Les modèles de Markov d’ordre 2 (HMM2) sont des modèles stochastiques qui ont démontré leur efficacité dans l’exploration de séquences génomiques. Cette thèse explore l’intérêt de modèles de différents types (M1M2, M2M2, M2M0) ainsi que leur couplage à des méthodes combinatoires pour segmenter les génomes bactériens sans connaissances a priori du contenu génétique. Ces approches ont été appliquées à deux modèles bactériens afin d’en valider la robustesse : Streptomyces coelicolor et Streptococcus thermophilus. Ces espèces bactériennes présentent des caractéristiques génomiques très distinctes (composition, taille du génome) en lien avec leur écosystème spécifique : le sol pour les S. coelicolor et le milieu lait pour S. thermophilus / Second-order Hidden Markov Models (HMM2) are stochastic processes with a high efficiency in exploring bacterial genome sequences. Different types of HMM2 (M1M2, M2M2, M2M0) combined to combinatorial methods were developed in a new approach to discriminate genomic regions without a priori knowledge on their genetic content. This approach was applied on two bacterial models in order to validate its achievements: Streptomyces coelicolor and Streptococcus thermophilus. These bacterial species exhibit distinct genomic traits (base composition, global genome size) in relation with their ecological niche: soil for S. coelicolor and dairy products for S. thermophilus. In S. coelicolor, a first HMM2 architecture allowed the detection of short discrete DNA heterogeneities (5-16 nucleotides in size), mostly localized in intergenic regions. The application of the method on a biologically known gene set, the SigR regulon (involved in oxidative stress response), proved the efficiency in identifying bacterial promoters. S. coelicolor shows a complex regulatory network (up to 12% of the genes may be involved in gene regulation) with more than 60 sigma factors, involved in initiation of transcription. A classification method coupled to a searching algorithm (i.e. R’MES) was developed to automatically extract the box1-spacer-box2 composite DNA motifs, structure corresponding to the typical bacterial promoter -35/-10 boxes. Among the 814 DNA motifs described for the whole S. coelicolor genome, those of sigma factors (B, WhiG) could be retrieved from the crude data. We could show that this method could be generalized by applying it successfully in a preliminary attempt to the genome of Bacillus subtilis

No description available.

No description available.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.:EIDESSTATTLICHE ERKLÄRUNG ... 2 CONTENT ... 4 SUMMARY ... 9 CHAPTER I ... 11 ORIGIN AND MICROBIOLOGY OF ACID MINE DRAINAGE ... 11 ACIDOPHILIC IRON OXIDIZING BACTERIA OF THE GENUS “FERROVUM” ... 12 APPLICATION OF OMICS-BASED APPROACHES TO CHARACTERIZE ACIDOPHILES ... 14 AIMS OF THE PRESENT WORK ... 15 CHAPTER II ... 17 ABSTRACT ... 18 INTRODUCTION ... 18 METHODS ... 19 GENOME PROJECT HISTORY ... 19 GROWTH CONDITIONS AND GENOMIC DNA PREPARATION ... 20 GENOME SEQUENCING AND ASSEMBLY ... 20 GENOME ANNOTATION ... 21 RESULTS ... 21 CLASSIFICATION AND FEATURES ... 21 GENOME PROPERTIES ... 24 INSIGHTS FROM THE GENOME SEQUENCE ... 24 COMPARATIVE GENOMICS ... 28 CONCLUSIONS ... 30 ACKNOWLEDGMENTS ... 32 AUTHOR CONTRIBUTIONS ... 32 CHAPTER III ... 33 ABSTRACT ... 34 INTRODUCTION ... 34 METHODS ... 36 ORIGIN AND CULTIVATION OF “FERROVUM” STRAIN JA12 ... 36 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 37 VISUALIZATION OF THE NEARLY COMPLETE GENOME ... 38 PHYLOGENETIC ANALYSIS ... 39 PREDICTION OF MOBILE GENETIC ELEMENTS ... 39 NUCLEOTIDE SEQUENCE ACCESSION NUMBER ... 39 RESULTS AND DISCUSSION ... 39 PHYLOGENETIC CLASSIFICATION OF “FERROVUM” STRAIN JA12 ... 39 GENOME PROPERTIES ... 40 NUTRIENT ASSIMILATION AND BIOMASS PRODUCTION ... 44 Carbon dioxide fixation ... 44 Central carbon metabolism ... 45 Nitrogen ... 47 Phosphate ... 49 Sulfate ... 50 ENERGY METABOLISM ... 50 Ferrous iron oxidation ... 50 Other redox reactions connected to the quinol pool ... 54 Predicted formate dehydrogenase ... 55 STRATEGIES TO ADAPT TO ACIDIC ENVIRONMENTS, HIGH METAL LOADS AND OXIDATIVE STRESS ... 55 Acidic environment ... 55 Strategies to cope with high metal and metalloid loads ... 58 Oxidative stress ... 59 HORIZONTAL GENE TRANSFER ... 60 CONCLUSIONS ... 61 ACKNOWLEDGMENTS ... 62 AUTHORS\' CONTRIBUTIONS ... 62 CHAPTER IV ... 63 ABSTRACT ... 64 INTRODUCTION ... 64 METHODS ... 66 ORIGIN AND CULTIVATION OF “FERROVUM” STRAINS PN-J185 AND Z-31 ... 66 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 66 PREDICTION OF MOBILE GENETIC ELEMENTS ... 67 COMPARATIVE GENOMICS ... 68 Phylogenomic analysis ... 68 Assignment of protein-coding genes to the COG classification ... 68 Identification of orthologous proteins ... 68 Comparison and analysis of genome architectures ... 69 RESULTS ... 69 GENERAL GENOME FEATURES AND PHYLOGENETIC RELATIONSHIP OF THE FOUR “FERROVUM” STRAINS ... 69 COMPARISON OF INFERRED METABOLIC TRAITS ... 71 Identification of core genes and flexible genes ... 71 Comparison of the central metabolism ... 74 Central carbon metabolism ... 74 Nitrogen metabolism ... 77 Energy metabolism ... 78 Cell mobility and chemotaxis ... 78 Diversity of predicted stress tolerance mechanisms ... 78 Maintaining the intracellular pH homeostasis ... 78 Coping with high metal loads ... 79 Oxidative stress management ... 79 IDENTIFICATION OF POTENTIAL DRIVING FORCES OF GENOME EVOLUTION ... 80 Prediction of mobile genetic elements ... 81 Linking the differences in the predicted metabolic profiles to the genome architectures ... 82 Gene cluster associated with flagella formation and chemotaxis in “F. myxofaciens” ... 84 Gene clusters associated with the utilization of alternative nitrogen sources ... 86 Gene cluster associated with carboxysome formation in “F. myxofaciens” and OTU-2 strain JA12 ... 87 Putative genomic islands in the OTU-strain JA12 ... 89 CRISPR/Cas in “F. myxofaciens” Z-31: a defense mechanism against foreign DNA ... 91 DISCUSSION ... 92 THE COMPARISON OF THEIR METABOLIC PROFILES INDICATES THE EXISTENCE OF OTU- AND STRAIN-SPECIFIC FEATURES ... 92 GENOME EVOLUTION OF THE “FERROVUM” STRAINS APPEARS TO BE DRIVEN BY HORIZONTAL GENE TRANSFER AND GENOME REDUCTION ... 94 Horizontal gene transfer ... 94 Mechanisms of genome reduction ... 95 CONCLUDING REMARKS ... 98 ACKNOWLEDGMENTS ... 98 AUTHOR CONTRIBUTIONS ... 98 CHAPTER V ... 99 ABSTRACT ... 100 INTRODUCTION ... 100 METHODS ... 102 CULTIVATION OF THE “FERROVUM”-CONTAINING MIXED CULTURE JA12 ... 102 Up-scaling of pre-cultures for the transcriptome study ... 103 Experimental setup of the transcriptome study ... 103 Cell harvest from large culture volumes ... 106 EXTRACTION OF TOTAL RNA ... 106 LIBRARY CONSTRUCTION AND SEQUENCING ... 107 DATA ANALYSIS ... 107 Processing of raw data ... 107 Quantification of gene expression levels ... 108 Functional analysis ... 108 RESULTS ... 108 CULTIVATION OF THE MIXED CULTURE JA12 IN THE MULTIPLE BIOREACTOR SYSTEM ... 108 Growth monitoring ... 108 Microbial composition ... 111 RNA SEQUENCING (RNA-SEQ) ... 112 FUNCTIONAL CATEGORIZATION OF EXPRESSED GENES ... 113 Functional assignment of highly expressed genes ... 117 Functional assignment of poorly expressed genes ... 121 COMPARISON OF EXPRESSION LEVELS OF GENES PREDICTED TO BE INVOLVED IN OXIDATIVE STRESS MANAGEMENT ... 122 DISCUSSION ... 124 METABOLIC PATHWAYS RELEVANT UNDER CULTURE CONDITIONS MIMICKING THE NATURAL CONDITIONS IN THE MINE WATER TREATMENT PLANT ... 125 Novel insights into the energy metabolism of “Ferrovum” sp. JA12 ... 125 Insights from poorly expressed genes ... 126 VARIATION OF GENE EXPRESSION PATTERNS UNDER THE DIFFERENT CONDITIONS ... 128 EVALUATION OF THE EXPERIMENTAL SET-UP INVOLVING THE MULTIPLE BIOREACTOR SYSTEM ... 129 CONCLUDING REMARKS: SIGNIFICANCE OF THE PRESENT TRANSCRIPTOME STUDY ... 130 ACKNOWLEDGMENTS ... 131 AUTHOR CONTRIBUTIONS ... 131 CHAPTER VI ... 133 ABSTRACT ... 133 EXTENDED INSIGHTS INTO THE FERROUS IRON OXIDATION IN BETAPROTEOBACTERIA ... 133 MECHANISMS OF PHYLOGENETIC AND METABOLIC DIVERSIFICATION WITHIN THE GENUS “FERROVUM” ... 136 INFERRED ROLES OF “FERROVUM” SPP. IN THE MICROBIAL NETWORK OF THE MINE WATER TREATMENT PLANT ... 138 PERSPECTIVES ... 143 REFERENCES ... 145 SUPPLEMENTARY MATERIAL ... 170 DATA DVD ... 170 SUPPLEMENTARY MATERIAL FOR CHAPTER III ... 171 NUCLEOTIDE ACCESSION NUMBERS ... 171 PHYLOGENETIC ANALYSIS ... 171 GENOME PROPERTIES ... 173 NUTRIENT ASSIMILATION ... 174 Carbon metabolism ... 174 FERROUS IRON OXIDATION ... 176 HORIZONTAL GENE TRANSFER ... 179 SUPPLEMENTARY MATERIAL FOR CHAPTER IV ... 180 PHYLOGENETIC ANALYSIS ... 180 ASSIGNMENT OF PROTEIN-CODING GENES TO THE COG CLASSIFICATION ... 180 COMPARISON OF THE CENTRAL METABOLISM ... 181 Predicted metabolic potential of the four “Ferrovum” strains ... 181 Genes predicted to be involved in the central metabolism, energy metabolism, cell motility and stress management in the four “Ferrovum” strains ... 183 PREDICTED MOBILE GENETIC ELEMENTS IN THE GENOMES OF THE FOUR “FERROVUM” STRAINS ... 184 THE FLAGELLA AND CHEMOTAXIS GENE CLUSTER ... 184 THE UREASE GENE CLUSTER ... 185 THE CARBOXYSOME GENE CLUSTER ... 186 PUTATIVE GENOMIC ISLANDS IN “FERROVUM” SP. JA12 ... 187 Gene content of the genomic islands ... 187 Flanking sites of the putative genomic islands 1 and 2 ... 188 SUPPLEMENTARY MATERIAL FOR CHAPTER V ... 189 ORGANIZATION AND OPERATION OF THE LABFORS 5 MULTIPLE BIOREACTOR SYSTEM ... 189 INVESTIGATION OF THE MICROBIAL COMPOSITION IN THE IRON OXIDIZING MIXED CULTURE JA12 ... 192 SUPPLEMENTARY DATA OF THE TRANSCRIPTOME DATA ANALYSIS ... 193 RNA-Seq statistics ... 193 Expression strength of protein-coding genes ... 194 Expression of genes involved in carboxysome formation ... 197 Expression of a ribosomal proteins-encoding gene cluster ... 199 Expression of a gene cluster presumably involved in ferrous iron oxidation ... 202 Lowest expressed genes ... 205 Expression of genes predicted to be involved in oxidative stress response ... 206 ACKNOWLEDGMENTS ... 208 COLLEAGUES ... 208 ERFOLGSTEAM “JUNGE FRAUEN AN DIE SPITZE” (“YOUNG WOMEN TO THE TOP“) ... 208 FAMILY AND FRIENDS ... 209 FUNDING ... 209 CURRICULUM VITAE ... 210 LIST OF PUBLICATIONS ... 212 RESEARCH ARTICLES ... 212 CONFERENCE PROCEEDINGS ... 212 ORAL PRESENTATIONS AND POSTERS ... 213

Bien que l’évolution de la résistance aux médicaments soit l'un des plus grands défis dans la lutte contre les maladies infectieuses, beaucoup de phénomènes restent inexpliqués. Dans le cas de la leishmaniose, la résistance est un problème majeur depuis plusieurs années. Cette maladie zoonotique négligée est causée par le parasite protozoaire Leishmania, transmis par des phlébotomes lors du repas sanguin. Chez l’humain, elle se décline en trois formes principales : viscérale, cutanée et muco-cutanée. La leishmaniose peut aussi se développer chez les chiens infectés, qui constituent un réservoir majeur de transmission. Sans vaccin efficace, le contrôle de la maladie repose principalement sur la chimiothérapie, mais peu de molécules homologuées sont disponibles. En outre, les mêmes produits sont utilisés chez les chiens et les humains, ce qui favorise l'émergence et la propagation de souches résistantes aux médicaments. Leishmania est en effet connu pour détenir d’incroyables particularités génomiques (telles que la formation d’amplicons circulaires contenant des gènes de résistance) qui lui permettent de survivre dans des conditions de stress, telles que la pression médicamenteuse. Par ailleurs, Leishmania est un eucaryote qui a conservé la capacité de produire des vésicules extracellulaires (EVs) au cours de l’évolution. Ces particules de taille nanométrique sont produites naturellement par la majorité des cellules biologiques et ont un contenu riche en protéines, lipides et acides nucléiques. Bien que des caractéristiques clés des EVs du protozoaire aient été découvertes, aucune étude n’a encore été réalisée sur les EVs de souches résistantes. Ainsi, cet aspect constitue le cœur de mon projet de recherche de doctorat : l’étude des EVs de Leishmania et leur rôle dans le phénomène de la résistance aux médicaments. Nos travaux sont les premiers à démontrer que les mécanismes de résistance aux médicaments peuvent induire des changements dans la morphologie, la taille et la distribution des EVs de Leishmania. Nous avons identifié le protéome de base des EVs du parasite et nous avons mis en évidence des protéines enrichies dans les EVs libérées par des parasites résistants à l'antimoine, à la miltéfosine et à l'amphotéricine B. Nous avons également étudié le contenu en ADN des EVs de parasites résistants et confirmé l'enrichissement des amplicons porteurs de gènes de résistance aux médicaments, associés aux EVs. En complément, nos tests de transferts d’EVs ont prouvé que ces vésicules permettent le transfert horizontal de gènes de résistance : un mécanisme alternatif de résistance aux médicaments. Finalement, nous avons montré que les EVs des parasites résistants améliorent la croissance des promastigotes et réduisent l'accumulation de ROS, ce qui favorise la survie et la propagation des populations résistantes aux médicaments. En conclusion, ces découvertes permettront le développement de nouveaux tests diagnostiques et de nouvelles approches thérapeutiques pour certaines maladies infectieuses zoonotiques, basés sur les profils des EVs. De plus, elles ont une importance majeure dans la compréhension de la résistance médicamenteuse et prouve que les EVs fonctionnent comme des médiateurs efficaces dans le transfert horizontal de gène. Ce nouveau mécanisme facilite ainsi la transmission des gènes de résistance aux médicaments entre les parasites et favorise leur survie lorsqu'ils sont confrontés à des environnements stressants. / Although the evolution of drug resistance is one of the greatest challenges in the fight against infectious diseases, many phenomena remain unexplained. In the case of leishmaniasis, resistance has been a major problem for several years. This neglected zoonotic disease is caused by the protozoan parasite Leishmania, transmitted by sandflies during the blood meal. In humans, it comes in three main forms: visceral, cutaneous, and mucocutaneous. Leishmaniasis can also develop in infected dogs, which constitute a major reservoir of transmission. Without an effective vaccine, the control of the disease relies mainly on chemotherapy, but few approved molecules are available. Furthermore, the same products are used in dogs and humans, which promotes the emergence and spread of drug-resistant strains. Leishmania is indeed known to possess incredible genomic peculiarities (such as the formation of circular amplicons containing resistance genes) which allow it to survive under stressful conditions, such as drug pressure. Furthermore, Leishmania is a eukaryote that has retained the ability to produce extracellular vesicles (EVs) during evolution. These nano-sized particles are produced naturally by most biological cells and have a rich content of proteins, lipids, and nucleic acids. Although key characteristics of the protozoan EVs have been discovered, no studies have yet been performed on the EVs of resistant strains. Thus, this aspect constitutes the heart of my doctoral research project: the study of Leishmania EVs and their role in the phenomenon of drug resistance. Our work is the first to demonstrate that drug resistance mechanisms can induce changes in the morphology, size, and distribution of Leishmania EVs. We have identified the basic proteome of parasite EVs, and we have demonstrated enriched proteins in EVs released by parasites resistant to antimony, miltefosine and amphotericin B. We also studied the DNA content of EVs from resistant parasites and confirmed the enrichment of amplicons carrying drug resistance genes associated with EVs. In addition, our EV transfer tests have proven that these vesicles allow the horizontal transfer of resistance genes: an alternative mechanism of drug resistance. Finally, we showed that EVs from resistant parasites enhance promastigote growth and reduce ROS accumulation, which promotes the survival and spread of drug-resistant populations. In conclusion, these discoveries will allow the development of new diagnostic tests and new therapeutic approaches for certain zoonotic infectious diseases, based on the profiles of EVs. Moreover, these findings are of major importance in the understanding of drug resistance and prove that EVs function as effective mediators in horizontal gene transfer. This new mechanism thus facilitates the transmission of drug resistance genes between parasites and promotes their survival when confronted with stressful environments.

Les champignons mycorhiziens à arbuscules (CMA) sont des organismes microscopiques du sol qui jouent un rôle crucial dans les écosystèmes naturels et que l’on retrouve dans tous les habitats de la planète. Ils vivent en relation symbiotique avec la vaste majorité des plantes terrestres. Ils sont des biotrophes obligatoires, c'est-à-dire qu'ils ne peuvent croître qu'en présence d'une plante hôte. Cette symbiose permet entre autres à la plante d'acquérir des nutriments supplémentaires, en particulier du phosphore et du nitrate. Malgré le fait que cette symbiose apporte des services importants aux écosystèmes, la richesse des espèces, la structure des communautés, ainsi que la diversité fonctionnelle des CMA sont mal connues et l'approfondissement des connaissances dans ces domaines dépend d’outils de diagnostic moléculaire. Cependant, la présence de polymorphisme nucléaire intra-isolat combiné à un manque de données génomiques dans différents groupes phylogénétique de ces champignons complique le développement de marqueurs moléculaires et la détermination de l'affiliation évolutive à hauts niveaux de résolution (c.a.d. entre espèces génétiquement similaires et/ou isolats de la même espèce). . Pour ces raisons, il semble une bonne alternative d’utiliser un système génétique différent en ciblant le génome mitochondrial, qui a été démontré homogène au sein d'un même isolat de CMA. Cependant, étant donné le mode de vie particulier de ces organismes, une meilleure compréhension des processus évolutifs mitochondriaux est nécessaire afin de valoriser l'utilisation de tels marqueurs dans des études de diversité et en génétique des populations. En ce sens, mon projet de doctorat consistait à investiguerétudier: i) les vecteurs de divergences inter-isolats et -espèces génétiquement rapprochéesphylogénétiquement apparentées, ii) la plasticité des génomes mitochondriaux, iii) l'héritabilité mitochondriale et les mécanismes potentiels de ségrégation, ainsi que iv) la diversité mitochondriale intra-isolat in situ. À l'aide de la génomique mitochondriale comparative, en utilisant le séquençage nouvelle génération, on a démontré la présence de variation génétique substantielle inter-isolats et -espèces, engendrées par l'invasion d'éléments mobiles dans les génomes mitochondriaux des CMA, donnant lieu à une évolution moléculaire rapide des régions intergéniques. Cette variation permettait de développer des marqueurs spécifiques à des isolats de la même espèce. Ensuite, à l'aide d'une approche analytique par réseaux de gènes sur des éléments mobiles, on a été en mesure de démontrer des évènements de recombinaisons homologues entre des haplotypes mitochondriaux distincts, menant à des réarrangements génomiques. Cela a permis d'ouvrir les perspectives sur la dynamique mitochondriale et l'hétéroplasmie dans un même isolatsuggère une coexistence de différents haplotypes mitochondriaux dans les populations naturelles et que les cultures monosporales pourraient induirent une sous-estimation de la diversité allélique mitochondriale. Cette apparente contradiction avec l'homogénéité mitochondriale intra-isolat généralement observée, a amené à investiguer étudier les échanges génétiques à l'aide de croisements d'isolats génétiquement distincts. Malgré l'observation de quelques spores filles hétéroplasmiques, l'homoplasmie était le statut par défaut dans toutes les cultures monosporales, avec un biais en faveur de l'un des haplotypes parentaux. Ces résultats suggèrent que la ségrégation opère durant la formation de la spore et/ou le développement de la coloniedu mycélium. De plus, ils supportent la présence d'une machinerie protéique de ségrégation mitochondriale chez les CMAAMF, où l'ensemble des gènes impliqués dans ce mécanisme ont été retrouvé et sont orthologues aux autres champignons. Finalement, on est revenue aux sources avecon a étudié le polymorphisme mitochondrial intra-isolat à l'aide d'une approche conventionnelle de PCR en utilisant une Taq polymérase de haute fidélité, suivie de clonage et de séquençage Sanger, sur deux isolats de R. irregularis. Cela a permis l'observation d'hétéroplasmie in situ, ainsi que la co-expression de variantes de variantes de protéines'ARNm dans une souche in vitro. Les résultats suggèrent que d'autres études basées sur le séquençage nouvelle génération aurait potentiellement ignorée cette variation, offrant ainsi plusieurs nouveaux arguments permettant de considérer les CMA comme des organismes possédant une population de génomes mitochondriaux et nucléaires distincts. / The association between arbuscular mycorrhizal fungi (AMF) and plant roots is one of the most widespread symbioses involving plants, and thus has an important role in terrestrial ecosystems. In exchange for carbohydrates, AMF improve plant fitness by enhancing mineral nutrient uptake, especially in particular phosphate and nitrate. Although this symbiosisDespite the fact that these symbioses contribute provides to important services toin ecosystems, the species richness, community structure and functional diversity of AMF is not well understood due to a lack of reliable molecular tools. The intra-isolate genetic polymorphism of nuclear DNA observed in AMF, combined with a lack of genomic data in a broad range of phylogenetic groups, has made it difficult to develop molecular markers and to determine evolutionary relatedness at high levels of resolution (i.e. between genetically-similar species and/or isolates). For these reasons, it seems a good alternative to use a different genetic system by targeting the mitochondrial genome, which have been shown to be homogeneous within AMF isolates. However, given the peculiar lifestyle of these organisms, a better understanding of the mitochondrial evolutionary processes and dynamics were is necessary in order to validate the usefulness of such markers in diversity and population genetics studies. In that regard, the objectives of my PhD project were to investigate: i) the divergence between closely related species and isolates, ii) mitochondrial genomes plasticity, iii) mitochondrial heritability and potential segregation mechanisms and iv) in situ mitochondrial intra-isolate allelic diversity. With Using comparative mitochondrial genomics using and next generation sequencing (NGS) sequencing, we found substantial sequence variation in intergenic regions caused by the invasion of mobile genetic elements. This variation gives risecontributes to rapid mitochondrial genome evolution among closely related isolates and species, which makes it possible to design reliable intra- and inter-specific markers. Also, an extensive gene similarity network-based approach allowed us to provide strong evidence of inter-haplotype recombination in AMF, leading to a reshuffled mitochondrial genome. These findings suggest the coexistence of distinct mtDNA haplotypes in natural populations and raise questions as to whether AMF single spore cultivations artificially underestimates mitochondrial genetic diversity in natural population.. This apparent contradiction with the intra-isolate mtDNA homogeneity usually observed in these fungi, led to the investigation of mitochondrial heritability in the spore progeny resulting from crossed-cultures. Although an heteroplasmic state was observed in some daughter spores, we found that homoplasmy was the dominant state in all monosporal cultures, with an apparent bias towards one of the parental haplotypes. These results strongly support the presence of a putative mitochondrial segregation proteic machinery in AMF, whose complete set of genes were orthologous with those found in other fungi. Our findings suggest that segregation takes place either during spore formation or colony mycelium development. Finally, we performed a conventional PCR based approach with a high fidelity Taq polymerase, followed by downstream cloning and Sanger sequencing using the model organism Rhizophagus irregularis. We found in situ heteroplasmy along with substantial intra-isolate allelic variation within the mtDNA that persists in the transcriptome. Our study also suggest that genetic variation in Glomeromycota is higher than meets the eye and might be critically underestimated in most NGS based-AMF studies both in nuclei and mitochondria.

Το βακτήριο Wolbachia είναι ένα ενδοκυττάριο και μητρικά κληρονομούμενο συμβιωτικό βακτήριο. Ανήκει στην ομοταξία των Alphaproteobacteria και την τάξη των Rickettsiales. Αποτελεί ίσως τον πιο διαδεδομένο ενδοκυττάριο συμβιωτικό οργανισμό στον πλανήτη, καθώς έχει εντοπιστεί μέχρι στιγμής σε πληθώρα αρθροπόδων και νηματωδών της φιλαρίασης. Πρόσφατες μελέτες εκτιμούν ότι πάνω από το 40% των ειδών αρθροπόδων είναι μολυσμένα με το βακτήριο Wolbachia. Το συμβιωτικό αυτό βακτήριο επηρεάζει τις βιολογικές λειτουργίες και ιδιότητες των ξενιστών του και είναι υπεύθυνο για μια σειρά αναπαραγωγικών ανωμαλιών, όπως η κυτταροπλασματική ασυμβατότητα, η παρθενογένεση, η θανάτωση των αρσενικών εμβρύων και η θηλυκοποίηση. Τα μοναδικά αυτά βιολογικά χαρακτηριστικά του βακτηρίου Wolbachia προσελκύουν όλο και περισσότερο το ενδιαφέρον διαφόρων ερευνητών τόσο για το ρόλο του βακτηρίου σε εξελικτικές διαδικασίες (κυρίως ειδογένεση) όσο και για τη χρησιμοποίησή του σε περιβαλλοντικά φιλικές εφαρμογές καταπολέμησης οργανισμών που είναι επιβλαβείς στους τομείς του γεωργικού και δασικού περιβάλλοντος, και της υγείας. Τα είδη του γένους Glossina (Diptera: Glossinidae), γνωστά και ως μύγες τσε-τσε, αποτελούν ξενιστές του βακτηρίου Wolbachia. Η μύγα τσε-τσε είναι ο σημαντικότερος φορέας των παθογόνων τρυπανοσωμάτων στην τροπική Αφρική, τα οποία προκαλούν την ασθένεια του ύπνου (sleeping sickness) στον άνθρωπο και την αντίστοιχη τρυπανοσωμίαση, γνωστή ως nagana, στα ζώα. Η χρησιμοποίηση του βακτηρίου Wolbachia σε μεθόδους βιολογικής καταπολέμησης της μύγας τσε-τσε προαπαιτεί την πλήρη γνώση της γενετικής του ταυτότητας και των αλληλεπιδράσεων του με το ξενιστή. Προς την κατεύθυνση αυτή, και στα πλαίσια της παρούσας διατριβής, πραγματοποιήθηκε η ανίχνευση του συμβιωτικού βακτηρίου Wolbachia σε περισσότερα από 5300 άτομα από φυσικούς και εργαστηριακούς πληθυσμούς 11 διαφορετικών ειδών μύγας τσε-τσε από 13 Αφρικανικές χώρες. Τα αποτελέσματα έδειξαν τεράστια απόκλιση της παρουσίας του βακτηρίου τόσο μεταξύ ειδών όσο και μεταξύ πληθυσμών του ίδιου είδους. Επίσης, πραγματοποιήθηκε ο γενετικός χαρακτηρισμός των στελεχών Wolbachia από συνολικά 29 αντιπροσωπευτικά δείγματα διαφόρων πληθυσμών και ειδών μύγας τσε-τσε, ενώ σε αρκετά από αυτά παρατηρήθηκαν πολλαπλά στελέχη του βακτηρίου. Διαπιστώθηκε εντυπωσιακή γενετική ποικιλότητα στελεχών Wolbachia που απαντούν στα διάφορα είδη μύγας τσε-τσε καθώς και ασυμφωνία μεταξύ των φυλογενειών των στελεχών Wolbachia και των μυγών τσε-τσε ξενιστών της, γεγονός που σημαίνει οριζόντια μετακίνηση του συμβιωτικού βακτηρίου κατά την εξέλιξη. Επιπρόσθετα, εντοπίστηκαν για πρώτη φορά εκτεταμένα γεγονότα οριζόντιας μεταφοράς βακτηριακών γονιδίων στο γονιδίωμα τριών ειδών μύγας τσε-τσε: στο Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. Από εξελικτικής σκοπιάς, κρίσιμα ερωτήματα προκύπτουν από τα παραπάνω ευρήματα, και πιο συγκεκριμένα σχετικά με: την προέλευση-μηχανισμό αυτών των γεγονότων οριζόντιας μεταφοράς, τον χρονικό προσδιορισμό τους, τον πιθανό ρόλο τους σε διαδικασίες ειδογένεσης και την επιλεκτική εμφάνισή τους σε ορισμένα μόνο είδη Glossina π.χ. στo υποείδos Glossina morsitans centralis που είναι πολύ συγγενικό του Glossina morsitans morsitans δεν παρατηρήθηκε το φαινόμενο. Εξίσου σημαντική και επιβεβλημένη κρίνεται η διεξοδική διερεύνηση του ενδεχομένου τα βακτηριακά γονίδια που ενσωματώθηκαν στο ευκαρυωτικό γονιδίωμα της μύγας τσε-τσε να ευθύνονται για την έκφραση νέων λειτουργιών-ιδιοτήτων (ή να μεταβάλλουν τις ήδη υπάρχουσες), ιδίως μάλιστα εάν αυτές συνδέονται με την αποδοτικότητα μετάδοσης της νόσου της τρυπανοσωμίασης μέσω του φορέα της, δηλαδή της μύγας τσε-τσε. Τέλος, διαπιστώθηκε πιθανή αρνητική συσχέτιση της παρουσίας του βακτηρίου Wolbachia με τον παθογόνο ιό Salivary Gland hypertrophy Virus (SGHV), γεγονός που συζητείται στα πλαίσια βιολογικών εφαρμογών καταπολέμησης του εντόμου-φορέα και της τρυπανοσωμίασης. Παράλληλα, μεγάλο ενδιαφέρον παρουσιάζει η προοπτική χρησιμοποίησης του βακτηρίου Wolbachia για τη βιολογική καταπολέμηση εντόμων αγροτικής ή /και περιβαλλοντικής σημασίας, όπως είναι οι αφίδες και η καρπόκαψα καστανιάς. Το γεγονός αυτό προϋποθέτει την ανίχνευση και τη γενετική ταυτοποίηση του βακτηρίου σε φυσικούς πληθυσμούς εντόμων. Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε ανίχνευση και χαρακτηρισμός του βακτηρίου Wolbachia σε 78 συνολικά άτομα από 22 είδη αφίδων, από 26 φυσικούς πληθυσμούς από την Ελλάδα. Από αυτούς τους 26 πληθυσμούς, μόλις οι 4 βρέθηκαν να είναι μολυσμένοι με το βακτήριο Wolbachia και συγκεκριμένα πληθυσμοί των ειδών: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. Τα αποτελέσματα αυτά δείχνουν για πρώτη φορά ότι η παρουσία του βακτηρίου Wolbachia στις αφίδες είναι πιθανά πιο διαδεδομένη από ότι προέκυπτε από προηγούμενες μελέτες. Επίσης, μελετήθηκε η ανίχνευση και ο χαρακτηρισμός του βακτηρίου Wolbachia στα είδη Cydia splendana, Cydia fagiglandana και Pammene fasciana. Το βακτήριο Wolbachia ανιχνεύθηκε για πρώτη φορά στα συγκεκριμένα είδη και μάλιστα διαπιστώθηκε ότι η συχνότητα εμφάνισής του ποικίλει τόσο μεταξύ των δύο ειδών Cydia όσο και μεταξύ των πληθυσμών του κάθε είδους. Στο είδος Pammene fasciana, το βακτήριο ανιχνεύθηκε σε όλα τα άτομα που μελετήθηκαν. Τα αποτελέσματα της παρούσας διατριβής συζητούνται από τη σκοπιά τόσο της οικολογικής και εξελικτικής σημασίας τους όσο και της προοπτικής χρησιμοποίησης του συμβιωτικού βακτηρίου Wolbachia για τον πληθυσμιακό έλεγχο επιβλαβών εντόμων όπως οι μύγες τσε-τσε, οι αφίδες και η καρπόκαψα καστανιάς. / Wolbachia is an intracellular and maternally inherited symbiotic bacterium that belongs to the class of Alphaproteobacteria and the order of Rickettsiales. It is the most ubiquitous intracellular symbiotic organism of the planet, since it has been estimated that over 40% of insect species, in addition to filarial nematodes, crustaceans, and arachnids are infected with Wolbachia. In arthropods Wolbachia affects the biological functions and properties of its hosts and it is responsible for a number of reproductive abnormalities, such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males and male killing. These unique biological characteristics of Wolbachia are attracting the interest of various researchers for: (a) decyphering the role of Wolbachia in evolutionary processes (mainly speciation), and (b) for its use in environmentally friendly applications for the control of agricultural pests and disease vectors. The species of genus Glossina (Diptera: Glossinidae) known as tsetse flies, have been found to be infected with Wolbachia. Tsetse flies are the sole vectors of pathogenic trypanosomes in tropical Africa, causing the “sleeping sickness” in humans and the “nagana” in animals. The potential use of Wolbachia for the control of tsetse flies, prerequisite a thorough knowledge of its genetic identity and the interactions with the host. To further characterize the prevalence of Wolbachia in tsetse flies an extensive screen of more than 5300 specimens from natural and laboratory populations of 11 different Glossina species originating from 13 African countries was carried out. Our results indicated a huge divergence in the prevalence of Wolbachia, both among the species and among populations of the same species. Further characterization by MLST and wsp genotyping was carried out for the Wolbachia strains of 29 representative populations and species of tsetse flies. An impressive genetic diversity of Wolbachia strains in tsetse flies was revealed. Interestingly, disconcordance between the phylogeny of Wolbachia and that of the tsetse flies was observed, suggesting horizontal transmission of Wolbachia during the evolution. Moreover, extended horizontal gene transfer events were detected for first time in Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. These results raise critical questions concerning: (a) the origin/mechanism of these horizontal gene transfer events, (b) their temporal determination, (c) their potential role as agents of speciation and (d) their selective appearance in only some Glossina species e.g in the subspecies Glossina morsitans centralis which is closely related with Glossina morsitans morsitans the phenomenon was not observed. Equally important will be to examine if genes from the chromosomal insertions were potentially expressed and examine if these genes are associated with the vectorial capacity of tsetse flies for the trypanosoma transmission. Finally, a negative correlation between the presence of Wolbachia with the Salivary Gland Hypertrophy Virus (SGHV) was identified. This is further discussed in the context of biological applications for control of tsetse fly-vector and trypanosomiasis. Finally in this thesis, the detection and characterization of Wolbachia in 78 specimens of 22 aphids species, from 26 natural populations, from Greece was examined. Only 4 out of 26 populations were found to be infected with Wolbachia, and specifically the species: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. These results indicated that the presence of Wolbachia in aphids is probably more prevalent than it was derived from previous studies. Also, detection and characterization of Wolbachia in the Cydia splendana, Cydia fagiglandana and Pammene fasciana was carried out. Wolbachia was detected for first time in these species, and it was found that the prevalence of Wolbachia varies between the two species of Cydia and among populations of each species, with the infection in Pammene fasciana being fixed. At the end the ecological and evolutionary importance of Wolbachia, together with the use of the bacterium for the population control of harmful insects like tsetse flies, aphids and moths is further discussed.