Jean-Michel Fayard

Different Levels of Transcriptional Regulation Due to Trophic Constraints in the Reduced Genome of Buchnera aphidicola APS

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.

Systemic analysis of the symbiotic function of Buchnera aphidicola, the primary endosymbiont of the pea aphid Acyrthosiphon pisum

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.

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

BackgroundGenomic 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.ResultsUsing 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.ConclusionDespite 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.

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

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.

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

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.

Protein kinase C inhibitors stimulate arachidonic and docosahexaenoic acids release from uterine stromal cells through a Ca2+-independent pathway

The mechanisms underlying arachidonic acid (AA) release by uterine stromal (UIII) cells were studied. Stimulation of AA release by calcium ionophore and PMA are inhibited by various PKC inhibitors and by calcium deprivation. These results suggest the involvement of an AA‐specific cPLA2 as the release of docosahexaenoic acid (DHA) from prelabelled cells is much lower than the release of AA. The results also show a more original stimulation of AA and DHA release induced by PKC inhibitors, which is insensitive to calcium deprivation. This stimulation is not due to acyltransferase inhibition, suggesting the participation of a Ca2+‐independent PLA2 (iPLA2). However, iPLA2 activity measured in UIII cells is inhibited by the specific iPLA2 inhibitor, BEL, and is not stimulated by PKC inhibitors, in contrast with the AA and DHA release. It seems therefore that this iPLA2 cannot be involved in this mechanism. The participation of another iPLA2, BEL‐insensitive, is discussed.

Control of cell proliferation via transduction of sPLA2-I activity and possible PPAR activation at the nuclear level

Pancreatic phospholipase A2 (PLA2‐I) stimulates UIII cells proliferation, a rat uterine cell line, after binding to membrane receptors, internalization and translocation. Here, we demonstrate that during these steps of internalization, PLA2‐I retains its hydrolytic activity and thus could exert its proliferative effect via nuclear phospholipids hydrolysis. Since fatty acids and eicosanoids released by such activity are known to be ligands of PPAR, we study the expression of these nuclear receptors and demonstrate that, in the experimental conditions where PLA2‐I stimulates UIII cells proliferation, PLA2‐I also regulates PPAR expression indicating a possible mechanism of its proliferative effect.

Self-adaptation of genome size in artificial organisms

In this paper we investigate the evolutionary pressures influencing genome size in artificial organisms. These were designed with three organisation levels (genome, proteome, phenotype) and are submitted to local mutations as well as rearrangements of the genomic structure. Experiments with various per-locus mutation rates show that the genome size always stabilises, although the fitness computation does not penalise genome length. The equilibrium value is closely dependent on the mutational pressure, resulting in a constant genome-wide mutation rate and a constant average impact of rearrangements. Genome size therefore self-adapts to the variation intensity, reflecting a balance between at least two pressures: evolving more and more complex functions with more and more genes, and preserving genome robustness by keeping it small.