Déchiffrer le "code OPR" pour une meilleure compréhension du rôle physiologique des protéines OPR

Following endosymbiosis, the chloroplast genome shrunk and became reliant on the host genome for its expression. In Chlamydomonas reinhardtii, Octotricopeptide repeat proteins (OPR), encoded in the nucleus, control the expression of a specific organellar mRNA. The OPR repeat is a degenerate motif of 38 amino-acids, folding into a tandem of antiparallel α-helices which can bind to RNA. An individual OPR repeat is predicted to interact with one given nucleotide thanks to specificity-conferring residues at defined positions within the repeat. OPR proteins contain tracks of successive OPR motifs, thus they can bind to a specific RNA “target” sequence and act on it. I aimed to study this specificity, called the “OPR code”, starting with a draft code based on known OPR protein/mRNA couples. I mutated in vivo the chloroplast targets of some OPR factors to disrupt the OPR/RNA interaction, and then tried to restore it by mutating the specificity-conferring residues in the corresponding repeats. Surprisingly, OPR/RNA interactions seem very resilient, challenging our view of how the specificity is established in vivo. Complementary functional studies that I performed on the OPR factors MDB1 and MTHI1 revealed that chloroplast gene expression might rely on complex networks of nuclear factors. By cooperating those putative systems would be both more specific and more resilient.À la suite de l’endosymbiose, le génome chloroplastique a rétréci et dépend maintenant du génome nucléaire pour son expression. Chez Chlamydomonas reinhardtii, les protéines Octotricopeptide repeat (OPR), codées dans le noyau, contrôlent l’expression d’ARNm chloroplastiques spécifiques. La répétition OPR est un motif dégénéré de 38 acides aminés, qui forme un tandem d’hélices α antiparallèles qui lient l’ARN. Une répétition OPR est prédite pour interagir avec un nucléotide spécifique grâce à des résidus variables à des positions précises. La succession de répétitions permet aux protéines OPR de se lier à une séquence donnée. En partant d’un « code OPR » théorique, j’ai cherché à étudier cette spécificité de reconnaissance. J’ai mute in vivo les cibles chloroplastiques de facteurs OPR pour empêcher l’interaction OPR/ARN, puis j’ai tenté de la restaurer en mutant les résidus conférant la spécificité dans les répétitions correspondantes. Étonnamment, les interactions OPR/ARN sont très résilientes, ce qui a complétement changé notre vision de ces interactions in vivo. Des études fonctionnelles complémentaires que j’ai réalisées sur les facteurs OPR MDB1 and MTHI1 ont révélé que l’expression des gènes chloroplastiques dépend probablement de systèmes de facteurs nucléaires. En coopérant ces facteurs auraient une affinité combinée plus forte et seraient ainsi plus résilients

Deciphering the "OPR code" to further assess the physiological role of OPR proteins

À la suite de l’endosymbiose, le génome chloroplastique a rétréci et dépend maintenant du génome nucléaire pour son expression. Chez Chlamydomonas reinhardtii, les protéines Octotricopeptide repeat (OPR), codées dans le noyau, contrôlent l’expression d’ARNm chloroplastiques spécifiques. La répétition OPR est un motif dégénéré de 38 acides aminés, qui forme un tandem d’hélices α antiparallèles qui lient l’ARN. Une répétition OPR est prédite pour interagir avec un nucléotide spécifique grâce à des résidus variables à des positions précises. La succession de répétitions permet aux protéines OPR de se lier à une séquence donnée. En partant d’un « code OPR » théorique, j’ai cherché à étudier cette spécificité de reconnaissance. J’ai mute in vivo les cibles chloroplastiques de facteurs OPR pour empêcher l’interaction OPR/ARN, puis j’ai tenté de la restaurer en mutant les résidus conférant la spécificité dans les répétitions correspondantes. Étonnamment, les interactions OPR/ARN sont très résilientes, ce qui a complétement changé notre vision de ces interactions in vivo. Des études fonctionnelles complémentaires que j’ai réalisées sur les facteurs OPR MDB1 and MTHI1 ont révélé que l’expression des gènes chloroplastiques dépend probablement de systèmes de facteurs nucléaires. En coopérant ces facteurs auraient une affinité combinée plus forte et seraient ainsi plus résilients.Following endosymbiosis, the chloroplast genome shrunk and became reliant on the host genome for its expression. In Chlamydomonas reinhardtii, Octotricopeptide repeat proteins (OPR), encoded in the nucleus, control the expression of a specific organellar mRNA. The OPR repeat is a degenerate motif of 38 amino-acids, folding into a tandem of antiparallel α-helices which can bind to RNA. An individual OPR repeat is predicted to interact with one given nucleotide thanks to specificity-conferring residues at defined positions within the repeat. OPR proteins contain tracks of successive OPR motifs, thus they can bind to a specific RNA “target” sequence and act on it. I aimed to study this specificity, called the “OPR code”, starting with a draft code based on known OPR protein/mRNA couples. I mutated in vivo the chloroplast targets of some OPR factors to disrupt the OPR/RNA interaction, and then tried to restore it by mutating the specificity-conferring residues in the corresponding repeats. Surprisingly, OPR/RNA interactions seem very resilient, challenging our view of how the specificity is established in vivo. Complementary functional studies that I performed on the OPR factors MDB1 and MTHI1 revealed that chloroplast gene expression might rely on complex networks of nuclear factors. By cooperating those putative systems would be both more specific and more resilient

Metagenomics reveal contrasted and dynamic responses of microbial soil communities to in situ wheat straw amendment in croplands or grasslands

National audienceSoil microbial communities respond quickly to agricultural land uses and management prac- tices. To assess the effects of contrasted land-use history on microbial diversity dynamics induced by a wheat straw amendment, an in situ experiment was conducted on adjacent plots with dif- ferent land use-history (20 years croplands or 17 years grasslands in Lusignan, France). A metabarcoding study conducted previously1 highlighted the compositional differences in bacte- rial, archaeal and fungal successions in the two types of soils. To explore the whole microbial communities’ responses (especially viral and eukaryotic dynamics), a global metagenomic ap- proach was used on the same samples. About 2 billion paired-ends reads (Illumina 2*150bp) were obtained for a total of 48 samples. After quality control steps, Kaiju v.1.7.0 was used to taxo- nomically assign the reads. These taxonomic counts were then used to follow the dynamics of the total microbial communities. Metagenomics and metabarcoding results were concordant, with similar dynamics for bacterial and fungal genera, which confirms the validity of both approaches. Then, we defined clusters of genera: (i) specific to either grassland or cropland systems, and/or (ii) responding at different time points after the amendment. A high increase of viral sequences shortly after amendment, alongside stimulation of particular bacterial and fungal fast-growing populations, might suggest a regulatory role of viruses in soils. Differential responses of micro- bial groups provide new insights into the ecology of the communities involved in C turnover in soil. This metagenomic dataset will also be used to study the functional potential of these communities in carbon cycling

Complete genome of Sphingomonas aerolata PDD-32b-11, isolated from cloud water at the summit of puy de Dôme, France

International audienceThe complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy

The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii

International audienc

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water

Abstract The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dôme (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment

Exploring metabolic acclimation of the cloud microflora to contrasting summer day and winter night conditions using metatranscriptomics and fluxomics approaches

International audienceMetabolically active microorganisms are increasingly acknowledged as actors of cloud chemical reactivity able to use organic compounds present in clouds (e.g. organic acids, aldhedydes) for their metabolism (Vaïtilingom et al., 2013). Uncharacterized biological activity may play a major role especially during the night, while during daytime the abiotic degradation of organic compounds would be driven and dominated by hydroxyl radical (•OH) chemistry (Vaïtilingom et al., 2011). To better understand and predict the impact of biological activity on atmospheric chemical reactivity, the metabolic pathways of the whole cloud microbiome and their modulations by environmental conditions (temperature, light, oxidants) must now be assessed.The METACLOUD project addresses metabolic acclimatation of cloud microorganisms under two contrasted situations simulating a summer day (17°C, with solar light and presence of hydrogen peroxide) and a winter night (at 5°C, in the dark and without hydrogen peroxide). A focus is made on formaldehyde assimilations as this compound is a key intermediate both in cloud radical chemistry and in many C1 biological pathway, using fluxomics (LC-HRMS and IC-HRMS) on 13C-formaldehyde supplemented samples. Experiments were conducted in specially designed photobioreactors, either on (1) freshly sampled cloud water from the research station at the top of the puy de Dôme station (1465m asl, PUY, France) including naturally present microorganisms, or (2) an artificial consortium assembled from microbial strains isolated from cloud water sampled at PUY and resuspended in an artificial medium mimicking the composition of marine cloud water (major inorganic and organic compounds).Metatranscriptomes and metabolomes indicate metabolic acclimations of the cloud microbiome to model summer/winter conditions, especially linked with fatty acid regulation and central metabolism (e.g. citrate cycle). First results with 13C-formaldehyde showed carbon incorporation from this molecule into several classes of metabolites (e.g. nucleotides, amino acids, central metabolites), illustrating the complex biological fate of this compound in the environment. The data will be used to implement biological activity on cloud chemistry models. Vaïtilingom M. et al. (2011) Atmospheric chemistry of carboxylic acids: microbial implication versus photochemistry. Atmos. Chem. Phys. 11, 8721-8733. doi: 10.5194/acp-11-8721-2011.Vaïtilingom M. et al. (2013) Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds. Proc. Nat. Acad. Sci. USA 110, 559-564. doi: 10.1073/pnas.1205743110