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Molecular Characterization of the Life Support Bacterium Rhodospirillum Rubrum S1H Cultivated Under Space Related Environmental Conditions

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Molecular Characterization of the Life Support Bacterium Rhodospirillum Rubrum S1H Cultivated Under Space Related Environmental Conditions Université de Mons-Hainaut Faculté des Sciences Service de Protéomie et Biochimie des Protéines Centre d'étude de l'Energie Nucléaire Unité de Microbiologie Molecular characterization of the life support bacterium Rhodospirillum rubrum S1H cultivated under space related environmental conditions Dissertation originale présentée par Felice Mastroleo en vue de l'obtention du grade de docteur en Sciences Directeur de thèse: Prof. R. Wattiez Co-directeur: Dr. Ir. R. Van Houdt (SCK•CEN) Janvier 2009 "De la Terre à la Lune". Jules Verne. Paris, 1865. REMERCIEMENTS Mes premiers mots seront pour honorer la mémoire du Dr. Larissa Hendrickx, qui m'a fait confiance, voici bientôt 5 ans, et m'a donné la chance de commencer cette aventure. Je tiens à remercier tout particulièrement les professeurs Ruddy Wattiez et Max Mergeay qui m'ont accueillit au sein de leur service respectif. Ils m'ont permis d'aborder un sujet captivant pour lequel mon intérêt ne s'est pas démenti, que du contraire, au cours de ces années. Leur emploi du temps surchargé ne les a pas empêché d'être là pour moi lorsque le besoin s'en faisait sentir, je leur en suis pour cela très reconnaissant. J'exprime également toute ma gratitude envers les Drs. Natalie Leys et Rob Van Houdt qui ont su reprendre le flambeau de leur prédécesseur avec panache et enthousiasme, malgré un contexte difficile. Leur soutien m'a été d'un grand secours. Je remercie également le SCK •CEN qui a permis le financement de cette thèse grâce à une bourse AWM. Un grand merci également à tous les membres du service de Protéomie et Biochimie des Protéines de l'UMH, ainsi qu'à tout le groupe de Biologie Moléculaire et Cellulaire du SCK•CEN. Je dédie ce travail à ma famille et à mes parents en particulier qui m'ont permis d'entreprendre et de mener à leur terme ces études universitaires. CONTENT LIST OF ABBREVIATIONS RESUME SUMMARY 1 – INTRODUCTION 1 2 – OBJECTIVES OF THE WORK 33 3 – WHOLE PROTEOME STUDY 35 4 – STUDY OF R. RUBRUM S1 H IN SPACE RELATED ENVIRONMENTAL CONDITIONS 75 A – THE MESSAGE 2 EXPERIMENT 75 B – THE BASE-A EXPERIMENT 101 C – R. RUBRUM S1H LIQUID CULTURE IN MODELED MICROGRAVITY 121 5 – GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES 145 REFERENCES 151 ADDITIONAL DATA ARE PRESENTED IN THE ATTACHED CD-ROM ABBREVIATIONS 2D-PAGE Bi-dimensional polyacrylamide gel electrophoresis aaRNA Amino allyl RNA ACLAME A classification of mobile genetic elements ACN Acetonitrile AHL N-acyl-L-homoserine lactone APS Ammonium persulfate aRNA Amplified RNA ATCC American type culture collection BASE-A Bacterial adaptation to the space environment – part A BCHL Bacteriochlorophyll BLiSS Bioregenerative life support system CDS Coding sequence CELSS Closed ecological life support system CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CME Coronal mass ejection COG Cluster of orthologous group CRT Carotenoid DAE Dark aerobic DSB Double strand break DTE Dithioerythritol EDTA ethylenediaminetetraacetic acid emPAI Exponentially modified protein abundance index ERB Earth radiation belt ECF Extracytoplasmic function ESA European space agency FDR False discovery rate GCR Galactic cosmic rays Gy Gray H/L Heavy (H) to light (L) ICPL reagent form ratio HARV High aspect to ratio vessel HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-performance liquid chromatography HZE high charge (Z) and high energy (E) particles ICC Ion charge current ICM Intracytoplasmic chromatophore membrane ICPL Isotope-coded protein label IEF Isoelectric focusing IPG Immobilized pH gradient IR Ionizing radiation ISS International space station LAN Light anaerobic LEO Low Earth orbit LET Linear energy transfer LSMMG Low shear modeled microgravity MaGe Magnifiying genome MALDI-ToF Matrix assisted laser desorption ionization-time of flight MELiSSA Micro-ecological life support system alternative MESSAGE 2 Microbial experiment in the space station about gene expression - part 2 MS Mass spectrometry MS/MS Tandem mass spectrometry MudPIT Multidimensional protein identification technology MW Molecular weight NASA National aeronautics and space administration NL Non linear OD optical density OSLD Optically stimulated luminescence detector PHB Polyhydroxybutyrate pI isoelectric point RBE Relative biological effectiveness RC Reaction center complex ROS Reactive oxygen species RP Reverse phase RPM Random positioning machine RT-qPCR Real time-quantitative polymerase chain reaction RuBisCO ribulose bisphosphate carboxylase RWV Rotating wall vessel SAA South atlantic anomaly SB3-10 Sulfobetaine-10 SCX Strong cation exchange SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis SILE Stable isotope labeling experiment SimRAD Simulation of ionizing radiation SPE Solar particle event SPY Sistrom peptone yeast Sv Sievert TBP Tributyl phosphine TED Track-etch detector TEMED N,N,N',N'-Tetramethylethylenediamine TFA Trifluoroacetic acid TGS Tris-glycine-SDS TLD Thermoluminescent detector RESUME Introduction . MELiSSA (acronyme pour Micro-Ecological Life Support System Alternative) est un système clos, développé par l'Agence Spatiale Européenne pour la régénération des consommables, et qui servira de support de vie pour les futurs voyages spatiaux de longue distance. Il est composé de processus interconnectés (bioréacteurs, compartiment pour plantes supérieures, unités de filtration, etc ) ayant pour objectif de recycler les déchets organiques en oxygène, en eau et en nourriture. Au sein de la boucle MELiSSA, l' α-protéobactérie pourpre non-sulfureuse R. rubrum S1H est utilisée pour convertir les acides gras volatiles, en provenance du digesteur situé en amont, en CO 2 et en biomasse. De plus, elle complète la + minéralisation des acides aminés en NH 4 , le tout étant ensuite transmis au compartiment nitrifiant situé en aval. Parmi les nombreux défis du projet, la stabilité fonctionnelle des bioréacteurs en conditions de vol spatial est d'une importance capitale pour assurer l'efficacité du système et par conséquent la sécurité de l'équipage. Lors de cette étude, nous avons caractérisé les changements induits chez R. rubrum S1H par les conditions environnementales rencontrées pendant un vol spatial. Matériel et méthodes . R. rubrum , inoculée sur milieu agar solide en condition d'obscurité et aérobie, a séjourné dans la Station Spatiale Internationale (ISS) en octobre 2003 (expérience MESSAGE 2) et en septembre 2006 (expérience BASE-A). De plus, dans un effort pour identifier une réponse spécifique de R. rubrum au vol spatial, nous avons réalisé des simulations terrestres des paramètres de radiation ionisante et de gravité rencontrés dans l'espace, utilisant des conditions de culture et de croissance identiques à celle rencontrées lors du vol spatial proprement dit. Ces cultures ont ensuite été analysées et comparées à leur contrôle respectif au niveau transcriptomique et protéomique. Pour se faire, nous avons développé et utilisé respectivement une puce à ADN de type oligonucléotidique, couvrant l'ensemble du génome de R. rubrum , et une approche protéomique différentielle à haut débit et sans gels couplée à un marquage isotopique de type ICPL (acronyme pour Isotope-Coded Protein Label). A côté de cela, l'entièreté du contenu en protéines de R. rubrum a été caractérisée à l'aide de la technologie MudPIT (acronyme pour Multidimensional Protein Identification Technology). Résultats . Nous avons pu mettre en évidence l'importance de la composition du milieu et du dispositif de culture dans la réponse de cette bactérie aux conditions environmentales liées au vol spatial. De plus, nous avons montré pour la première fois qu'une faible dose de radiation ionisante (2 mGy) pouvait induire une réponse significative au niveau transcriptomique, mais sans altérer la viabilité de la cellule et en induisant un nombre restreint de protéines significativement régulées. A côté de cela, nous avons mis en évidence l'expression de gènes liés à la détection du quorum (quorum sensing) et à l'appareil photosynthétique de R. rubrum , au niveau transcriptomique et protéomique, lorsque la bactérie est cultivée dans l'obscurité et en condition aérobie, dans 2 dispositifs différents simulant la microgravité. Enfin, la technique MudPIT nous a permis d'identifier environ 25 % (c'est-à-dire 1.007 protéines) du protéome de R. rubrum . Associés à l'ensemble des données obtenues en étudiant une variété de conditions environmentales, ces derniers résultats seront particulièrement précieux pour l'annotation experte du génome de R. rubrum , qui compte toujours 25 % de gènes annotés comme codant pour des protéines hypothétiques. Conclusion . Notre approche, qui a intégré des données transcriptomiques et protéomiques, a permis d'évaluer la sensibilité de R. rubrum S1H à différentes conditions environmentales (vol spatial, radiation ionisante et microgravité simulée). De plus, dans la perspective d'une utilisation de cette bactérie dans un écosystème clos en conditions de lumière et anaérobie, une étude plus approfondie de la détection du quorum doit être envisagée. Finalement, des simulations terrestres impliquant la culture en bioréacteur nous apparaissent cruciales pour confirmer des changements métaboliques chez R. rubrum . SUMMARY Background . MELiSSA, which stands for 'Micro-Ecological Life Support System Alternative', is a closed regenerative life support system for future space flights under development by the European Space Agency. It consists of interconnected processes ( i.e. bioreactors, higher plant compartments, filtration units, etc.) targeting
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