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Characterization of the Endosymbiotic Forms of Sinorhizobium Sp. Strain

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Characterization of the Endosymbiotic Forms of Sinorhizobium Sp. Strain UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de Botanique et Biologie Végétale Dr. Xavier Perret, MER ___________________________________________________________________ Characterization of the Endosymbiotic Forms of Sinorhizobium sp. Strain NGR234 THÈSE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie par Nadia BAKKOU de Salé (Maroc) Thèse N° 4286 Genève Atelier d’impression ReproMail 2011 Remerciements Cette thèse a été réalisée au sein de l’Unité de Microbiologie dans le département de Botanique et Biologie végétale à l’université de Genève. Je tiens à remercier en premier lieu le Dr. Xavier Perret qui a dirigé cette thèse, pour sa disponibilité et pour sa confiance, qui m’ont permis de mener à bien ce projet. Je tiens également à remercier le Prof. Jurek Paszkowski, pour m’avoir permis de réaliser ma thèse dans ce département. Je remercie les membres du jury: le Dr. Cornelia Reimmann et le Prof. Hans-Martin Fischer, pour m’avoir fait l’honneur d’accepter de juger mon travail, pour leur remarques et conseils pertinents. Je tiens à associer à ces remerciements toutes les personnes qui ont participé à ce projet et sans qui ce travail n’aurait pas été possible. Je remercie donc le Dr. Michèle Crèvecœur, le Dr. Peter Mergaert et le Prof. Eva Kondorosi pour leur collaboration et leur aide scientifique et technique lors de la caractérisation morphologique et physiologique des bactéroides de Sinorhizobium sp. NGR234. Je remercie sincèrement Madame Yin Aung, le Dr. William Deakin et le Prof. Bill Broughton pour leur disponibilité, leurs conseils avisés et leur soutien tout au long de ce travail. Toute l’équipe du LBMPS et les personnes qui sont passées par l’unité de Microbiologie avec qui j’ai eu beaucoup de plaisir à travailler pendant toutes ces années, en particulier j’aimerai remercier: Kumi, Silvia, Olivier, Greg, Antoine, Maged, Abder, Loic, Coralie, Natalia, Karl, Rosa, et toutes les autres personnes que j’ai oublié. Vous m’avez permis d’accomplir ce travail dans des conditions agréables et chaleureuses donc je vous adresse un grand merci pour votre compétence, pour votre efficacité, mais surtout pour votre bonne humeur, pour vos encouragements, pour votre sympathie et pour votre amitié. Je clos enfin ces remerciements en dédiant cette thèse de doctorat à mes parents, à mes sœurs, à dabdoub, à mes «apéro girls» et à tous mes amis que j'ai eus la chance d'avoir à mes côtés et qui m'ont soutenue tout au long de ces années. i ii Table of contents Page Remerciements i Résumé en français 1 English summary 3 List of abbreviations 5 List of figures 7 List of Tables 9 Chapter 1. General introduction 11 1.1. Biological nitrogen fixation 13 1.2. Legumes, rhizobia and nitrogen fixation 15 1.3. From a free-living to an endosymbiotic lifestyle 18 1.4. Symbiosome formation and bacteroid differentiation 25 1.5. Nitrogenase and the regulation of nitrogen fixation 28 1.6. The promiscuous Sinorhizobium sp. strain NGR234 33 1.7. Aims of the PhD project 41 Chapter 2. Morphological characterization of NGR234 bacteroids 43 2.1. Distinct hosts and different nodule types 45 2.2. Percoll gradients to standardize cell populations 48 2.3. Sizes and shapes of free-living cells and bacteroids of NGR234 51 2.4. Physiological characteristics of bacteroids of NGR234 53 2.5. Nodule-specific modulation of the cell cycle in bacteroids of NGR234 57 2.6. Survival of bacteroids of NGR234 isolated from nitrogen-fixing nodules 60 2.7. Conclusions to Chapter 2 61 Chapter 3. From local to global transcription profiles of the versatile NGR234 63 3.1. Generalities on transcription in prokaryotes 65 3.2. Down-regulation of housekeeping functions in bacteroids 72 iii 3.3. Characterization of selected NGR234 promoter regions 74 3.4. Assessing promoter activities using transcriptional Gus-fusions 76 3.5. Implementation of qRT-PCR to follow gene expression in NGR234 80 3.6. Do numbers of replicons vary during the NGR234 lifestyles? 82 3.7. Mapping the transcriptional start sites of reference promoters 84 3.8. Exploring the transcriptome using high-throughput sequencing 86 3.9. Conclusion to Chapter 3 96 Chapter 4. Characterization of the BxqH regulon of NGR234 99 4.1. Genetics of quorum sensing (QS) 101 4.2. QS in Sinorhizobium sp. strain NGR234 106 4.3. BxqH is structurally similar to QS-dependent regulators 108 4.4. bxqH expression inside bacteroids is 54 dependent 110 4.5. Analysis of the BxqH-regulon of NGR234 112 4.6. Role of BxqH in symbiosis 114 4.7. Construction of reporter systems dedicated to the analysis of BxqH 117 4.8. Conclusion to Chapter 4 121 Chapter 5. Functional analysis of the T3SS-II locus of NGR234 123 5.1. A symbiotic role for Type-Three Secretion Systems (T3SS) 125 5.2. pNGR234b codes for a second and complete T3SS locus 128 5.3. Transcription analysis of the T3SS-II locus 130 5.4. Construction of the T3SS-II deletion strains 132 5.5. Symbiotic phenotype of T3SS-II deleted mutants of NGR234 135 5.6. T3SS-II sequences are conserved in various strains of Sinorhizobium 136 5.7. Conclusion to Chapter 5 138 Chapter 6. Discussion 141 6.1. T3SS-II, a tool for another and yet unknown lifestyle of NGR234? 143 6.2. Many bacteroid « differentiation » states for many symbioses 147 6.3. Symbiosis and modulation of gene expression in NGR234 151 iv 6.4. Concluding remarks 160 Chapter 7. Material and Methods 165 7.1. Nodulation tests 167 7.2. Microbiology techniques 167 7.3. Preparation of RNA templates for qRT-PCR and 5’-RACE PCR 172 7.4. cDNA synthesis and Quantitative Real-Time PCR 173 7.5. Mapping of transcriptional sites with 5’ RACE-PCR 175 7.6. Microscopical analysis of nodule sections 176 7.7. Isolation of bacteroids via Percoll gradient 177 7.8. Staining and observation of endosymbiotic or free-living cells of NGR234 177 7.9. Flow cytometry and cell cycle analysis 178 7.10. Construction of gusA-reporter fusions 178 7.11. Fluorometric β-glucuronidase (Gus) assay 180 7.12. Construction of T3SS-II deleted mutant strains 180 References 183 Appendix 205 v vi Résumé en français La plupart des plantes appartenant à la famille des légumineuses ont la capacité de former des symbioses fixatrices d’azote avec des bactéries du sol appelées rhizobia. La fixation d’azote se déroule uniquement à l’intérieur d’organes spécialisés appelés nodules (ou nodosités) qui ne se développent sur les racines, et plus rarement sur les tiges des plantes hôtes, qu’en présence d’une souche bactérienne compatible. Pour fixer l’azote, les rhizobia doivent coloniser le cytoplasme des cellules végétales des nodosités et s’y différentier en bactéroïdes fixateurs d’azote. Dans les légumineuses d’importance agronomique on distingue deux principaux types de nodosités : les nodules de type déterminé (DNs) et indéterminé (IDNs) qui se distinguent notamment par leur profil de croissance et la persistance ou non de zones méristématiques. Notre souche modèle Sinorhizobium sp. NGR234, possède un spectre d’hôte particulièrement étendu. En effet, NGR234 peut induire la formation de nodules sur plus de 120 genres de légumineuses et fixe l’azote atmosphérique dans des nodules DNs ou IDNs de plus de 150 plantes appartenant à toutes les sous-familles des légumineuses. Cette extraordinaire capacité fait de NGR234 un modèle de choix pour étudier les mécanismes moléculaires nécessaires à l’établissement de symbioses fonctionnelles sur un aussi grand nombre de plantes hôtes. Encore récemment, les recherches se focalisaient essentiellement sur les mécanismes moléculaires permettant à NGR234 d’induire la formation de nodules puis de les coloniser (processus de nodulation), laissant la fixation de l’azote per se pratiquement inexplorée. Afin de combler cette importante lacune, ce travail de thèse a porté sur l’analyse comparative de caractères morphologiques et physiologiques des bactéroïdes fixateurs d’azote de NGR234, et dans le décryptage détaillé des mécanismes moléculaires qui régissent la régulation génique lors de la fixation de l’azote. Nous avons aussi tenté d’examiner la fonction du deuxième système de sécrétion de type III (T3SS-II) qui a été récemment identifié suite au séquençage du génome de NGR234. Seuls les bactéroïdes différentiés à l’intérieur des nodules sont capables de réduire l’azote atmosphérique grâce à la nitrogénase, enzyme irréversiblement inactivée par la présence d’oxygène libre. Ce processus de différenciation peut être irréversible, comme dans le cas de Sinorhizobium meliloti en symbiose avec les espèces Medicago (nodules IDNs). Les bactéries endosymbiotiques sont alors incapables de former des colonies une fois isolées des nodosités. En examinant les bactéroïdes de NGR234 isolés à partir de nodules des plantes Vigna unguiculata (DNs), Leuceana leucocephala (IDNs) ou Tephrosia vogelii (IDNs), nous avons montré que cette différenciation était réversible pour les cellules isolées des nodules DNs et irréversible pour celles isolées des nodosités IDNs. L’utilisation de colorants fluorescents en combinaison avec des techniques de microscopie et de cytométrie de flux démontra que l’irréversibilité de la différenciation n’était pas, comme c’est le cas chez -1- S. meliloti, liée à une endoréduplication du génome. En revanche, les bactéroïdes de NGR234 incapables de se multiplier bloqués en phase G0/G1 du cycle cellulaire, possédaient une perméabilité membranaire notablement altérée. Ces résultats ouvrent la porte à l’identification dans des plantes autres que les Galegae de nouveaux composés végétaux susceptibles de bloquer la croissance bactérienne. La faible pression partielle en oxygène qui règne à l’intérieur des nodules est responsable de l’expression spécifique des gènes bactériens de la fixation de l’azote.
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