benzoate .....................................................</a><a href="#0_0"> </a><a href="#0_0">123 </a><a href="#0_31">2. Genome sequencing of </a><a href="#0_31"><em>P. stutzeri </em></a><a href="#0_31">BZ4D ..................................................................... 125 </a><a href="#0_32">3. Proteomic analysis of BZ4D .........................................................................................</a><a href="#0_32"> </a><a href="#0_32">128 </a><a href="#0_30">4. Complementation of AN10 with BZ4D benzoate genes ..............................................</a><a href="#0_30"> </a><a href="#0_30">131 </a><br><a href="#0_6"><strong>GENERAL DISCUSSION</strong></a><a href="#0_6"><strong> </strong></a><a href="#0_6">............................................................................................................ 133 </a><a href="#0_33"><strong>CONCLUSIONS </strong></a><a href="#0_33">......................................................................................................................... 139 </a><a href="#0_33"><strong>REFERENCES </strong></a><a href="#0_33">............................................................................................................................ 143 </a><a href="#0_33"><strong>SUPPLEMENTARY INFORMATION </strong></a><a href="#0_33">.......................................................................................... 165 </a></p><p>II </p><p><strong>List of Figures </strong></p><p><a href="#28_0"><strong>Figure I1. </strong></a><a href="#28_0">Example of aerobic aromatic hydrocarbon degradatio</a><a href="#28_0">n</a><a href="#28_0">.</a><a href="#28_0">..........................................</a><a href="#28_0"> </a><a href="#28_0">10 </a><a href="#0_34"><strong>Figure I2. </strong></a><a href="#0_34">Organization of the </a><a href="#0_34"><em>nar</em></a><a href="#0_34">, </a><a href="#0_34"><em>nir</em></a><a href="#0_34">, </a><a href="#0_34"><em>nor</em></a><a href="#0_34">, and </a><a href="#0_34"><em>nos </em></a><a href="#0_34">genes in </a><a href="#0_34"><em>P. stutzeri </em></a><a href="#0_34">.................................. 13 </a></p><p><a href="#0_35"><strong>Figure I3. </strong></a><a href="#0_35">Representation of the </a><a href="#0_35"><em>P. stutzeri </em></a><a href="#0_35">A1501 </a><a href="#0_35"><em>nif </em></a><a href="#0_35">genes compared with </a><a href="#0_35"><em>A. vinelandii </em></a></p><p><a href="#0_35">AvOP, </a><a href="#0_35"><em>K. pneumoniae </em></a><a href="#0_35">M5a1, </a><a href="#0_35"><em>Azoarcus </em></a><a href="#0_35">sp. BH72, and </a><a href="#0_35"><em>A. brasilense </em></a><a href="#0_35">Sp7 ..................................</a><a href="#0_35"> </a><a href="#0_35">14 </a><a href="#0_36"><strong>Figure I4. </strong></a><a href="#0_36">Aerobic catabolism of aromatic compounds in </a><a href="#0_36"><em>P. stutzeri </em></a><a href="#0_36">........................................ 15 </a><a href="#0_37"><strong>Figure I5. </strong></a><a href="#0_37">Naphthalene degradation pathway and the genes involved in </a><a href="#0_37"><em>P. stutzeri </em></a><a href="#0_37">AN10 ..... 16 </a></p><p><a href="#0_38"><strong>Figure I6. </strong></a><a href="#0_38">The catechol and protocatechuate branches of the β</a><a href="#0_38">-ketoadipate pathway and </a></p><p><a href="#0_38">its regulation in </a><a href="#0_38"><em>P. stutzeri </em></a><a href="#0_38">A1501 ..............................................................................................</a><a href="#0_38"> </a><a href="#0_38">17 </a><a href="#0_39"><strong>Figure I7. </strong></a><a href="#0_39">Number of genomic sequences submitted to NCBI and GOLD per year</a><a href="#0_39"> </a><a href="#0_39">.................. 18 </a></p><p><a href="#0_40"><strong>Figure M1. </strong></a><a href="#0_40">pCatA and pBenR constructions used to complement AN10 and primer </a><a href="#0_40">localization respect from </a><a href="#0_40"><em>catA </em></a><a href="#0_40">and </a><a href="#0_40"><em>benR </em></a><a href="#0_40">genes......................................................................... 37 </a></p><p><a href="#0_41"><strong>Figure 1.1. </strong></a><a href="#0_41">Structure of genes involved in the flagellar assembly system in </a><a href="#0_41"><em>P. stutzeri </em></a><a href="#0_41">and </a><a href="#0_41"><em>P. balearica </em></a><a href="#0_41">................................................................................................................................ 49 </a><a href="#0_42"><strong>Figure 1.2. </strong></a><a href="#0_42">Structure of genes involved in denitrification in </a><a href="#0_42"><em>P. stutzeri </em></a><a href="#0_42">and </a><a href="#0_42"><em>P. balearica</em></a><a href="#0_42">.......... 49 </a><a href="#0_43"><strong>Figure 1.3. </strong></a><a href="#0_43">Structure of genes involved in nitrogen fixation in </a><a href="#0_43"><em>P. stutzeri </em></a><a href="#0_43">................................ 50 </a><a href="#0_44"><strong>Figure 1.4. </strong></a><a href="#0_44">Neighbor-Joining tree calculated from the concatenation of 69 shared COGs </a><a href="#0_44">in the 18 </a><a href="#0_44"><em>P. stutzeri </em></a><a href="#0_44">strains and </a><a href="#0_44"><em>P. balearica </em></a><a href="#0_44">SP1402</a><sup style="top: -0.33em;"><a href="#0_44">T </a></sup>................................................................ 54 <a href="#0_45"><strong>Figure 1.5. </strong></a><a href="#0_45">Number of core-COGs assigned to each group of biological functions according </a><a href="#0_45">to KEGG categories ....................................................................................................................</a><a href="#0_45"> </a><a href="#0_45">56 </a><a href="#0_46"><strong>Figure 1.6. </strong></a><a href="#0_46">Percentage of TnpAs classified on each IS-family ...................................................</a><a href="#0_46"> </a><a href="#0_46">58 </a><a href="#0_47"><strong>Figure 1.7. </strong></a><a href="#0_47">Number of putative TnpAs of each IS-family identified in the different studied </a><a href="#0_47">strains ........................................................................................................................................ 59 </a></p><p><a href="#0_48"><strong>Figure 2.1. </strong></a><a href="#0_48">4-Hydroxyphenylpyruvate degradation pathway in </a><a href="#0_48"><em>Pseudomonas putida </em></a><a href="#0_48">U ......... 66 </a><a href="#0_49"><strong>Figure 2.2. </strong></a><a href="#0_49">Structure of genes involved in 4-hydroxyphenylpyruvate degradation in </a><a href="#0_49"><em>Pseudomonas putida </em></a><a href="#0_49">U ............................................................................................................. 68 </a><a href="#0_50"><strong>Figure 2.3. </strong></a><a href="#0_50">4-Hydroxybenzoate degradation pathway in </a><a href="#0_50"><em>P. stutzeri </em></a><a href="#0_50">A1501 ..............................</a><a href="#0_50"> </a><a href="#0_50">69 </a><a href="#0_17"><strong>Figure 2.4. </strong></a><a href="#0_17">Structure of genes involved in 4-hydroxybenzoate degradation in </a><a href="#0_17"><em>P. stutzeri </em></a><a href="#0_17">A1501 .........................................................................................................................................</a><a href="#0_17"> </a><a href="#0_17">71 </a><a href="#0_51"><strong>Figure 2.5. </strong></a><a href="#0_51">Benzoate degradation pathway in </a><a href="#0_51"><em>P. stutzeri </em></a><a href="#0_51">A1501................................................ 72 </a><a href="#0_52"><strong>Figure 2.6. </strong></a><a href="#0_52">Structure of genes involved in benzoate degradation in </a><a href="#0_52"><em>P. stutzeri </em></a><a href="#0_52">A1501 ............</a><a href="#0_52"> </a><a href="#0_52">74 </a><a href="#0_53"><strong>Figure 2.7. </strong></a><a href="#0_53">Homoprotocatechuate degradation pathway in </a><a href="#0_53"><em>P. putida </em></a><a href="#0_53">U ................................. 76 </a><a href="#0_54"><strong>Figure 2.8. </strong></a><a href="#0_54">Structure of genes involved in homoprotocatechuate degradation in </a><a href="#0_54"><em>P. putida </em></a><a href="#0_54">U </a><a href="#0_54">and </a><a href="#0_54"><em>P. stutzeri </em></a><a href="#0_54">KOS6 .................................................................................................................. 77 </a><a href="#0_55"><strong>Figure 2.9. </strong></a><a href="#0_55">Carbazole degradation pathway in </a><a href="#0_55"><em>P. resinovorans </em></a><a href="#0_55">CA10 ...................................... 78 </a><a href="#0_56"><strong>Figure 2.10. </strong></a><a href="#0_56">Structure of genes involved in carbazole degradation in </a><a href="#0_56"><em>P. resinovorans </em></a><a href="#0_56">CA10, </a><a href="#0_56">and </a><a href="#0_56"><em>P. stutzeri </em></a><a href="#0_56">XLDN-R ...............................................................................................................</a><a href="#0_56"> </a><a href="#0_56">80 </a><a href="#0_1"><strong>Figure 2.11. </strong></a><a href="#0_1">Phenol degradation pathway in </a><a href="#0_1"><em>Pseudomonas </em></a><a href="#0_1">sp. CF60</a><a href="#0_1">0</a><a href="#0_1">.</a><a href="#0_1">....................................</a><a href="#0_1"> </a><a href="#0_1">81 </a><a href="#0_57"><strong>Figure 2.12. </strong></a><a href="#0_57">Structure of genes involved in phenol degradation in </a><a href="#0_57"><em>Pseudomonas </em></a><a href="#0_57">sp. CF600 </a><a href="#0_57">and </a><a href="#0_57"><em>P. stutzeri </em></a><a href="#0_57">KOS6 and TS44 strains........................................................................................</a><a href="#0_57"> </a><a href="#0_57">83 </a><a href="#0_58"><strong>Figure 2.13. </strong></a><a href="#0_58">Naphthalene degradation pathway in </a><a href="#0_58"><em>P. stutzeri </em></a><a href="#0_58">AN10 ....................................... 84 </a></p>
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