Graph-based analysis of the metabolic exchanges between two co-resident intracellular symbionts, baumannia cicadellinicola and sulcia muelleri with their insect host, homalodisca coagulata

International audienceEndosymbiotic bacteria from different species can live inside cells of the same eukaryotic organism. Metabolic exchanges occur between host and bacteria but also between different endocytobionts. Since a complete genome annotation is available for both, we built the metabolic network of two endosymbiotic bacteria, Sulcia muelleri and Baumannia cicadellinicola, that live inside specific cells of the sharpshooter Homalodisca coagulata and studied the metabolic exchanges involving transfers of carbon atoms between the three. We automatically determined the set of metabolites potentially exogenously acquired (seeds) for both metabolic networks. We show that the number of seeds needed by both bacteria in the carbon metabolism is extremely reduced. Moreover, only three seeds are common to both metabolic networks, indicating that the complementarity of the two metabolisms is not only manifested in the metabolic capabilities of each bacterium, but also by their different use of the same environment. Furthermore, our results show that the carbon metabolism of S. muelleri may be completely independent of the metabolic network of B. cicadellinicola. On the contrary, the carbon metabolism of the latter appears dependent on the metabolism of S. muelleri, at least for two essential amino acids, threonine and lysine. Next, in order to define which subsets of seeds (precursor sets) are sufficient to produce the metabolites involved in a symbiotic function, we used a graph-based method, PITUFO, that we recently developed. Our results highly refine our knowledge about the complementarity between the metabolisms of the two bacteria and their host. We thus indicate seeds that appear obligatory in the synthesis of metabolites are involved in the symbiotic function. Our results suggest both B. cicadellinicola and S. muelleri may be completely independent of the metabolites provided by the co-resident endocytobiont to produce the carbon backbone of the metabolites provided to the symbiotic system (., thr and lys are only exploited by B. cicadellinicola to produce its proteins)

The Symbiome of Llaveia Cochineals (Hemiptera: Coccoidea: Monophlebidae) Includes a Gammaproteobacterial Cosymbiont Sodalis TME1 and the Known Candidatus Walczuchella monophlebidarum

The genome and transcriptome of the endosymbiotic flavobacterium Candidatus Walczuchella monophlebidarum revealed its role in the synthesis of essential amino acids for its host, the wax cochineal Llaveia axin axin. There were, however, missing genes in the endosymbiont for some biosynthetic pathways. Here, we characterized TME1, another cochineal symbiont that may metabolically complement Walczuchella. TME1 was ascribed to the gammaproteobacterial genus Sodalis on a phylogenomic basis using gene sequences from 143 proteins core genome sequences and the core average nucleotide identity (ANI) confirmed its position. Additionally, we describe Sodalis as a coherent genus. TME1 genome is around 3.4 Mb and has complete gene sequences for the biosynthesis of 10 essential amino acids, for polyamines, flagella, nitrate respiration, and detoxification among many others. Transcripts from ovaries and bacteriomes allowed the identification of differentially transcribed genes from the endosymbionts and host. Highly transcribed genes were identified in TME1 and transcripts involved in amino acid biosynthesis were found. We review here that cosymbionts that derived from different bacterial classes and genera seem to be advantageous for insects that have Flavobacteria as the primary endosymbionts

Symbionts associated with the salivary glands of the potato leafhopper, Empoasca fabae, and their function when feeding on leguminous hosts

Many species of phloem feeding insects are host to symbiotic bacteria, which provide their hosts with nutrients lacking from phloem. The potato leafhopper, Empoasca fabae, feeds on the phloem and cell contents of a wide variety of plants. In this study, I identified two taxa of symbiotic bacteria present in the salivary glands, midgut, bacteriomes and saliva of the potato leafhopper: Sulcia muelleri and Wolbachia. Treatment of the leafhoppers with 0.01% oxytetracycline-HCl resulted in the creation of aposymbiotic leafhoppers. Longevity and fecundity of aposymbiotic leafhoppers on alfalfa, Medicago sativa, and fava bean, Vicia faba, were significantly lower than that of symbiotic leafhoppers. In addition, aposymbiotic leafhoppers caused less of a decrease in photosynthesis rates on both alfalfa and fava bean in comparison to symbiotic leafhoppers. The salivary gland transcriptome of the potato leafhopper revealed the presence of potential salivary components, including lipase, pectin lyase and alkaline phosphatase, all of which were expressed at higher levels in salivary glands than in midgut or hind femur tissue. In addition, transcripts attributed to Wolbachia were discovered in the sialotranscriptome, providing more evidence that this bacterium is present in the salivary glands of the potato leafhopper. Finally, expression of alfalfa wound response genes after exposure to potato leafhopper saliva was measured. Endo 1-3 ß-D-glucanase, isoflavone reductase, chalcone synthase and phenylalanine ammonia-lyase gene expression were higher in plants exposed to leafhopper saliva than in unexposed controls. Treatment of saliva with heat, filter sterilization, DTT, EDTA and K2HPO4 led to different plant wound response gene expression patterns. I conclude that the symbionts present in the potato leafhopper are necessary for the normal development and reproduction of this species, in addition to playing a potential role in plant wound response to feeding

Vliv endosymbiotických baktérií na schopnost pavouků (Araneae) poskytovat ekosystémové služby

The impact of the microbial community as well as individual strains of endosymbiotic bacteria on spider hosts has only begun to be increasingly studied in the last 20 years. This is primarily due to new molecular methods and devices, but on the other hand also to a growing desire to unravel the mechanisms behind the ability of spiders to provide ecosystem services. Their unique contribution lies in their high diversity, their generalist hunting strategy, and their efficient long-distance dispersal called ballooning. However, their abilities are possibly affected by the tiny organisms inhabiting their tissues - from the haemolymph to the digestive tract and the reproductive organs. In this work, attention is paid to the aspects that are influenced or manipulated by microbes for the purpose of their own transmission. Studies investigating known reproductive manipulators such as Wobachia, Cardinium or Rickettsia are summarized. The work summarizes recent findings in the areas of the impact of endosymbiotic bacteria on metabolic pathways and nutritional support, but also on behavioral aspects such as predatory/antipredatory behavior, defense against parasitoids or pathogens, and dispersal or avoidance of stress. Information about spiders is also related to knowledge about other invertebrates. Keywords:...Vliv mikrobiálního společenstva i jednotlivých kmenů endosymbiotických bakterií na aspekty pavouka jako hostitele se začal intenzivněji studovat teprve v posledních 20 letech. Je to především díky novým molekulárním metodám a přístrojům, ale na druhé straně také díky rostoucí snaze odhalit mechanismy, které stojí za schopností pavouků poskytovat člověku užitečné ekosystémové služby. Jejich přínos spočívá ve vysoké diverzitě, v jejich generalistické strategii lovu a v efektivním šíření na velké vzdálenosti, tzv. ballooningu. Všechny tyto vlastnosti jsou však na pozadí dost možná ovlivňovány drobnými organismy obývajícími tkáně jejich hostitelů - od hemolymfy po trávicí soustavu a reprodukční orgány. V této práci je věnována pozornost právě těm životním aspektům, které jsou ovlivňovány nebo manipulovány mikroby za účelem jejich vlastní proliferace. Jsou zde shrnuty studie zkoumající notoricky známé manipulátory reprodukce, jako jsou Wobachia, Cardinium nebo Rickettsia. Práce shrnuje recentní poznatky v oblastech vlivu endosymbiotických bakterií a jejich společenstev na metabolické dráhy a výživu, ale také na behaviorální oblasti, jako je predační/anti-predační chování, obrana proti patogenům, disperse a vyhýbáni se stresu. Informace ze skupiny pavouků jsou dávány do kontextu poznatků o ostatních...Department of EcologyKatedra ekologieFaculty of SciencePřírodovědecká fakult

Genome evolution and systems biology in bacterial endosymbionts of insects

Gene loss is the most important event in the process of genome reduction that appears associated with bacterial endosymbionts of insects. These small genomes were derived features evolved from ancestral prokaryotes with larger genome sizes, consequence of a massive process of genome reduction due to drastic changes in the ecological conditions and evolutionary pressures acting on these prokaryotic lineages during their ecological transition to host-dependent lifestyle. In the present thesis, the process of genome reduction is studied from different perspectives. In the first chapter, genome rearrangements have been studied in a set of 31 complete γ-proteobacterial genomes that includes five genomes of bacterial endosymbionts of insects. This is carried out by comparing the order of a subset of 244 single-copy orthologous genes presents in all the genomes and calculating the number of inversions and breakpoints between each genome pair. This reveals that inversions were the main rearrangement event in γ-proteobacteria evolution, with a progressive increase in the number of rearrangements with increased evolutionary distance. However, significant heterogeneity in different γ-proteobacterial lineages was also detected, with a significant acceleration in the rates of genome rearrangements in bacterial endosymbionts of insects at initial stages of the association. In the second chapter, the structure and functional capabilities of Sodalis glossinidius has been studied. S. glossinidius is the secondary endosymbiont of tsetse flies, and it´s at very initial stages of genome reduction process. It´s genome is experiencing a massive process of gene inactivation, with 972 pseudogenes (inactivated genes) that were described but not annotated in the original annotation of the genome. In this chapter, a complete functional re-annotation of this genome was carried out, that includes the characterization of 1501 pseudogenes though analysis of S. glossinidius intergenic regions. A massive presence of CDSs related with mobile genetic elements and surface proteins were detected, being also the functional classes most affected by pseudogenization. The reconstruction of the metabolic map of S. glossinidius revealed a functional profile very similar to that of free-living enterics, with inactivation of L-arginine biosynthesis pathway, whereas the comparison with Wigglesworthia glossinidia (tsetse primary endosymbiont) reveals possible cases of metabolic complementation between both tsetse endosymbionts at thiamine, coenzyme A and tetrahydrofolate biosynthesis level. Finally, in the third chapter of the thesis, the complete reductive evolution process associated with S. glossinidius was studied from a systems biology perspective through the reconstruction of their genome-scale metabolic networks at different stages of this process and the prediction of their internal reaction fluxes under different external conditions through Flux Balance Analysis. This revealed the decisive role of the pseudogenization of genes involved in L-arginine and glycogen biosynthesis and specially the pseudogenization of the key anaplerotic enzyme phosphoenolpyruvate carboxylase in the ecological transition to a host-dependent lifestyle experienced by S. glossinidius. A progressive decrease in network robustness to gene deletion events and to changes in particular reaction fluxes were detected. Finally, reductive evolution simulations over the functional network of S. glossinidius under different external conditions revealed a higher plasticity in minimal networks evolved in a nutrient-rich environment, and allow defining different sets of essential and disposable genes based on their presence or absence in minimal metabolic networks. These essential genes had more optimized patterns of codon usage and more restricted patterns of sequence evolution than disposable genes that could be lost without affecting the functionality of the network. However, lineage-specific estimates of dN and dS in S. glossinidius and Escherichia coli revealed that common features of ancient bacterial endosymbionts like acceleration in the rates of sequence evolution and the loss of adaptative codon usage were starting to affect S. glossinidius evolution.En esta tesis doctoral, el proceso de reducción genómica característico de bacterias endosimbiontes de insectos ha sido estudiado utilizando diferentes aproximaciones computacionales basadas en la genómica comparada y la biología de sistemas. Por un lado, las dinámicas de reordenaciones genómicas han sido estudiadas en un subconjunto de 31 genomas completos de γ-proteobacterias que incluyen 5 genomas completos de endosimbiontes bacterianos de insectos, revelando una aceleración significativa de las tasas de reordenaciones en estos genomas en etapas iniciales del proceso de reducción. Posteriormente, el genoma de Sodalis glossinidius, el endosimbionte secundario de la mosca tsétsé, fue re-anotado con el objetivo de evaluar el impacto de los procesos de inactivación génica y proliferación de elementos genéticos móviles en etapas tempranas del proceso de reducción, asi como su impacto sobre las capacidades funcionales de la bacteria en el contexto ecológico de su coexistencia con el endosimbionte primario ancestral Wigglesworthia glossinidia. Finalmente, el proceso completo de reducción genómica en S. glossinidius ha sido estudiado a través de la reconstrucción de su red metabólica a diferentes etapas de este proceso y su análisis funcional mediante Análisis de Balance de Flujos, evaluando la robustez de las redes frente a sucesos de deleción asi como las dinámicas evolutivas de genes esenciales y no esenciales en base a su presencia en redes mínimas evolucionadas a partir de la red funcional. Este análisis permitió identificar sucesos de inactivación génica con efectos drásticos sobre las capacidades funcionales del sistema como los genes implicados en la biosíntesis de arginina y glicógeno, y especialmente la inactivación de la enzima fosfoenolpiruvato carboxilasa, asi como una disminución progresiva de la robustez de las redes frente a diferentes sucesos mutacionales asociada al proceso de pérdida génica. Finalmente, simulaciones de evolución reductiva sobre la red funcional bajo diferentes condiciones de entorno ha permitido definir conjuntos de genes esenciales y delecionables en base a su presencia o ausencia en las redes mínimas producto de las simulaciones, revelando una mayor conservación a nivel de secuencia y un uso de codones más optimizado en genes esenciales frente a genes cuya pérdida no afecta a la funcionalidad del sistema