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1. Compartmentalized into Bacteriocytes but Highly Invasive: the Puzzling Case of the Co-Obligate Symbiont Serratia symbiotica in the Aphid Periphyllus lyropictus .

Author

Renoz F, Lopes MR, Gaget K, Duport G, Eloy MC, Geelhand de Merxem B, Hance T, and Calevro F

Subjects
Animals, Phylogeny, Serratia genetics, Symbiosis, Aphids genetics, Aphids microbiology, Buchnera genetics
Abstract

Dependence on multiple nutritional symbionts that form a metabolic unit has evolved many times in insects. Although it has been postulated that host dependence on these metabolically interconnected symbionts is sustained by their high degree of anatomical integration (these symbionts are often housed in distinct symbiotic cells, the bacteriocytes, assembled into a common symbiotic organ, the bacteriome), the developmental aspects of such multipartner systems have received little attention. Aphids of the subfamilies Chaitophorinae and Lachninae typically harbor disymbiotic systems in which the metabolic capabilities of the ancient obligate symbiont Buchnera aphidicola are complemented by those of a more recently acquired nutritional symbiont, often belonging to the species Serratia symbiotica. Here, we used microscopy approaches to finely characterize the tissue tropism and infection dynamics of the disymbiotic system formed by B. aphidicola and S. symbiotica in the Norway maple aphid Periphyllus lyropictus (Chaitophorinae). Our observations show that, in this aphid, the co-obligate symbiont S. symbiotica exhibits a dual lifestyle: intracellular by being housed in large syncytial bacteriocytes embedded between B. aphidicola -containing bacteriocytes in a well-organized compartmentalization pattern, and extracellular by massively invading the digestive tract and other tissues during embryogenesis. This is the first reported case of an obligate aphid symbiont that is internalized in bacteriocytes but simultaneously adopts an extracellular lifestyle. This unusual infection pattern for an obligate insect symbiont suggests that some bacteriocyte-associated obligate symbionts, despite their integration into a cooperative partnership, still exhibit invasive behavior and escape strict compartmentalization in bacteriocytes. IMPORTANCE Multipartner nutritional endosymbioses have evolved many times in insects. In Chaitophorinae aphids, the eroded metabolic capabilities of the ancient obligate symbiont B. aphidicola are complemented by those of more recently acquired symbionts. Here, we report the atypical case of the co-obligate S. symbiotica symbiont associated with P. lyropictus . This bacterium is compartmentalized into bacteriocytes nested into the ones harboring the more ancient symbiont B. aphidicola , reflecting metabolic convergences between the two symbionts. At the same time, S. symbiotica exhibits highly invasive behavior by colonizing various host tissues, including the digestive tract during embryogenesis. The discovery of this unusual phenotype for a co-obligate symbiont reveals a new face of multipartner nutritional endosymbiosis in insects. In particular, it shows that co-obligate symbionts can retain highly invasive traits and suggests that host dependence on these bacterial partners may evolve prior to their strict compartmentalization into specialized host structures.

Published
2022
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2. Bacteriocyte cell death in the pea aphid/ Buchnera symbiotic system.

Author

Simonet P, Gaget K, Balmand S, Ribeiro Lopes M, Parisot N, Buhler K, Duport G, Vulsteke V, Febvay G, Heddi A, Charles H, Callaerts P, and Calevro F

Subjects
Animals, Cell Death, Lysosomes, Aphids microbiology, Buchnera physiology, Symbiosis physiology
Abstract

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Published
2018
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3. Genomic analysis of the regulatory elements and links with intrinsic DNA structural properties in the shrunken genome of Buchnera.

Author

Brinza L, Calevro F, and Charles H

Subjects
Bacterial Proteins genetics, Gene Expression Regulation, Bacterial genetics, Genome, Bacterial genetics, Buchnera genetics, DNA, Bacterial genetics, Genome Size genetics, Genomics, Regulatory Sequences, Nucleic Acid genetics
Abstract

Background: Buchnera aphidicola is an obligate symbiotic bacterium, associated with most of the aphididae, whose genome has drastically shrunk during intracellular evolution. Gene regulation in Buchnera has been a matter of controversy in recent years as the combination of genomic information with the experimental results has been contradictory, refuting or arguing in favour of a functional and responsive transcription regulation in Buchnera.The goal of this study was to describe the gene transcription regulation capabilities of Buchnera based on the inventory of cis- and trans-regulators encoded in the genomes of five strains from different aphids (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, Cinara cedri and Cinara tujafilina), as well as on the characterisation of some intrinsic structural properties of the DNA molecule in these bacteria., Results: Interaction graph analysis shows that gene neighbourhoods are conserved between E. coli and Buchnera in structures called transcriptons, interactons and metabolons, indicating that selective pressures have acted on the evolution of transcriptional, protein-protein interaction and metabolic networks in Buchnera. The transcriptional regulatory network in Buchnera is composed of a few general DNA-topological regulators (Nucleoid Associated Proteins and topoisomerases), with the quasi-absence of any specific ones (except for multifunctional enzymes with a known gene expression regulatory role in Escherichia coli, such as AlaS, PepA and BolA, and the uncharacterized hypothetical regulators YchA and YrbA). The relative positioning of regulatory genes along the chromosome of Buchnera seems to have conserved its ancestral state, despite the genome erosion. Sigma-70 promoters with canonical thermodynamic sequence profiles were detected upstream of about 94% of the CDS of Buchnera in the different aphids. Based on Stress-Induced Duplex Destabilization (SIDD) measurements, unstable σ70 promoters were found specifically associated with the regulator and transporter genes., Conclusions: This genomic analysis provides supporting evidence of a selection of functional regulatory structures and it has enabled us to propose hypotheses concerning possible links between these regulatory elements and the DNA-topology (i.e., supercoiling, curvature, flexibility and base-pair stability) in the regulation of gene expression in the shrunken genome of Buchnera.

Published
2013
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4. Multimodal dynamic response of the Buchnera aphidicola pLeu plasmid to variations in leucine demand of its host, the pea aphid Acyrthosiphon pisum.

Author

Viñuelas J, Febvay G, Duport G, Colella S, Fayard JM, Charles H, Rahbé Y, and Calevro F

Subjects
Amino Acids, Essential genetics, Amino Acids, Essential metabolism, Animals, Aphids growth & development, Aphids microbiology, Buchnera genetics, Crops, Agricultural, DNA Copy Number Variations, Genome, Bacterial, Leucine biosynthesis, Metabolic Networks and Pathways genetics, Plasmids, Symbiosis genetics, Symbiosis physiology, Aphids metabolism, Buchnera metabolism
Abstract

Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the γ-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions., (© 2011 Blackwell Publishing Ltd.)

Published
2011
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5. A genomic reappraisal of symbiotic function in the aphid/Buchnera symbiosis: reduced transporter sets and variable membrane organisations.

Author

Charles H, Balmand S, Lamelas A, Cottret L, Pérez-Brocal V, Burdin B, Latorre A, Febvay G, Colella S, Calevro F, and Rahbé Y

Subjects
Animals, Biological Transport, Buchnera cytology, Buchnera ultrastructure, Cell Membrane ultrastructure, Genes, Bacterial genetics, Hydrogen-Ion Concentration, Metabolic Networks and Pathways genetics, Microscopy, Confocal, Aphids microbiology, Buchnera physiology, Cell Membrane metabolism, Genomics methods, Membrane Transport Proteins genetics, Symbiosis genetics
Abstract

Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. In this study, we reappraised the transport function of different Buchnera strains, from the aphids Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistaciae and Cinara cedri, using the re-annotation of their transmembrane proteins coupled with an exploration of their metabolic networks. Although metabolic analyses revealed high interdependencies between the host and the bacteria, we demonstrate here that transport in Buchnera is assured by low transporter diversity, when compared to free-living bacteria, being mostly based on a few general transporters, some of which probably have lost their substrate specificity. Moreover, in the four strains studied, an astonishing lack of inner-membrane importers was observed. In Buchnera, the transport function has been shaped by the distinct selective constraints occurring in the Aphididae lineages. Buchnera from A. pisum and S. graminum have a three-membraned system and similar sets of transporters corresponding to most compound classes. Transmission electronic microscopic observations and confocal microscopic analysis of intracellular pH fields revealed that Buchnera does not show any of the typical structures and properties observed in integrated organelles. Buchnera from B. pistaciae seem to possess a unique double membrane system and has, accordingly, lost all of its outer-membrane integral proteins. Lastly, Buchnera from C. cedri revealed an extremely poor repertoire of transporters, with almost no ATP-driven active transport left, despite the clear persistence of the ancestral three-membraned system.

Published
2011
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6. Structure and dynamics of the operon map of Buchnera aphidicola sp. strain APS.

Author

Brinza L, Calevro F, Duport G, Gaget K, Gautier C, and Charles H

Subjects
Base Sequence, Codon genetics, Conserved Sequence genetics, DNA, Intergenic genetics, Escherichia coli genetics, Evolution, Molecular, Gene Expression Regulation, Bacterial, Genes, Bacterial, Models, Genetic, Oligonucleotide Array Sequence Analysis, Open Reading Frames genetics, Promoter Regions, Genetic genetics, ROC Curve, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Terminator Regions, Genetic genetics, Transcription, Genetic, Buchnera genetics, Operon genetics
Abstract

Background: Gene expression regulation is still poorly documented in bacteria with highly reduced genomes. Understanding the evolution and mechanisms underlying the regulation of gene transcription in Buchnera aphidicola, the primary endosymbiont of aphids, is expected both to enhance our understanding of this nutritionally based association and to provide an intriguing case-study of the evolution of gene expression regulation in a reduced bacterial genome., Results: A Bayesian predictor was defined to infer the B. aphidicola transcription units, which were further validated using transcriptomic data and RT-PCR experiments. The characteristics of B. aphidicola predicted transcription units (TUs) were analyzed in order to evaluate the impact of operon map organization on the regulation of gene transcription.On average, B. aphidicola TUs contain more genes than those of E. coli. The global layout of B. aphidicola operon map was mainly shaped by the big reduction and the rearrangements events, which occurred at the early stage of the symbiosis. Our analysis suggests that this operon map may evolve further only by small reorganizations around the frontiers of B. aphidicola TUs, through promoter and/or terminator sequence modifications and/or by pseudogenization events. We also found that the need for specific transcription regulation exerts some pressure on gene conservation, but not on gene assembling in the operon map in Buchnera. Our analysis of the TUs spacing pointed out that a selection pressure is maintained on the length of the intergenic regions between divergent adjacent gene pairs., Conclusions: B. aphidicola can seemingly only evolve towards a more polycistronic operon map. This implies that gene transcription regulation is probably subject to weak selection pressure in Buchnera conserving operons composed of genes with unrelated functions.

Published
2010
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7. Systemic analysis of the symbiotic function of Buchnera aphidicola, the primary endosymbiont of the pea aphid Acyrthosiphon pisum.

Author

Brinza L, Viñuelas J, Cottret L, Calevro F, Rahbé Y, Febvay G, Duport G, Colella S, Rabatel A, Gautier C, Fayard JM, Sagot MF, and Charles H

Subjects
Amino Acids metabolism, Animals, Aphids cytology, Aphids physiology, Bacterial Proteins genetics, Bacterial Proteins physiology, Buchnera classification, Buchnera genetics, Chromosomes, Bacterial genetics, Evolution, Molecular, Gene Expression Regulation, Bacterial, Gene Regulatory Networks, Genes, Bacterial, Genetic Drift, Metabolic Networks and Pathways genetics, Phylogeny, Symbiosis genetics, Aphids microbiology, Buchnera physiology, Genome, Bacterial, Genomics, Pisum sativum parasitology, Symbiosis physiology
Abstract

Buchnera aphidicola is the primary obligate intracellular symbiont of most aphid species. B. aphidicola and aphids have been evolving in parallel since their association started, about 150 Myr ago. Both partners have lost their autonomy, and aphid diversification has been confined to smaller ecological niches by this co-evolution. B. aphidicola has undergone major genomic and biochemical changes as a result of adapting to intracellular life. Several genomes of B. aphidicola from different aphid species have been sequenced in the last decade, making it possible to carry out analyses and comparative studies using system-level in silico methods. This review attempts to provide a systemic description of the symbiotic function of aphid endosymbionts, particularly of B. aphidicola from the pea aphid Acyrthosiphon pisum, by analyzing their structural genomic properties, as well as their genetic and metabolic networks.

Published
2009
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8. Impact of host developmental age on the transcriptome of the symbiotic bacterium Buchnera aphidicola in the pea aphid (Acyrthosiphon pisum).

Author

Bermingham J, Rabatel A, Calevro F, Viñuelas J, Febvay G, Charles H, Douglas A, and Wilkinson T

Subjects
Aging, Animals, Embryo, Nonmammalian metabolism, Gene Expression Regulation, Bacterial, Genes, Bacterial genetics, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Reproducibility of Results, Up-Regulation, Aphids microbiology, Buchnera genetics, Buchnera metabolism, Gene Expression Profiling, Symbiosis physiology
Abstract

Of the 617 genes from Buchnera aphidicola, the obligate bacterial symbiont of the pea aphid, 23% were differentially expressed in embryos compared to adults. Genes involved in flagellar apparatus and riboflavin synthesis exhibited particularly robust upregulation in embryos, suggesting functional differences between the symbiosis in the adult and embryo insect.

Published
2009
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9. Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of Buchnera aphidicola.

Author

Viñuelas J, Calevro F, Remond D, Bernillon J, Rahbé Y, Febvay G, Fayard JM, and Charles H

Subjects
Analysis of Variance, Animals, Aphids microbiology, DNA, Bacterial, Evolution, Molecular, Genes, Bacterial, Oligonucleotide Array Sequence Analysis, RNA, Messenger genetics, Reproducibility of Results, Buchnera genetics, Chromosomes, Bacterial genetics, Genome, Bacterial genetics, Transcription, Genetic
Abstract

Background: Genomic studies on bacteria have clearly shown the existence of chromosomal organization as regards, for example, to gene localization, order and orientation. Moreover, transcriptomic analyses have demonstrated that, in free-living bacteria, gene transcription levels and chromosomal organization are mutually influenced. We have explored the possible conservation of relationships between mRNA abundances and chromosomal organization in the highly reduced genome of Buchnera aphidicola, the primary endosymbiont of the aphids, and a close relative to Escherichia coli., Results: Using an oligonucleotide-based microarray, we normalized the transcriptomic data by genomic DNA signals in order to have access to inter-gene comparison data. Our analysis showed that mRNA abundances, gene organization (operon) and gene essentiality are correlated in Buchnera (i.e., the most expressed genes are essential genes organized in operons) whereas no link between mRNA abundances and gene strand bias was found. The effect of Buchnera genome evolution on gene expression levels has also been analysed in order to assess the constraints imposed by the obligate symbiosis with aphids, underlining the importance of some gene sets for the survival of the two partners. Finally, our results show the existence of spatial periodic transcriptional patterns in the genome of Buchnera., Conclusion: Despite an important reduction in its genome size and an apparent decay of its capacity for regulating transcription, this work reveals a significant correlation between mRNA abundances and chromosomal organization of the aphid-symbiont Buchnera.

Published
2007
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10. Different levels of transcriptional regulation due to trophic constraints in the reduced genome of Buchnera aphidicola APS.

Author

Reymond N, Calevro F, Viñuelas J, Morin N, Rahbé Y, Febvay G, Laugier C, Douglas A, Fayard JM, and Charles H

Subjects
Amino Acids, Essential metabolism, Animals, Aphids growth & development, Aphids metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Buchnera genetics, Gene Expression Profiling, Oligonucleotide Array Sequence Analysis, Sucrose metabolism, Symbiosis, Aphids microbiology, Buchnera metabolism, Gene Expression Regulation, Bacterial, Genome, Bacterial, Transcription, Genetic
Abstract

Symbiotic associations involving intracellular microorganisms and animals are widespread, especially for species feeding on poor or unbalanced diets. Buchnera aphidicola, the obligate intracellular bacterium associated with most aphid species, provides its hosts with essential amino acids (EAAs), nutrients in short supply in the plant phloem sap. The Buchnera genome has undergone severe reductions during intracellular evolution. Genes for EAA biosynthesis are conserved, but most of the transcriptional regulatory elements are lost. This work addresses two main questions: is transcription in Buchnera (i) regulated and (ii) scaled to aphid EAA demand? Two microarray experiments were designed for profiling the gene expression in Buchnera. The first one was characterized by a specific depletion of tyrosine and phenylalanine in the aphid diet, and the second experiment combined a global diminution of EAAs in the aphid diet with a sucrose concentration increase to manipulate the aphid growth rate. Aphid biological performance and budget analysis (the balance between EAAs provided by the diet and those synthesized by Buchnera) were performed to quantify the nutritional demand from the aphids toward their symbiotic bacteria. Despite the absence of known regulatory elements, a significant transcriptional regulation was observed at different levels of organization in the Buchnera genome: between genes, within putative transcription units, and within specific metabolic pathways. However, unambiguous evidence for transcriptional changes underpinning the scaling of EAA biosynthesis to aphid demand was not obtained. The phenotypic relevance of the transcriptional response from the reduced genome of Buchnera is addressed.

Published
2006
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11. Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum.

Author

Charles H, Calevro F, Vinuelas J, Fayard JM, and Rahbe Y

Subjects
Animals, Anticodon, Buchnera metabolism, Cytosine analysis, Diet, Guanine analysis, Oligonucleotide Array Sequence Analysis, Phenylalanine metabolism, RNA, Transfer metabolism, Tyrosine metabolism, Aphids microbiology, Buchnera genetics, Codon, Gene Expression Regulation, Bacterial, RNA, Transfer genetics
Abstract

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon-anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera.

Published
2006
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12. Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.

Author

Calevro F, Charles H, Reymond N, Dugas V, Cloarec JP, Bernillon J, Rahbé Y, Febvay G, and Fayard JM

Subjects
Base Pair Mismatch, DNA, Complementary chemistry, DNA, Complementary genetics, Fluorescence, Nucleic Acid Hybridization methods, Oligonucleotide Probes, RNA, Bacterial genetics, RNA, Bacterial isolation & purification, Sensitivity and Specificity, Buchnera genetics, Gene Expression Profiling methods, Oligonucleotide Array Sequence Analysis methods
Abstract

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.

Published
2004
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13. A novel cell death process eliminates both bacteriocytes and their symbionts in the pea aphid/Buchnera symbiotic system

Author

Ribeiro Lopes, Mélanie, Simonet, Pierre, Gaget, Karen, Balmand, Severine, Parisot, Nicolas, Buhler, Kurt, Duport, Marie-Gabrielle, Vulsteke, Veerle, Febvay, Gérard, Heddi, Abdelaziz, Charles, Hubert, Callaerts, Patrick, Calevro, Federica, Charles, Hubert, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Université de Lyon, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Unité de recherche Productions végétales (CRAG ANT PROD V), Institut National de la Recherche Agronomique (INRA), laboratory of Developmental Genetics, VIB-KUL, Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon

Subjects
[SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV]Life Sciences [q-bio], Acyrthosiphon pisum, apoptosis, bacteriocyte, artificial diet, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], symbiosis, [SDV] Life Sciences [q-bio], transcriptomics, nutrition, cell death, Buchnera, ComputingMilieux_MISCELLANEOUS
Abstract

International audience; Symbiosis is a key source of ecological and evolutionary diversification of eukaryotic organisms throughout the animal and plant kingdoms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells, the bacteriocytes, have evolved for harboring beneficial microbial partners. These cells constitute a fascinating riddle in developmental cell biology, as their embryonic origin and the molecular mechanisms governing their development and organogenesis, as well as their elimination in response to host physiology, remain largely unsolved.Here we report the discovery of a hitherto unknown cell-death process involved in the degeneration of bacteriocytes in the pea aphid Acyrthosiphon pisum/Buchnera aphidicola symbiotic system, which emerged in recent years as a powerful model for studying symbiotic relationships. This new form of cell death is activated progressively throughout aphid adulthood and exhibits morphological features distinct from evolutionary conserved pathways, including apoptosis- or autophagy-dependent cell deaths. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of non-autophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, swelling of bacteriocyte mitochondria, activation of reactive oxygen species, and Buchnera aphidicola endosymbiont degradation by the lysosomal system. This multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization in these cells, probably imposed by the presence of millions of Buchnera endosymbionts in each bacteriocyte (Simonet et al., 2018, PNAS, doi: 10.1073/pnas.1720237115). Our findings reveal novel mechanisms by which both bacteriocyte cell and symbiotic bacterial numbers are controlled to maintain organismal homeostasis. They also shed light on previously unknown consequences of persistent obligatory symbiotic bacteria on the cellular organization and functioning of bacteriocytes in their eukaryotic hosts.

Published
2019

14. « ArthropodaCyc » : une collection de bases de données enrichies à l’aide de CycADS pour étudier et comparer le métabolisme des arthropodes

Author

Baa-Puyoulet, Patrice, Colella, Stefano, Vellozo, Augusto, Huerta-Cepas, Jaime, Febvay, Gérard, Calevro, Federica, Gabaldon, Toni, Sagot, Marie-France, Charles, Hubert, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), An algorithmic view on genomes, cells, and environments (BAMBOO), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Centre for Genomic Regulation [Barcelona] (CRG), Universitat Pompeu Fabra [Barcelona] (UPF)-Centro Nacional de Analisis Genomico [Barcelona] (CNAG), Unité de recherche Productions végétales (CRAG ANT PROD V), Institut National de la Recherche Agronomique (INRA), Barcelona Supercomputing Center - Centro Nacional de Supercomputacion (BSC - CNS), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Inria Grenoble - Rhône-Alpes, and Institut National de Recherche en Informatique et en Automatique (Inria)

Subjects
transcriptomics, Buchnera, system biology, Acyrthosiphon pisum, microarray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]
Abstract

International audience; « ArthropodaCyc » : une collection de bases de données enrichies à l’aide de CycADS pour étudier et comparer le métabolisme des arthropodes.Patrice Baa-Puyoulet1, Augusto F. Vellozo2,3, Jaime Huerta-Cepas4, Gérard Febvay1, Federica Calevro1, Toni Gabaldon4, Marie-France Sagot2,3, Hubert Charles1,3 et Stefano Colella1,*.1 UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, INRA, INSA-Lyon, Université de Lyon, Villeurbanne, France; 2 Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France; 3 BAMBOO, INRIA Rhône-Alpes, France; 4 Center for Genomic Regulation, Barcelona, Spain ; * contact : Stefano.Colella@lyon.inra.frAprès la publication de plusieurs génomes d’arthropodes, de nombreuses autres espèces vont être séquencées dans un futur proche, en particulier à travers l’initiative i5ki. La disponibilité de ces multiples séquences ouvre la voie à des études comparatives entre espèces afin de mieux comprendre les différents aspects de la biologie des arthropodes. Ces études nécessitent l’intégration des données génomiques et fonctionnelles en évolution constante : les séquences génomiques et l’annotation fonctionnelle des protéines devant être collectées de sources et méthodes variées et mises à jour régulièrement.Durant l’annotation du génome du puceron du pois (Acyrthosiphon pisum) nous avons développé CycADSii (Cyc Annotation Database System), un système de gestion d’annotations automatisé permettant l’intégration des informations qui sont utilisées pour la reconstruction des réseaux métaboliques. Les données issues de Genbank et/ou de bases de données génomiques spécifiques, ainsi que les annotations fonctionnelles obtenues par les méthodes KAAS-KEGG, PRIAM, Blast2GO, PhylomeDB et MetaPhOrs, sont collectées dans CycADS. Ces annotations sont ensuite extraites, avec possibilité d’appliquer différents filtres de qualité, pour produire les fichiers d’entrée de PathwayTools, un logiciel qui génère et/ou met à jour des bases de données métaboliques de type BioCyc. Nous avons utilisé CycADS pour créer ArthropodaCyciii, une collection de bases de données métaboliques qui, à ce jour, contient 22 arthropodes: [Arachnida] Ixodes scapularis ; [Coleoptera] Tribolium castaneum ; [Crustacea] Daphnia pulex ; [Diptera] Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster, Glossina morsitans ; [Hemiptera] Acyrthosiphon pisum, Rhodnius prolixus ; [Hymenoptera] Apis mellifera, Nasonia vitripennis, Atta cephalotes, Acromyrmex echinatior, Camponotus floridanus, Harpegnathos saltator, Linepithema humile, Pogonomyrmex barbatus, Solenopsis invicta, [Lepidoptera] Danaus plexippus, Heliconius melpomene ; [Phthiraptera] Pediculus humanus. Dans ces bases, nous avons ajouté de nombreux liens externes vers les données génomiques de chaque organisme (AphidBase, BeetleBase, VectorBase, Hymenoptera Genome, FlyBase, wFleaBase, MonarchBase, ButterflyGenome) et d’annotation fonctionnelle (Brenda-Enzyme, Gene Ontology, KEGG Orthology et PhylomeDB). Notre collection de bases métaboliques permet alors de réaliser des analyses comparatives en utilisant les fonctionnalités web interactives d’ArthropodaCyc.Dans le futur, nous prévoyons d’ajouter l’annotation métabolique d’autres génomes en cours de séquençage et d’implémenter des passerelles vers des outils d’analyses de réseaux (tels que MetExplore). Nous sommes aussi ouverts à des collaborations sur des projets de séquençage d’arthropodes en cours, pour participer à la première annotation fonctionnelle des protéines et réaliser la reconstruction de leur métabolisme.i http://arthropodgenomes.org/wiki/i5K ii http://www4.inra.fr/cycads/iii http://arthropodacyc.cycadsys.org/

Published
2013

15. Diminution de fécondité et défauts de développement induits par l’inactivation fonctionnelle par RNAi du gène codant la phénylalanine hydroxylase chez le puceron du pois, Acyrthosiphon

Author

Gaget, Karen, Duport, Marie-Gabrielle, Rey, Marjolaine, Balmand, Severine, Charles, Hubert, Febvay, Gérard, Colella, Stefano, Calevro, Federica, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Unité de recherche Productions végétales (CRAG ANT PROD V), Institut National de la Recherche Agronomique (INRA), and Charles, Hubert

Subjects
[SDV] Life Sciences [q-bio], parthénogenèse, Buchnera, développement embryonnaire, RNAi, [SDV]Life Sciences [q-bio], Acyrthosiphon pisum, métabolisme, tyrosine, symbiosis
Abstract

International audience; Le puceron du pois est un ravageur important des cultures en région tempérée. Le grand potentiel invasif de cet insecte est essentiellement dû à son système de reproduction, basé pendant le printemps et l’été sur une parthénogenèse vivipare, et à la symbiose obligatoire avec la bactérie Buchnera aphidicola, qui fournit au puceron certains acides aminés essentiels peu présents dans son alimentation. Une étude transcriptomique, récemment conduite dans notre laboratoire, nous a permis de dégager une voie métabolique clé dans le développement parthénogénétique du puceron du pois: la voie de la tyrosine. Afin de mieux caractériser l’importance de cette voie, nous avons étudié l’effet de l’inactivation fonctionnelle par RNAi du gène codant la phénylalanine hydroxylase, enzyme qui transforme la phénylalanine en tyrosine, sur la reproduction parthénogénétique du puceron du pois. L’injection d’ARN doubles-brins dirigés contre ce gène cible de manière privilégiée les compartiments symbiotiques (embryons vivipares et bactériocytes), où nous avons observé une diminution significative de l’expression du gène 1 et 3 jours après l’injection. Les pucerons traités montrent une fécondité réduite (-37 %) et une augmentation (dix fois supérieure) de la mortalité larvaire et de l’apparition de défauts de développement par rapport aux contrôles. Des analyses HPLC sont en cours pour vérifier si les phénotypes observés sont liés à une diminution de la production de tyrosine dans les pucerons traités.

Published
2013

16. Multimodal dynamic response of the Buchnera aphidicola pLeu plasmid to variations in leucine demand of its host, the pea aphid Acyrthosiphon pisum

Author

Viñuelas, José, Febvay, Gérard, Duport, Gabrielle, Colella, Stefano, Fayard, Jean-Michel, Charles, Hubert, Rahbé, Yvan, Calevro, Federica, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), ANR-Genoplante programme 2008-2010, GPLA07-02 Aphicibles project, Equipe de recherche européenne en algorithmique et biologie formelle et expérimentale (ERABLE), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), ANR-07-GPLA-0020,GENOPLANTE,Utilisation du déséquilibre de liaison par le logiciel MCQTL(2007), and Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon

Subjects
Crops, Agricultural, DNA Copy Number Variations, food and beverages, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Buchnera, Leucine, Aphids, Animals, Amino Acids, Essential, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Symbiosis, Research Articles, Genome, Bacterial, Metabolic Networks and Pathways, Plasmids, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the gamma-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.

Published
2011
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17. Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola

Author

Wilson, A. C. C., Ashton, P. D., Calevro, Federica, Charles, H., Colella, Stefano, Febvay, G., Jander, G., Kushlan, P. F., Macdonald, S. J., Schwartz, J. F., Thomas, G. H., Douglas, A. E., Department of Biology [Miami], University of Miami [Coral Gables], Department of Biology [York], University of York [York, UK], Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon, An algorithmic view on genomes, cells, and environments (BAMBOO), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Boyce Thompson Institute [Ithaca], Cornell University [New York], BBSRC, ANR BB/F005342/1, USDA 2005-35604-15446, University of Miami Start-up Funds, Sarkaria Institute for Insect Physiology and Toxicology, University of Miami Honors Summer Research Program, Biotechnology and Biological Sciences Research Council BB/F005342/1, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), and Cornell University

Subjects
MESH: Amino Acids, MESH: Buchnera, Genome, Insect, ACYRTHOSIPHON PISUM, MESH: Peas, MESH: Genome, Bacterial, Models, Biological, GENOME ANNOTATION, HEMIPTERA, Bacterial Proteins, Buchnera, MESH: Transaminases, MESH: Insect Proteins, Animals, MESH: Animals, Amino Acids, MESH: Bacterial Proteins, Transaminases, GENOME EVOLUTION, MESH: Genetic Complementation Test, MESH: Symbiosis, MESH: Genome, Insect, Genetic Complementation Test, ESSENTIAL AMINO ACID, SYMBIOSIS, Peas, MESH: Models, Biological, food and beverages, biochemical phenomena, metabolism, and nutrition, BUCHNERA APHICOLA, Aphids, GENOMIC COMPLEMENTARITY, Insect Proteins, AMINO ACID METABOLISM, MESH: Aphids, Genome, Bacterial, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract

International audience; The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched-chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

Published
2010
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18. Chromosome organisation in Buchnera: a dynamic active structure involved in gene expression regulation

Author

Brinza, Lilia, Calevro, Federica, Vinuelas, J., Gautier, Christian, Charles, Hubert, Immunité et lymphocytes cytotoxiques – Immunity and cytotoxic lymphocytes, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Baobab, Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
transcriptomics, Buchnera, system biology, Acyrthosiphon pisum, microarray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]
Abstract

International audience; Most bacterial chromosomes consist of a single closed-circular DNA molecule folded into a compact and dynamic structure called the nucleoid. Variations of chromosome 3D-structure act as a global regulatory factor of gene expression. More particularly, the modulation of genome architecture is admitted to belong to the class of mechanisms allowing genome-wide transcriptional profile variation in response to environmental changes [1, 2].We searched for evidences of spatial organization of the chromosome in an extremely intriguing bacterial model: Buchnera aphidicola. Associated with most agricultural pest aphids and being partly responsible for their harmfulness, Buchnera are one of the most studied intracellular symbiotic bacteria of insects. Their genomes present all the characteristics of intracellular bacteria: (1) small size of 400 - 600 kb depending on aphid species, (2) highly biased base composition towards A and T and (3) high evolutionary rate due to the isolation of Buchnera populations within the host cells combined with the drastic bottlenecks that occur in the population dynamics of the bacteria during their transmission to the aphid progeny.A crucial stage for the symbiosis comprehension passes through the understanding of symbiont gene expression regulation, and yet little is known about the transcriptional regulation capabilities of the bacteria. Given the “poor” catalogue of transcriptional factors it was suggested that the bacteria are no longer able to regulate their gene expression. Also, Buchnera conserved target genes for regulatory proteins absent in their genome. Nevertheless, recent works using a dedicated microarray showed that Buchnera respond specifically to somenutritional stresses imposed to their host [3].The aim of this work was to study potential structural units of the Buchnera chromosome and the impact they could have on the gene expression regulation. For this purpose, we analysed the potential structural domains of the chromosome at different scales and the main contributor proteins for the organization and maintenance of these domains. Our study brings evidences for (1) the existence of structural chromosomal units at several scales, (2) a functionally complete set of proteins essential for nucleoid organization and (3) the tight interdependence between these proteins, the chromosome architecture and the gene expression profile.Basic genomic structural elements in bacteria participating to the chromosome organization are transcription units (operons). A transcription unit contains one or several adjacent genes transcribed as a single mRNA. Thus, genes belonging to the same transcriptional unit display strong correlated transcription levels. A first annotation of Buchnera transcription units was available in BioCyc (http://biocyc.org/). We found that some of these transcriptional units were not consistent with the analysis of the gene expression profile. Thus, we decided to re-annotate the transcription units of Buchnera taking into account gene expression levels, gene order conservation, sequence features of Buchnera, like Rho-independent terminators inferred by the bioinformatics tool (TransTermHP, [4]) and specific intergenic distances. We tested this new annotation with microarray gene expression data.A higher level of structural units in bacterial genomes is represented by the organization in topological domains (~10kbp in E. coli [5]). These structures are mainly organized and maintained by Nucleoid Associated Proteins (NAPs). Analysis of the NAP set of Buchnera pointed out that, despite genome reduction, the bacteria retains the most important members of the group (IHF, H-NS, Fis, DnaA and HU). Our bioinformatics analysis of these proteins confirms a strong conservation of structural domains and 3D structure. Moreover, key amino acids (their mutation compromises NAPs function in E. coli) are also well conserved. As NAPs must frequently bind DNA (every 10 kbp) to form dynamic inter-domains barriers, they rather recognize specific topologic structures (i.e., bend DNA) than specific motive binding sites. By using Curvature program [6]), we showed that Buchnera have a more curved DNA than E. coli (this result may be partially explained by the strong A-T bias) , that might influence the size and shape of their topological domains.Combination of topological domains and DNA fold are at the origin of a third class of larger structural units in bacterial chromosomes. Previous results of our team pointed out a periodic transcriptional pattern that supports the existence of these kinds of structures in Buchnera [7]. We completed this work by using a more realistic distance on the chromosome (i.e., physical distance (bp), instead of the “gene number” distance).Our work brings several evidences that Buchnera chromosome is a functional structure probably playing an active role in gene expression regulation. This kind of regulation was often neglected in free-living bacteria but might be central in shrunken genomes of endosymbionts. A short-term perspective of our work will be to inactivate in vivo specific NAP proteins of Buchnera and analyse the induced modifications within the gene expression profile of the symbiotic bacteria.

Published
2009

19. A preliminary methodological approach to study the transcriptome of Buchnera, the intracellular symbiotic bacteria of aphids

Author

Calevro, Federica, Charles, Hubert, Reymond, Nancie, Dugas, Vincent, Cloarec, J.P., Bernillon, Jacques, Rahbé, Yvan, Febvay, Gérard, Fayard, Jean-Michel, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Sciences Analytiques (SA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), INL - Chimie et Nanobiotechnologies (INL - C&N), Institut des Nanotechnologies de Lyon (INL), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie Analytique et Synthèse Bioorganique (LBASB), Université de Lyon-Université de Lyon, Unité de recherche Productions végétales (CRAG ANT PROD V), and Institut National de la Recherche Agronomique (INRA)

Subjects
transcriptomics, Buchnera, system biology, [SDV]Life Sciences [q-bio], Acyrthosiphon pisum, microarray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2003

20. Bioinformatique du transcriptome de Buchnera, symbiote intracellulaire des pucerons

Author

Charles, Hubert, Calevro, Federica, Reymond, Nancie, Laforest, Frederique, Tchounikine, Anne, Beslon, Guillaume, Soula, Hédi, Dugas, Vincent, Cloarec, J.P., Bernillon, Jacques, Rahbé, Yvan, Febvay, Gérard, Fayard, Jean-Michel, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Laboratoire d'InfoRmatique en Image et Systèmes d'information (LIRIS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École Centrale de Lyon (ECL), Université de Lyon-Université Lumière - Lyon 2 (UL2), Laboratoire de Productique et Informatique des systèmes manufacturiers (PRISMA), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Sciences Analytiques (SA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), INL - Chimie et Nanobiotechnologies (INL - C&N), Institut des Nanotechnologies de Lyon (INL), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie Analytique et Synthèse Bioorganique (LBASB), Université de Lyon-Université de Lyon, Unité de recherche Productions végétales (CRAG ANT PROD V), and Institut National de la Recherche Agronomique (INRA)

Subjects
transcriptomics, Buchnera, system biology, [SDV]Life Sciences [q-bio], Acyrthosiphon pisum, [INFO]Computer Science [cs], microarray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]
Abstract

International audience; Buchnera est une bactérie symbiotique intracellulaire associée à la plupart des pucerons d'importance économique. Cette bactérie incultivable est obligatoire pour le puceron qui se nourrit exclusivement de sève phloémienne. Buchnera est caractérisée par la taille extrêmement réduite de son génome (400 à 600 kb) séquencé entièrement chez trois espèces. Une des particularités fonctionnelles tout à fait originale de cette bactérie est la conservation des voies de biosynthèse des acides aminés essentiels, alors que les gènes des voies de synthèse des acides aminés non essentiels (ceux que l'hôte est capable de synthétiser) sont perdus. Jusqu'à présent l'analyse expérimentale des réponses métaboliques et moléculaires ne pouvait s'effectuer que "voie par voie". Le développement de la technique des puces à ADN permet maintenant d'envisager une approche globale. Dans l'UMR BF2I, nous avons développé une puce à ADN dédiée à l'analyse du transcriptome de Buchnera APS (associée au puceron Acyrthosiphon pisum). Cette puce devrait nous permettre d’étudier les mécanismes de régulation de l’expression des gènes de cette bactérie symbiotique en condition de stress trophique de son hôte (pucerons élevés sur des milieux déprimés en acides aminés essentiels par exemple).Ce travail correspond à l'émergence d'une thématique de recherche en bioinformatique à l'INSA associée au développement de la plate-forme transcriptome de la Génopole Rhône-Alpes sur le campus de la Doua. Notre approche s'est voulue verticale, impliquant la création d'outils bioinformatiques lorsqu'ils faisaient défaut. Quatre points seront présentés ici : Le développement d'un Système d'Information pour l'Analyse du Transcriptome (SI-Trans) Le développement d'un logiciel de choix d'oligonucléotides (ROSO) La validation d’un protocole expérimental grâce au développement d’une « minipuce » Buchnera L’intégration des résultats d'expression dans une base de connaissance (Genome Expert Bacteria) pour la représentation des réseaux de régulation

Published
2003

21. Analyse du transcriptome de Buchnera: développement d'une première puce à ADN

Author

Calevro, Federica, Charles, Hubert, Reymond, Nancie, Cloarec, J.P., Rahbé, Yvan, Heddi, Abdelaziz, Febvay, Gérard, Fayard, Jean-Michel, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), INL - Chimie et Nanobiotechnologies (INL - C&N), Institut des Nanotechnologies de Lyon (INL), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Centrale de Lyon (ECL), Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE), Unité de recherche Productions végétales (CRAG ANT PROD V), and Institut National de la Recherche Agronomique (INRA)

Subjects
transcriptomics, Buchnera, system biology, [SDV]Life Sciences [q-bio], Acyrthosiphon pisum, microarray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]
Abstract

International audience; Les pucerons d’importance économique appartiennent presque tous à la famille des Aphididae (Hemiptera: Aphidoidea: Aphididae). Ils vivent en symbiose avec une bactérie intracellulaire, du genre Buchnera, proche d’Escherichia coli et qui est transmise par voie maternelle. L’association Buchnera / puceron est très ancienne (environ 200 - 250 M d’années) et elle est devenue obligatoire pour les deux partenaires. Les pucerons rendus expérimentalement aposymbiotiques montrent une fécondité nulle et Buchnera ne semble pas cultivable in vitro. L’intervention de Buchnera dans la physiologie de son hôte a été très étudiée au laboratoire notamment pour ce qui concerne le métabolisme des acides aminés. Il a été montré que la bactérie est capable de fournir la totalité des acides aminés essentiels nécessaires au puceron lorsque ceux-ci sont absents de son alimentation. Cette synthèse est réalisée à partir de quelques précurseurs glucidiques ou de quelques acides aminés.Jusqu’au séquençage complet de Buchnera, l’analyse expérimentale des réponses métaboliques et moléculaires ne pouvait s'effectuer que “ voie par voie ”. Le développement de nouveaux outils d’analyse moléculaire (notamment les puces à ADN) et la taille réduite du génome de Buchnera (583 ORF) adaptée à la mise en œuvre de puces de moyenne densité, offrent la possibilité d’explorer globalement l'ensemble des réponses métaboliques de cet organisme.Le but de ce travail est d’explorer par la technologie des puces à ADN le fonctionnement global du génome de Buchnera, et plus particulièrement la régulation du métabolisme des acides aminés jouant un rôle clé dans la relation symbiotique Buchnera / puceron.

Published
2002

22. Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis

Author

Pierre Simonet, Stefano Colella, Abdelaziz Heddi, José Viñuelas, Hubert Charles, Gérard Febvay, Federica Calevro, Gabrielle Duport, Karen Gaget, Michèle Weiss-Gayet, Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Equipe de recherche européenne en algorithmique et biologie formelle et expérimentale (ERABLE), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), INRA, INSA-Lyon, French (IMetSym) program grant [ANR-13-BSV7-0016-01], French Ministry of Research, ANR-13-BSV7-0016,IMetSym,Contrôles immunitaire et métabolique dans les symbioses intracellulaires d'insectes(2013), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Simonet, Pierre, and Calevro, Federica

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, Population, Biology, Article, 03 medical and health sciences, Buchnera, Symbiosis, Animals, education, 2. Zero hunger, education.field_of_study, Aphid, Multidisciplinary, Obligate, Ecology, Host (biology), Bacteriocyte, fungi, food and beverages, biochemical phenomena, metabolism, and nutrition, Flow Cytometry, biology.organism_classification, Bacterial Load, Acyrthosiphon pisum, 030104 developmental biology, Evolutionary biology, Aphids
Abstract

International audience; Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum, and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry, and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development, and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, 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. Intracellular symbioses (endosymbioses) between prokaryotic and metazoan organisms play a central role in multicellular life, significantly impacting the evolution and shaping the ecology of countless species 1. In insects, which account for a great proportion of planet biodiversity, the exploitation of the metabolic capabilities of intra-cellular symbiotic bacteria (endosymbionts) enables the hosts to thrive on nutritionally unbalanced diets such as plant sap, grains, wood or vertebrate blood 2–4. The sustainability of these endosymbiotic relationships largely relies on the compartmentalization of bacterial endosymbionts into specialized host cells (or organs), called bac-teriocytes (or bacteriomes), whose functions are adapted to the tolerance and regulation of symbiotic populations 5,6. A detailed description of the interplay between bacteriocytes and endosymbionts across the host life cycle, and in response to an ever-changing environment, is expected to provide a better understanding of how microorganisms interact with eukaryotic cells, and, in turn, to contribute to the development of novel strategies for controlling pest and disease-vector insects. The relationship between aphids (Hemiptera: Aphididae) and the gamma-3-proteobacterium Buchnera aphidicola, represents the best-studied model among endosymbiotic associations. In the A. pisum/B. aphidicola

Published
2016
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