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Hereditary bacterial symbiosis is a common mechanism by which eukaryotic hosts can acquire traits beneficial for their fitness. Many insects have symbiotic associations with bacteria that trace back millions of years, whose function and evolution are well characterized. Insects can also possess more recently derived symbionts that are closely related to free-living bacteria, and often play a role in host defense. Serratia symbiotica is a recently derived symbiont that infects aphids and provides protection against heat stress, and possibly also plays a nutritional role. Many aspects of the biology of recent symbionts are less well studied, including the diversity of functional roles and evolution among hosts for single lineages of symbionts, the molecular mechanisms that contribute to defense, the early stages of symbiont genome evolution, and interactions with hosts. This dissertation focuses upon S. symbiotica to contribute research addressing each of these themes. Functional studies revealed that S. symbiotica lysis during heat-shock is correlated with protection of the nutritional symbiont Buchnera , and that S. symbiotica has a large effect upon aphid metabolite pools. Despite this large metabolic effect, S. symbiotica does not seem to dramatically influence expression of aphid genes, including those involved in immunity. Analysis of the evolution of S. symbiotica lineages in different aphid hosts revealed this symbiont is common in the aphid subfamily Lachninae, but did not support the obligate nutritional role hypothesized in the literature for this group. Finally, comparison of the S. symbiotica genome to close free-living relatives revealed a genome undergoing massive decay, and provided a rare opportunity to examine the evolution of a recently acquired symbiont.

Thèse doctorat : Bioinformatique : Villeurbanne, INSA : 2004. / Titre provenant de l'écran-titre. Bibliogr. p. 273-306. Publications de l'auteur p. 269-270.

The use of stable nitrogen isotope data in ecological and physiological studies is based in the assumption that nitrogen fractionates predictably during metabolism, leading to a broadly conserved pattern whereby consumers are isotopically enriched with respect to their diets. The application of stable isotope data to such studies is limited is by our understanding of the factors in that cause variability in the Δ15N values of consumers. In particular, parasites and fluid-feeders have been shown to demonstrate isotopic depletion with respect to their food sources. One factor that has been suggested to influence the Δ15N values seen in fluid-feeding consumers is the presence of endosymbionts and their contribution to nitrogen metabolism. The experiments described in this thesis directly test the hypothesis that the endosymbiotic bacteria Buchnera aphidicola is influencing the Δ15N value of the pea aphid on host alfalfa plants. Here I find that although aphids cured of their bacterial symbionts are less isotopically depleted than untreated aphids, they are still not enriched with respect to their phloem sap diet, indicating that endosymbiont metabolism alone is not responsible for the isotopic depletion observed in pea aphids. Metabolism of nitrogen in the pea aphid-Buchnera symbiosis has been well described with decades of physiological studies and with the publication of the pea aphid and Buchnera genomes. The two key features of metabolism in the pea aphid-Buchnera symbiosis are the recycling of waste ammonia by the aphid and the upgrading of the nonessential amino acids found in phloem sap to essential amino acids through collaborative metabolism between the pea aphid and Buchnera. Consistent with the described role of Buchnera in nitrogen metabolism, amino acid analyses of symbiotic and aposymbiotic aphids demonstrates an accumulation of the nonessential amino acids glutamine and glutamate and lower amounts of essential amino acids in the aposymbiotic aphids. I tested the influence of dietary amino acid profile on the Δ15N value of pea aphids and found that aphids are only isotopically depleted when they feed on diets with unbalanced amino acid compositions and are isotopically enriched when fed on a diet with a balanced profile of amino acids. I used isotopically labeled fructose to determine whether the difference in Δ15N value of pea aphids on diets of varying amino acid profiles is correlated to the amount of de novo amino acid synthesis occurring in the aphid. I found that there was a significantly higher incorporation of the labeled carbon backbone in the protein of pea aphids feeding on the unbalanced diets, supporting the idea that increased de novo amino acid synthesis are responsible for the differences in Δ15N values among aphids feeding on the two diets. The findings of this study highlight the influence of endosymbionts on the Δ15N values for pea aphids, demonstrate that dietary amino acid composition can influence the Δ15N value of pea aphids through the demand for metabolic upgrading of amino acids, and provide a model for the study of Δ15N values in systems where metabolism has been well characterized by experimental and genomic data.

Intimate, mutualistic, association with microbes is a common mechanism for organisms to utilize certain niches. Insects are a particularly well-studied group in this respect, frequently forming long-term, obligate associations with symbiotic microbes. These symbioses are often nutritional in nature, with the symbiont providing the host with nutrients that are otherwise unavailable. Aphids are notable for their well-defined relationship with the symbiotic Bacteria Buchnera aphidicola. By synthesizing the amino acids the aphid is unable to produce itself, Buchnera permits its host to feed on plant phloem, which lacks sufficient quantities of these essential nutrients. Buchnera, as with many obligate intracellular symbionts, has a reduced effective population size (Nₑ) due to asexual reproduction and severe population bottlenecks experienced during transmission between generations. The reduction in Nₑ has facilitated the degradation of the symbiont genome through fixation of deleterious mutations via drift. The consequences of accelerated evolutionary rates has been examined primarily through genome sequencing and comparative studies of symbionts from different host species. The work detailed in this dissertation examines the role of deleterious mutations and drift at multiple taxonomic levels. Analysis of aphid amino acid requirements utilizing an artificial diet assay revealed variation in clones of the pea aphid, Acyrthosiphon pisum. In one clone, a mutation in the arginine biosynthesis pathway appears to underlie a host dietary requirement for arginine. Examination of the number of Buchnera within an A. pisum clone also revealed variation in symbiont titer between clones. When compared across F₁ offspring of cross between a low- and a high-titer clone, extensive variation was observed in titer that exceeded variation observed in field-collected clones. No maternal effects were observed, suggesting that Buchnera is not in control of its replication. At a broader taxonomic scale, the replacement of Buchnera in the aphid Cerataphis brasiliensis was examined by sequencing the genome of its fungal symbiont (YLS). The genome of the YLS revealed a much greater metabolic capacity than Buchnera, possibly due to its extracellular habitat. The YLS exhibited signatures of elevated evolutionary rates and intron gain consistent with a reduction in Nₑ due to its symbiotic niche.

L'objectif de cette thèse est l'étude par la technologie des puces à ADN, du transcriptome de la bactérie Buchnera aphidicola, qui vit en symbiose avec le puceron du pois Acyrthosiphon pisum. Le génome extrêmement réduit de Buchnera a perdu l'essentiel de ses gènes de régulation, mais conserve les voies de biosynthèse permettant la production des acides aminés essentiels pour son hôte. La première partie de cette thèse concerne le développement du logiciel ROSO, qui permet de déterminer les sondes oligonucléotidiques destinées aux puces. Une interface a été conçue pour son utilisation en ligne (http://pbil.univ-lyon1.fr/roso). Dans une seconde partie, la conception et l'utilisation d'une puce dédiée à Buchnera ont permis d'étudier le transcriptome de la bactérie, lorsque son hôte subit un stress nutritionnel et osmotique. Cette analyse montre que Buchnera est capable de réguler son expression génique et de réorienter son métabolisme, pour répondre à la demande changeante de son hôte.

Les progrès fulgurants de ces dix dernières années réalisés dans les domaines de la microinformatique et de la microfluidique associés au génie génétique (PCR et séquençage) ont permis un changement d'échelle dans la quantité des données acquises au cours d'une même expérience. La transcriptomique est directement issue de ces avancées technologiques. Ce mémoire présenté pour l'obtention d'une Habilitation à Diriger des Recherdes porte sur l'analyse du transcriptome de la bactérie intracellulaire obligatoire des pucerons, Buchnera aphidicola. Dans la première partie, les principales méthodes d'analyses statistiques différentielles et d'intégration des données transcriptomiques sont présentées sous la forme d'une analyse bibliographique. La deuxième partie est consacrée au développement d'outils bioinformatiques : ROSO, un logiciel d'optimisation des sondes oligonucléotidiques, la puce Buchnera et SITRANS, un système d'information pour la gestion et la publication des données d'expression. Enfin, la dernière partie est consacrée à la caractérisation du transcriptome de Buchnera en condition de stress trophique de son hôte, le puceron du pois Acyrthosiphon pisum. La régulation transcriptionnelle chez les bactéries symbiotiques intracellulaires à génome réduit est encore actuellement très mal connue. Cette question sera abordée chez Buchnera tout d'abord au niveau évolutif par l'étude de la relation entre l'expression des gènes et leur organisation dans le génome, puis au niveau fonctionnel, par la caractérisation de la réponse de la bactérie à une diminution de la quantité d'acides aminés essentiels dans le substrat nutritif du puceron, combinée à un stress osmotique.

Many bacteria live in close association with other organisms such as plants and animals, with important implications for both health and disease. This thesis investigates bacteria that are well adapted to live inside an animal host, and describes the molecular evolutionary processes underlying host-adaptation, based on bacterial genome comparisons. Insect-transmitted bacteria of the genus Bartonella infect the red blood cells of mammals, and we investigate host adaptation and genome evolution in this genus. In Bartonella, many host-interaction systems are encoded in a highly variable chromosomal segment previously shown to be amplified and packaged into bacteriophage particles. Among all genes imported into the Bartonella ancestor, we identify the short gene cluster encoding these phage particles as the most evolutionary conserved, indicating a strong selective advantage and a role in niche adaptation. We also provide an overview of the remarkable evolutionary dynamics of type IV and type V secretion systems, including a detailed analysis of the type IV secretion system trw. Our results highlight the importance of recombination and gene conversion in the evolution of host-adaptation systems, and reveal how these mutational mechanisms result in strikingly different outcomes depending on the selective constraints. In the insect endosymbionts Buchnera and Blochmannia, we show that genes frameshifted at poly(A) tracts can remain functional due to transcriptional slippage. Selection against poly(A) tracts is very inefficient in these genomes compared to other bacteria, and we discuss why this can lead to increased rates of gene loss. Using the human pathogen Helicobacter pylori as a model, we provide a deeper understanding of why highly expressed genes evolve slowly. This thesis emphasizes the power of using complete genome sequences to study evolutionary processes. In particular, we argue that knowledge about the complex evolution of duplicated gene segments is crucial to understand host adaptation in bacteria.

Since the introduction of advanced molecular methods the research on insect bacterial symbioses underwent a major focus shift towards large scale phylogenetics and comparative genomics. These new fields provided answers to several fundamental questions of symbiont evolution, functional capabilities of the host-associated bacteria, and the role of symbionts in the host?s biology. However, the vast diversity and complexity of symbiotic relationships still leaves gaps in our understanding to a rich mosaic of various symbiont types, effects and transitions from facultative association to obligate mutualism. The presented study focuses on distribution, diversity, phylogenetic patterns, evolutionary transitions and genome evolution of two less known but ecologically diverse bacterial genera, Arsenophonus and Sodalis. The thesis also takes advantage of the knowledge on a well established symbiotic model between aphids and Buchnera and reveals several evolutionary patterns in the host and symbiont.

Les associations symbiotiques constituent un moteur majeur de la diversification écologique et évolutive des organismes métazoaires. Chez les insectes, elles ont conduit, au cours de l’évolution, à l’émergence d’un nouveau type cellulaire spécialisé dans l’hébergement des bactéries symbiotiques, les bactériocytes. Ces cellules demeurent une énigme fascinante de la symbiose, les processus déterminant leur développement, leur morphogenèse et leur dégénérescence restant encore méconnus. Dans cette étude, nous avons utilisé la symbiose entre le puceron Acyrthosiphon pisum et son endosymbiote obligatoire, Buchnera aphidicola, comme système modèle. En combinant des approches inédites de cytométrie en flux et d’imagerie cellulaire, nous avons démontré une régulation fine et coordonnée des dynamiques de croissance et de dégénérescence des bactériocytes et des symbiotes bactériens, en accord avec les besoins physiologiques de l’insecte. De l’embryon à l’âge adulte, les cellules symbiotiques croissent de manière exponentielle, répondant aux besoins nutritionnels de l’hôte qui nécessite pour son développement de grandes quantités d’acides aminés essentiels produits par le métabolisme bactériocytaire. Avec la sénescence du puceron, les bactériocytes diminuent en nombre, en taille et subissent une dégénérescence progressive. Ce processus dégénératif ne montre pas les signes classiques de l’apoptose. Il résulte d’une hypervacuolisation cytoplasmique, dérivée du réticulum endoplasmique, déclenchant une cascade de réponses cellulaires dont l’activation des voies autophagique et lysosomale. Ce phénomène de mort cellulaire non-apoptotique en deux étapes, rappelant la paraptosis, n’a jamais été décrit chez les insectes et sa découverte ouvre la voie à l’étude des régulations agissant sur l’homéostasie bactériocytaire. Dans le dernier volet de cette thèse, nous avons procédé à l’étude fonctionnelle du gène PAH, fortement exprimé dans les bactériocytes et potentiellement impliqué dans la régulation de leur homéostasie. Les résultats obtenus n’ont pas révélé de phénotype bactériocytaire, après ARN interférence, mais ont permis de démontrer un rôle essentiel de ce gène dans la morphogenèse des insectes. / Symbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Over the evolution, they have led to the emergence, in insects, of a novel eukaryotic cell type, the bacteriocytes, specialized in harboring symbiotic bacteria. These cells constitute a fascinating enigma in cell biology, as the processes underpinning their development, morphogenesis and degeneration remain still unsolved. In my PhD thesis, we have used the nutritional symbiosis between the aphid, Acyrthosiphon pisum, and its obligate endosymbiont, Buchnera aphidicola, as a model system. We have first developed a novel approach for counting symbiotic bacteria, based on flow cytometry, and showed that the endosymbiont population increases exponentially throughout aphid nymphal development, with a growing rate that has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that bacteriocytes also increase significantly in number and size during nymphal development. Once adulthood is reached, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and bacteriocytes start to degenerate. These results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts, and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development. Interestingly, the degenerative process that bacteriocytes undergo with aging exhibits morphological features distinct from the evolutionary conserved apoptotic cell deaths. It originates from an extensive ER-derived hypervacuolation, triggering a cascade of cellular stress responses including the activation of autophagy and lysosomal pathways. This stepwise non-apoptotic cell death, sharing several features with paraptosis, has hitherto never been characterized in insects and its discovery opens the way to the identification of the molecular mechanisms acting on bacteriocyte homeostasis. In the last part of this PhD project, we have proceeded to the characterization of the PAH gene functions in aphid physiology, using an RNA interference (RNAi) approach. Our results show that, even though this gene is highly expressed in bacteriocytes, it is not involved in the regulation of their homeostasis. Nevertheless, we have demonstrated a new role for this metabolic gene in insect embryonic development and morphogenesis.