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Accueil > Publications

Publications plate-forme I2BC

2019


  • A. Baroin-Tourancheau, Y. Jaszczyszyn, X. Benigni, et L. Amar, « Evaluating and Correcting Inherent Bias of microRNA Expression in Illumina Sequencing Analysis », Frontiers in Molecular Biosciences, vol. 6, p. 17, 2019.
    Résumé : microRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3'-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3'-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3'-adaptor ratios displayed a steady value of 0.3 +/- 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum.
    Mots-clés : cerebellum, high-throughput sequencing, Illumina technology, ligation bias, miRNA abundance, miRNA expression profile, NGS, PF.

  • E. Bordet, M. Frétaud, E. Crisci, E. Bouguyon, S. Rault, J. Pezant, A. Pleau, P. Renson, E. Giuffra, T. Larcher, M. Bourge, O. Bourry, O. Boulesteix, C. Langevin, I. Schwartz-Cornil, et N. Bertho, « Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus », Frontiers in Immunology, vol. 10, p. 953, 2019.
    Résumé : Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.
    Mots-clés : antibodies, antigen, B cell, BCL6, CYTO, PF, PRRSV.

  • C. Bou-Nader, P. Barraud, L. Pecqueur, J. Perez, C. Velours, W. Shepard, M. Fontecave, C. Tisne, et D. Hamdane, « Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2 », Nucleic Acids Research, vol. 47, nᵒ 6, p. 3117-3126, avr. 2019.
    Résumé : Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.
    Mots-clés : complex reveals, evolution, extended dsrbd, mechanism, motif, nmr, PF, PIM, proteins, structural insights, trna(3)(lys).

  • V. Campanacci, A. Urvoas, S. Cantos-Fernandes, M. Aumont-Nicaise, A. - A. Arteni, C. Velours, M. Valerio-Lepiniec, B. Dreier, A. Plückthun, A. Pilon, C. Poüs, P. Minard, et B. Gigant, « Insight into microtubule nucleation from tubulin-capping proteins », Proceedings of the National Academy of Sciences of the United States of America, vol. 116, nᵒ 20, p. 9859-9864, avr. 2019.
    Résumé : Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or β-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.
    Mots-clés : artificial binding proteins, B3S, CopN, CRYOEM, MIKICA, MIP, PF, PIM.

  • P. Fernández Varela, C. Velours, M. Aumont-Niçaise, B. Pineau, P. Legrand, et I. Poquet, « Biophysical and structural characterization of a zinc-responsive repressor of the MarR superfamily », PloS One, vol. 14, nᵒ 2, p. e0210123, 2019.
    Résumé : The uptake of zinc, which is vital in trace amounts, is tightly controlled in bacteria. For this control, bacteria of the Streptococcaceae group use a Zn(II)-binding repressor named ZitR in lactococci and AdcR in streptococci, while other bacteria use a Zur protein of the Ferric uptake regulator (Fur) superfamily. ZitR and AdcR proteins, characterized by a winged helix-turn-helix DNA-binding domain, belong to the multiple antibiotic resistance (MarR) superfamily, where they form a specific group of metallo-regulators. Here, one such Zn(II)-responsive repressor, ZitR of Lactococcus lactis subspecies cremoris strain MG1363, is characterized. Size Exclusion Chromatography-coupled to Multi Angle Light Scattering, Circular Dichroism and Isothermal Titration Calorimetry show that purified ZitR is a stable dimer complexed to Zn(II), which is able to bind its two palindromic operator sites on DNA fragments. The crystal structure of ZitR holo-form (Zn(II)4-ZitR2), has been determined at 2.8 Å resolution. ZitR is the fourth member of the MarR metallo-regulator subgroup whose structure has been determined. The folding of ZitR/AdcR metallo-proteins is highly conserved between both subspecies (cremoris or lactis) in the Lactococcus lactis species and between species (Lactococcus lactis and Streptococcus pneumoniae or pyogenes) in the Streptococcaceae group. It is also similar to the folding of other MarR members, especially in the DNA-binding domain. Our study contributes to better understand the biochemical and structural properties of metallo-regulators in the MarR superfamily.
    Mots-clés : PF, PIM.

  • M. A. Hanson, A. Dostálová, C. Ceroni, M. Poidevin, S. Kondo, et B. Lemaitre, « Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach », eLife, vol. 8, févr. 2019.
    Résumé : Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete all known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 4 Cecropins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking all 14 AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.
    Mots-clés : AMP, D. melanogaster, DBG, Diptericin, Drosocin, EQYY, Imd, immunology, inflammation, PF, systemic immunity, Toll.

  • R. Le Bars, M. W. Bianchi, et C. Lefebvre, « Three-Dimensional Surface Rendering of ESCRT Proteins Microscopy Data Using UCSF Chimera Software », Methods in Molecular Biology (Clifton, N.J.), vol. 1998, p. 149-161, 2019.
    Résumé : Visualization of subcellular localization of ESCRT proteins and their interactions with different cellular compartments are critical to understand their function. This approach requires the generation of an important amount of 3D fluorescence microscopy data that is not always easy to visualize and apprehend.We describe a step-by-step protocol for 3D surface rendering of confocal microscopy acquisitions using the free software UCSF-Chimera, generating snapshots and animations to facilitate analysis and presentation of subcellular localization data.
    Mots-clés : 3D animation, 3D fluorescence confocal microscopy, BIOCELL, ESCRT, MINION, OTOFAG, PF, PHOT, Surface rendering, UCSF Chimera.

  • S. - Z. Li, A. Vigouroux, M. Ahmar, A. El Sahili, L. Soulere, L. Sago, D. Cornu, S. Morera, et Y. Queneau, « Synthesis of a non-natural glucose-2-phosphate ester able to dupe the acc system of Agrobacterium fabrum », Organic & Biomolecular Chemistry, vol. 17, nᵒ 5, p. 1090-1096, févr. 2019.
    Résumé : The first non-natural derivative of the rare D-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.
    Mots-clés : B3S, diesters, glycogen, hydrolysis, MESB3S, opines, PF, phosphate, plasmids, SICAPS.

  • L. Marty, A. Vigouroux, M. Aumont-Nicaise, F. Pelissier, T. Meyer, C. L. Lavire, Y. Dessaux, et S. Moréra, « Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens », The Biochemical Journal, vol. 476, p. 165-178, janv. 2019.
    Résumé : Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid binding mode through three crystal structures at 1.4, 1.74 and 1.9 Å resolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Å resolution defines a different agropinic acid binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.
    Mots-clés : affinity, agrobacterium tumefaciens, B3S, catabolism, crown-gall, crystal structures, cyclase, degradation, gene, mannopinic acid, MESB3S, MICROBIO, octopine, opine, PBI, periplasmic binding protein, PF, PIM, region, ti-plasmid, transformed plants.

  • L. Pelosi, C. - D. - T. Vo, S. S. Abby, L. Loiseau, B. Rascalou, M. Hajj Chehade, B. Faivre, M. Goussé, C. Chenal, N. Touati, L. Binet, D. Cornu, C. D. Fyfe, M. Fontecave, F. Barras, M. Lombard, et F. Pierrel, « Ubiquinone Biosynthesis over the Entire O2 Range: Characterization of a Conserved O2-Independent Pathway », mBio, vol. 10, nᵒ 4, juill. 2019.
    Résumé : Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O2 Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O2 range.IMPORTANCE In order to colonize environments with large O2 gradients or fluctuating O2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O2 In contrast, the long-described pathway for ubiquinone biosynthesis requires O2 as a substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2 range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O2 levels.
    Mots-clés : bioenergetics, facultative anaerobes, hydroxylases, iron-sulfur, oxygen, peptidase U32, PF, proteobacteria, quinones, respiration, SICAPS, ubiquinone.

  • Y. Raoul des Essarts, J. Pédron, P. Blin, E. Van Dijk, D. Faure, et F. Van Gijsegem, « Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host », Environmental Microbiology, vol. 21, nᵒ 3, p. 1004-1018, mars 2019.
    Résumé : Blackleg and soft rot are devastating diseases on potato stem and tuber caused by Pectobacterium and Dickeya pectinolytic enterobacteria. In European potato cultures, D. dianthicola and D. solani species successively emerged in the past decades. Ecological traits associated to their settlement remain elusive, especially in the case of the recent invader D. solani. In this work, we combined genomic, metabolic and transcriptomic comparisons to unravel common and distinctive genetic and functional characteristics between two D. solani and D. dianthicola isolates. The two strains differ by more than a thousand genes that are often clustered in genomic regions (GRs). Several GRs code for transport and metabolism functions that correlate with some of the differences in metabolic abilities identified between the two Dickeya strains. About 800 D. dianthicola and 1100 D. solani genes where differentially expressed in macerated potato tubers as compared to when growing in rich medium. These include several genes located in GRs, pointing to a potential role in host interaction. In addition, some genes common to both species, including virulence genes, differed in their expression. This work highlighted distinctive traits when D. dianthicola and D. solani exploit the host as a resource. This article is protected by copyright. All rights reserved.
    Mots-clés : biovar 3, dadantii 3937, MICROBIO, NGS, PBI, PF, sequence, strains, virulence genes.

  • G. Sanz, N. Daniel, M. - C. Aubrière, C. Archilla, L. Jouneau, Y. Jaszczyszyn, V. Duranthon, P. Chavatte-Palmer, et A. Jouneau, « Differentiation of derived rabbit trophoblast stem cells under fluid shear stress to mimic the trophoblastic barrier », Biochimica Et Biophysica Acta. General Subjects, juill. 2019.
    Résumé : BACKGROUND: The placenta controls exchanges between the mother and the fetus and therefore fetal development and growth. The maternal environment can lead to disturbance of placental functions, with consequences on the health of the offspring. Since the rabbit placenta is very close to that of humans, rabbit models can provide biomedical data to study human placental function. Yet, to limit the use of animal experiments and to investigate the mechanistic aspects of placental function, we developed a new cell culture model in which rabbit trophoblast cells are differentiated from rabbit trophoblast stem cells. METHODS: Rabbit trophoblast stems cells were derived from blastocysts and differentiated onto a collagen gel and in the presence of a flow of culture medium to mimic maternal blood flow. Transcriptome analysis was performed on the stem and differentiated cells. RESULTS: Our culture model allows the differentiation of trophoblast stem cells. In particular, the fluid shear stress enhances microvilli formation on the differentiated cell surface, lipid droplets formation and fusion of cytotrophoblasts into syncytiotrophoblasts. In addition, the transcriptome analysis confirms the early trophoblast identity of the derived stem cells and reveals upregulation of signaling pathways involved in trophoblast differentiation. CONCLUSION: Thereby, the culture model allows mimicking the in vivo conditions in which maternal blood flow exerts a shear stress on trophoblast cells that influences their phenotype. GENERAL SIGNIFICANCE: Our culture model can be used to study the differentiation of trophoblast stem cells into cytotrophoblasts and syncytiotrophoblasts, as well as the trophoblast function in physiological and pathological conditions.
    Mots-clés : Cell culture model, Differentiation, Fluid shear stress, NGS, PF, Rabbit, Syncytiotrophoblast, Trophoblast stem cell.

  • A. Vigouroux, M. Aumont-Nicaise, A. Boussac, L. Marty, L. Lo Bello, P. Legrand, K. Brillet, I. J. Schalk, et S. Moréra, « A unique ferrous iron binding mode is associated to large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa », The FEBS journal, juill. 2019.
    Résumé : Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Iron release from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore binding protein, respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa. This article is protected by copyright. All rights reserved.
    Mots-clés : B3S, iron, MESB3S, PF, PIM, PS2, Pseudomonas aeruginosa, pyoverdine, siderophore, solute-binding protein.

2018


  • N. Agier, S. Delmas, Q. Zhang, A. Fleiss, Y. Jaszczyszyn, E. van Dijk, C. Thermes, M. Weigt, M. Cosentino-Lagomarsino, et G. Fischer, « The evolution of the temporal program of genome replication », Nature Communications, vol. 9, nᵒ 1, p. 2199, juin 2018.
    Résumé : Genome replication is highly regulated in time and space, but the rules governing the remodeling of these programs during evolution remain largely unknown. We generated genome-wide replication timing profiles for ten Lachancea yeasts, covering a continuous evolutionary range from closely related to more divergent species. We show that replication programs primarily evolve through a highly dynamic evolutionary renewal of the cohort of active replication origins. We found that gained origins appear with low activity yet become more efficient and fire earlier as they evolutionarily age. By contrast, origins that are lost comprise the complete range of firing strength. Additionally, they preferentially occur in close vicinity to strong origins. Interestingly, despite high evolutionary turnover, active replication origins remain regularly spaced along chromosomes in all species, suggesting that origin distribution is optimized to limit large inter-origin intervals. We propose a model on the evolutionary birth, death, and conservation of active replication origins.
    Mots-clés : CHERDIR, NGS, PF.


  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 6, p. 1335-1353, mars 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Corrigendum: Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 12, p. 3173, mai 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • M. Blondeau, M. Sachse, C. Boulogne, C. Gillet, J. - M. Guigner, F. Skouri-Panet, M. Poinsot, C. Ferard, J. Miot, et K. Benzerara, « Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria », Frontiers in Microbiology, vol. 9, p. 1768, 2018.
    Résumé : The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomy and (ii) high-pressure freezing followed by cryo-ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
    Mots-clés : amorphous calcium carbonate, bacterial microcompartment, biomineralization, ca, calcification, carboxysome, CEMOVIS, MET, PF.

  • E. Bordet, F. Blanc, M. Tiret, E. Crisci, E. Bouguyon, P. Renson, P. Maisonnasse, M. Bourge, J. - J. Leplat, E. Giuffra, L. Jouneau, I. Schwartz-Cornil, O. Bourry, et N. Bertho, « Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells », Frontiers in Immunology, vol. 9, p. 2299, oct. 2018.
    Résumé : Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFN gamma secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.
    Mots-clés : blood, cDc1, cellular response, CYTO, dendritic cells, differentiation, dust mite allergen, europe, Lena, lung, macrophages, monocytes, PF, pigs, prrsv, prrsv strain, sialoadhesin, swine, Th1 response.

  • L. Brottier, C. Chaintreuil, P. Simion, C. Scornavacca, R. Rivallan, P. Mournet, L. Moulin, G. P. Lewis, J. Fardoux, S. C. Brown, M. Gomez-Pacheco, M. Bourges, C. Hervouet, M. Gueye, R. Duponnois, H. Ramanankierana, H. Randriambanona, H. Vandrot, M. Zabaleta, M. DasGupta, A. D'Hont, E. Giraud, et J. - F. Arrighi, « A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses », BMC plant biology, vol. 18, nᵒ 1, p. 333, déc. 2018.
    Résumé : BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. RESULTS: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. CONCLUSIONS: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.
    Mots-clés : Aeschynomene, ancient, CHERDIR, CYTO, diversity, evenia, evolutionary dynamics, fixing stem nodules, Genetics, Legumes, leguminosae, model, Nodulation, PF, Phylogenetics, Polyploidy, Symbiosis.


  • C. Chaintreuil, X. Perrier, G. Martin, J. Fardoux, G. P. Lewis, L. Brottier, R. Rivallan, M. Gomez-Pacheco, M. Bourges, L. Lamy, B. Thibaud, H. Ramanankierana, H. Randriambanona, H. Vandrot, P. Mournet, E. Giraud, et J. - F. Arrighi, « Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics », BMC Plant Biology, vol. 18, nᵒ 1, 2018.

  • C. Dard-Dascot, D. Naquin, Y. d'Aubenton-Carafa, K. Alix, C. Thermes, et E. van Dijk, « Systematic comparison of small RNA library preparation protocols for next-generation sequencing », BMC genomics, vol. 19, nᵒ 1, p. 118, 2018.
    Résumé : BACKGROUND: Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance. RESULTS: Among the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2' OMe RNAs both in the TruSeq as well as in the NEXTflex protocol. CONCLUSIONS: While it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2' OMe RNAs.
    Mots-clés : 2’-O-methyl RNA, Bias, CHERDIR, DBG, Library preparation, Next-generation sequencing, NGS, PF, Small RNA.


  • A. Doerflinger, N. N. Quang, E. Gravel, G. Pinna, M. Vandamme, F. Ducongé, et E. Doris, « Biotin-functionalized targeted polydiacetylene micelles », Chemical Communications, 2018.

  • A. Gonzalez-Mula, J. Lachat, L. Mathias, D. Naquin, F. Lamouche, P. Mergaert, et D. Faure, « The biotroph Agrobacterium tumefaciens thrives in tumors by exploiting a wide spectrum of plant host metabolites », The New Phytologist, vol. 222, nᵒ 1, p. 455-467, nov. 2018.
    Résumé : Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g., sucrose, glutamate) and enriched (e.g., opines, GABA, GHB, pyruvate) metabolites that A. tumefaciens could use as nutrients. Transposon-sequencing and transcriptomics pinpointed the genes that are crucial and/or up-regulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, tricarboxylic acid cycle and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge) and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and transposon-sequencing to decipher the lifestyle of a plant pathogen. This article is protected by copyright. All rights reserved.
    Mots-clés : binding, biotroph, ecological niche, fitness, gaba, gene-expression, genome, high specificity, MICROBIO, NGS, pathogen agrobacterium, PBI, PF, plant pathogen, quorum-sensing signal, sequence, structural basis, transcriptomics, transposon-sequencing (Tn-seq), virulence factor.

  • A. González-Mula, J. Lang, C. Grandclément, D. Naquin, M. Ahmar, L. Soulère, Y. Queneau, Y. Dessaux, et D. Faure, « Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant », The New Phytologist, vol. 219, nᵒ 1, p. 350-362, juill. 2018.
    Résumé : Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.
    Mots-clés : acc operon, Agrobacterium, Arabidopsis, arabidopsis-thaliana, genetic-analysis, homoserine lactone, horizontal transfer, inducible locus, iv secretion, MICROBIO, NGS, niche exploitation, nitric-oxide, opine, PBI, PF, plasmid, polar growth, quorum-sensing, ti-plasmid, tumor, virulence plasmid.


  • B. Gronenborn, J. W. Randles, D. Knierim, Q. Barrière, H. J. Vetten, N. Warthmann, D. Cornu, T. Sileye, S. Winter, et T. Timchenko, « Analysis of DNAs associated with coconut foliar decay disease implicates a unique single-stranded DNA virus representing a new taxon », Scientific Reports, vol. 8, nᵒ 1, p. 5698, 2018.
    Mots-clés : MICROBIO, PBI, PF, SICAPS.


  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, M. Desmadril, N. Nhiri, A. Van Dorsselaer, S. Cianferani, E. Jacquet, T. Meinnel, et C. Giglione, « Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts », Biochimica Et Biophysica Acta, vol. 1866, nᵒ 2, p. 348-355, févr. 2018.
    Résumé : Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.
    Mots-clés : AMIG, B3S, DBG, Enzyme mechanism, IMAPP, MIP, N-terminal methionine excision, Peptide deformylase, PF, PIM, PROMTI, Structure, Virus.

  • P. Hardouin, C. Velours, C. Bou-Nader, N. Assrir, S. Laalami, H. Putzer, D. Durand, et B. Golinelli-Pimpaneau, « Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding », Biophysical Journal, vol. 115, nᵒ 11, p. 2102-2113, déc. 2018.
    Résumé : Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b lambda x isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of alpha-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.
    Mots-clés : angle x-ray, B3S, bacillus-subtilis, complexes, decay, FAAM, messenger-rna, PF, PIM, protein secondary structure, reveals binding, scattering, sequence, v-lambda-x.

  • S. Malli, C. Bories, M. Bourge, P. M. Loiseau, et K. Bouchemal, « Surface-dependent endocytosis of poly(isobutylcyanoacrylate) nanoparticles by Trichomonas vaginalis », International Journal of Pharmaceutics, vol. 548, nᵒ 1, p. 276-287, sept. 2018.
    Résumé : Previous data from our research group showed that chitosan-coated poly(isobutylcyanoacrylate) nanoparticles (NPs) (denoted PIBCA/Chito20) exhibited intrinsic anti-Trichomonas vaginalis activity, while PIBCA/pluronic (R) F68 without chitosan (PIBCA/F68) were inactive. However, the mechanism of anti-T. vaginalis activity of chitosan-coated PIBCA NPs is still unknown. Our hypothesis is that chitosan-coated NPs are internalized by the parasite, contrarily to PIBCA/F68. In this investigation, the impact of NP surface on their internalization by the protozoan was studied using flow cytometry and parasite morphological changes after different incubation times with PIBCA/Chito20 NPs were monitored by electron microscopy. Flow-cytometry revealed that PIBCA/Chito20 NPs were uptaken by T. vaginalis as early as 10-min-incubation. Drastic cell morphological transformations were observed from scanning electron microscopy and transmission electron microscopy after incubation with PIBCA/Chito20 NPs. Numerous pits were seen on cell membrane since 10 min. Gradual increase in contact time increased NP endocytosis and induced proportional damages to T. vaginalis membrane. Then, investigation of whether PIBCA/Chito20 NPs can improve MTZ anti-T. vaginalis activity was studied using checkerboard experiment. Calculation of fractional inhibitory concentration index (FICI = 3.53) showed an additive effect between NPs and MTZ.
    Mots-clés : Chitosan, CYTO, Metronidazole, PF, Poly(isobutylcyanoacrylate), Trichomonas vaginalis.

  • T. Meyer, A. Vigouroux, M. Aumont-Nicaise, G. Comte, L. Vial, C. Lavire, et S. Moréra, « The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum », The Journal of Biological Chemistry, vol. 293, nᵒ 21, p. 7930-7941, mai 2018.
    Résumé : The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose-containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.
    Mots-clés : ABC transporter, agrobacterium, Agrobacterium fabrum, B3S, bacteria, crystal structure, galactinol, MESB3S, microbiology, periplasmic binding protein, PF, PIM, plant defense, RFOs, sugar transport.

  • D. Naquin, C. Panozzo, G. Dujardin, E. van Dijk, Y. D'Aubenton-Carafa, et C. Thermes, « Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252 », Microbiology Resource Announcements, vol. 6, nᵒ 17, p. UNSP e00219-18, avr. 2018.
    Résumé : The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology.
    Mots-clés : BIOCELL, BIOMIT, NGS, PF.

  • T. Q. Nguyen, M. Aumont-Niçaise, J. Andreani, C. Velours, M. Chenon, F. Vilela, C. Geneste, P. F. Varela, P. Llinas, et J. Menetrey, « Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1) », The Journal of Biological Chemistry, vol. 293, nᵒ 36, p. 13946-13960, juill. 2018.
    Résumé : JIP1 was first identified as scaffold protein for the MAP kinase JNK and is a cargo protein for the kinesin1 molecular motor. JIP1 plays significant and broad roles in neurons, mainly as a regulator of kinesin1-dependent transport, and is associated with human pathologies such as cancer and Alzheimer disease. JIP1 is specifically recruited by the kinesin-light chain 1 (KLC1) of kinesin1, but the details of this interaction are not yet fully elucidated. Here, using calorimetry, we extensively biochemically characterized the interaction between KLC1 and JIP1. Using various truncated fragments of the tetratricopeptide repeat (TPR) domain of KLC1, we narrowed down its JIP1-binding region and identified seven KLC1 residues critical for JIP1 binding. These ITC-based binding data enabled us to footprint the JIP1-binding site on KLC1-TPR. This footprint was used to uncover the structural basis for the marginal inhibition of JIP1 binding by the autoinhibitory LFP-acidic motif of KLC1, as well as for the competition between JIP1 and another cargo protein of kinesin1, the W-acidic motif-containing Alcadein-α. Also, we examined the role of each of these critical residues of KLC1 for JIP1 binding in the light of the previously reported crystal structure of the KLC1-TPR:JIP1 complex. Finally, sequence search in eukaryotic genomes identified several proteins, among which SH2D6 that exhibit similar motif to the KLC1-binding motif of JIP1. Overall, our extensive biochemical characterization of the KLC:JIP1 interaction, as well as identification of potential KLC1-binding partners improve the understanding of how this growing family of cargos is recruited to kinesin1 by KLC1.
    Mots-clés : Alcadein, AMIG, B3S, JNK-interacting protein 1, MIKICA, MST, PF, PIM, SH2D6, TorsinA, TPR domain, Y-acidic motif.

  • J. Pirrello, C. Deluche, N. Frangne, F. Gévaudant, E. Maza, A. Djari, M. Bourge, J. - P. Renaudin, S. Brown, C. Bowler, M. Zouine, C. Chevalier, et N. Gonzalez, « Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation », The Plant Journal: For Cell and Molecular Biology, vol. 93, nᵒ 2, p. 387-398, 2018.
    Résumé : As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.
    Mots-clés : Cell Nucleus, CYTO, data interpretation, DNA, Plant, Endoreduplication, Fruit, Gene Expression Profiling, Lycopersicon esculentum, PF, Ploidies, RNA, Plant, RNA-Seq profiling, Sequence Analysis, RNA, Solanum lycopersicum.

  • X. Raffoux, M. Bourge, F. Dumas, O. C. Martin, et M. Falque, « High-throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae », Yeast (Chichester, England), vol. 35, nᵒ 6, p. 431-442, 2018.
    Résumé : Allelic recombination owing to meiotic crossovers is a major driver of genome evolution, as well as a key player for the selection of high-performing genotypes in economically important species. Therefore, we developed a high-throughput and low-cost method to measure recombination rates and crossover patterning (including interference) in large populations of the budding yeast Saccharomyces cerevisiae. Recombination and interference were analysed by flow cytometry, which allows time-consuming steps such as tetrad microdissection or spore growth to be avoided. Moreover, our method can also be used to compare recombination in wild-type vs. mutant individuals or in different environmental conditions, even if the changes in recombination rates are small. Furthermore, meiotic mutants often present recombination and/or pairing defects affecting spore viability but our method does not involve growth steps and thus avoids filtering out non-viable spores.
    Mots-clés : crossing-over, CYTO, flow cytometry, fluorescent tag, meiosis, PF, yeast.

  • X. Raffoux, M. Bourge, F. Dumas, O. C. Martin, et M. Falque, « Role of Cis, Trans, and Inbreeding Effects on Meiotic Recombination in Saccharomyces cerevisiae », Genetics, vol. 210, nᵒ 4, p. 1213-1226, déc. 2018.
    Résumé : Meiotic recombination is a major driver of genome evolution by creating new genetic combinations. To probe the factors driving variability of meiotic recombination, we used a high-throughput method to measure recombination rates in hybrids between SK1 and a total of 26 Saccharomyces cerevisiae strains from different geographic origins and habitats. Fourteen intervals were monitored for each strain, covering chromosomes VI and XI entirely, and part of chromosome I. We found an average number of crossovers per chromosome ranging between 1.0 and 9.5 across strains ("domesticated" or not), which is higher than the average between 0.5 and 1.5 found in most organisms. In the different intervals analyzed, recombination showed up to ninefold variation across strains but global recombination landscapes along chromosomes varied less. We also built an incomplete diallel experiment to measure recombination rates in one region of chromosome XI in 10 different crosses involving five parental strains. Our overall results indicate that recombination rate is increasingly positively correlated with sequence similarity between homologs (i) in DNA double-strand-break-rich regions within intervals, (ii) in entire intervals, and (iii) at the whole genome scale. Therefore, these correlations cannot be explained by cis effects only. We also estimated that cis and trans effects explained 38 and 17%, respectively, of the variance of recombination rate. In addition, by using a quantitative genetics analysis, we identified an inbreeding effect that reduces recombination rate in homozygous genotypes, while other interaction effects (specific combining ability) or additive effects (general combining ability) are found to be weak. Finally, we measured significant crossover interference in some strains, and interference intensity was positively correlated with crossover number.
    Mots-clés : crossover interference, crossovers, CYTO, diversity, dna recombination, double-strand breaks, evolution, heterozygosity, interference, landscape, maize, meiosis, mismatch repair, PF, resolution, yeast.

  • D. Raoux-Barbot, A. Belyy, L. Worpenberg, S. Montluc, C. Deville, V. Henriot, C. Velours, D. Ladant, L. Renault, et U. Mechold, « Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin », PloS One, vol. 13, nᵒ 11, p. e0206133, 2018.
    Résumé : Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.
    Mots-clés : ACTIN, adenylate-cyclase, B3S, calmodulin, cytoskeleton disruption, domains, edema factor, exotoxin, PF, PIM, pseudomonas-aeruginosa exoy, structural basis, toxin, yersinia protein-kinase.

  • S. Srisuwan, D. Sihachakr, J. Martín, J. Vallès, A. Ressayre, S. C. Brown, et S. Siljak-Yakovlev, « Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas, Convolvulaceae) complex », Plant Biology (Stuttgart, Germany), nov. 2018.
    Résumé : Genome size evolution, and its relationships with pollen grain size, has been investigated in the sweet potato (Ipomoea batatas), an economically important crop, and closely related diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, 10 sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined by flow cytometry. Pollen grains have been studied at scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged over 3.12-3.29 pg, mean monoploid genome size being 0.539 pg (527 Mbp) much as for the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was respectively 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida, I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% of AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, genome size and ploidy levels were correlated with pollen size. Our results allow us proposing alternative or complementary hypotheses to the one currently proposed for the formation of hexaploid Ipomoea batatas. This article is protected by copyright. All rights reserved.
    Mots-clés : Convolvulaceae, CYTO, genome size, Ipomoea batatas, PF, pollen size, polyploidy, propidium iodide flow cytometry, sweet potato.

  • Á. Szabó, C. Papin, D. Cornu, E. Chélot, Z. Lipinszki, A. Udvardy, V. Redeker, U. Mayor, et F. Rouyer, « Ubiquitylation Dynamics of the Clock Cell Proteome and TIMELESS during a Circadian Cycle », Cell Reports, vol. 23, nᵒ 8, p. 2273-2282, mai 2018.
    Résumé : Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. In this study, we used an in vivo ubiquitin labeling assay to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. We find that cyclic ubiquitylation affects MEGATOR (MTOR), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, we show that the ubiquitin ligase subunits CULLIN-3 (CUL-3) and SUPERNUMERARY LIMBS (SLMB) cooperate for ubiquitylating the TIMELESS protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system.
    Mots-clés : circadian clock, Drosophila, oscillation, PF, protein degradation, proteomics, SICAPS, ubiquitin, ubiquitin ligase.

  • A. Tubbs, S. Sridharan, N. van Wietmarschen, Y. Maman, E. Callen, A. Stanlie, W. Wu, X. Wu, A. Day, N. Wong, M. Yin, A. Canela, H. Fu, C. Redon, S. C. Pruitt, Y. Jaszczyszyn, M. I. Aladjem, P. D. Aplan, O. Hyrien, et A. Nussenzweig, « Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse », Cell, vol. 174, nᵒ 5, p. 1127-1142.e19, août 2018.
    Résumé : Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.
    Mots-clés : DNA breaks, fragile sites, genome instability, NGS, PF, poly(dA:dT) tracts, replication fork barrier, replication origins, replication stress, ribosomal DNA.

  • E. L. van Dijk, Y. Jaszczyszyn, D. Naquin, et C. Thermes, « The Third Revolution in Sequencing Technology », Trends in Genetics, vol. 34, nᵒ 9, p. 666-681, sept. 2018.
    Résumé : Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.
    Mots-clés : disease, dna methylation, human genome, long-read sequencing, n-6-adenine, nanopore, nanopore sequencing, next-generation sequencing, NGS, PF, real-time, single-molecule, single-molecule real-time sequencing, structural variation, surveillance, synthetic long-read sequencing, third-generation sequencing, transcriptome.

  • X. Wu, H. Kabalane, M. Kahli, N. Petryk, B. Laperrousaz, Y. Jaszczyszyn, G. Drillon, F. - E. Nicolini, G. Perot, A. Robert, C. Fund, F. Chibon, R. Xia, J. Wiels, F. Argoul, V. Maguer-Satta, A. Arneodo, B. Audit, et O. Hyrien, « Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions », Nucleic Acids Research, vol. 46, nᵒ 19, p. 10157-10172, nov. 2018.
    Résumé : The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repliseq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.
    Mots-clés : barrier, chronic myelogenous leukemia, damage-response, domains, gene, landscape, model, NGS, part, PF, reveals, stress.

2017


  • L. Becker, S. Bellow, V. Carré, G. Latouche, A. Poutaraud, D. Merdinoglu, S. C. Brown, Z. G. Cerovic, et P. Chaimbault, « Correlative Analysis of Fluorescent Phytoalexins by Mass Spectrometry Imaging and Fluorescence Microscopy in Grapevine Leaves », Analytical Chemistry, juin 2017.
    Résumé : Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.
    Mots-clés : IMAGIF, PF, PHOT.

  • C. Bou-Nader, D. Cornu, V. Guerineau, T. Fogeron, M. Fontecave, et D. Hamdane, « Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes », Angewandte Chemie (International Ed. in English), août 2017.
    Résumé : To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.
    Mots-clés : artificial enzymes, flavoenzyme mechanism, Methylation, Nucleotides, PF, reaction intermediates, SICAPS.

  • C. Bou-Nader, L. Pecqueur, D. Cornu, M. Lombard, M. Dezi, M. Nicaise, C. Velours, M. Fontecave, et D. Hamdane, « Power of protein/tRNA functional assembly against aberrant aggregation », Physical chemistry chemical physics: PCCP, oct. 2017.
    Résumé : Understanding the mechanisms of protein oligomerization and aggregation is a major concern for biotechnology and medical purposes. However, significant challenges remain in determining the mechanism of formation of these superstructures and the environmental factors that can precisely modulate them. Notably the role that a functional ligand plays in the process of protein aggregation is largely unexplored. We herein address these issues with an original flavin-dependent RNA methyltransferase (TrmFO) used as a protein model since this protein employs a complex set of cofactors and ligands for catalysis. Here, we show that TrmFO carries an unstable protein structure that can partially mis-unfold leading to either formation of irregular and nonfunctional soluble oligomers endowed with hyper-thermal stability or large amorphous aggregates in the presence of salts. Mutagenesis confirmed that this peculiarity is an intrinsic property of a polypeptide and it is independent of the flavin coenzyme. Structural characterization and kinetic studies identified several regions of the protein that enjoy conformational changes and more particularly pinpointed the N-terminal subdomain as being a key element in the mechanisms of oligomerization and aggregation. Only stabilization of this region via tRNA suppresses these aberrant protein states. Although protein chaperones emerged as major actors against aggregation, our study emphasizes that other powerful mechanisms exist such as the stabilizing effect of functional assemblies that provide an additional layer of protection against the instability of the proteome.
    Mots-clés : PF, PIM, SICAPS.

  • S. C. Brown, M. Bourge, N. Maunoury, M. Wong, M. W. Bianchi, S. Lepers-Andrzejewski, P. Besse, S. Siljak-Yakovlev, M. Dron, et B. Satiat-Jeunemaître, « DNA remodelling by Strict Partial Endoreplication in orchids, an original process in the plant kingdom », Genome Biology and Evolution, avr. 2017.
    Résumé : DNA remodelling during endoreplication appears to be a strong developmental characteristic in orchids. In this study, we analysed DNA content and nuclei in 41 species of orchids to further map the genome evolution in this plant family. We demonstrate that the DNA remodelling observed in 36 out of 41 orchids studied corresponds to strict partial endoreplication. Such process is developmentally regulated in each wild species studied. Cytometry data analyses allowed us to propose a model where nuclear states 2C, 4E, 8E, etc. form a series comprising a fixed proportion, the euploid genome 2C, plus 2 to 32 additional copies of a complementary part of the genome. The fixed proportion ranged from 89% of the genome in Vanilla mexicana down to 19% in V. pompona, the lowest value for all 148 orchids reported. Insterspecific hybridisation did not suppress this phenomenon. Interestingly, this process was not observed in mass-produced epiphytes. Nucleolar volumes grow with the number of endocopies present, coherent with high transcription activity in endoreplicated nuclei. Our analyses suggest species-specific chromatin rearrangement. Towards understanding endoreplication, V. planifolia constitutes a tractable system for isolating the genomic sequences that confer an advantage via endoreplication from those that apparently suffice at diploid level.
    Mots-clés : BIOCELL, CYTO, cytogenetics, cytometry, DYNBSJ, endoreplication, genome imbalance, Genome Size, IMAGIF, MINION, PF, PHOT, Vanilla.


  • E. Dambroise, M. Simion, T. Bourquard, S. Bouffard, B. Rizzi, Y. Jaszczyszyn, M. Bourge, P. Affaticati, A. Heuzé, J. Jouralet, J. Edouard, S. Brown, C. Thermes, A. Poupon, E. Reiter, F. Sohm, F. Bourrat, et J. - S. Joly, « Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile: Features of Neuroepithelial Cells in Fish », STEM CELLS, 2017.
    Mots-clés : BMgif, CYTO, IMAGIF, NGS, PF, PHOT.

  • T. Di Meo, W. Ghattas, C. Herrero, C. Velours, P. Minard, J. - P. Mahy, R. Ricoux, et A. Urvoas, « αRep A3: A versatile artificial scaffold for metalloenzyme design », Chemistry (Weinheim an Der Bergstrasse, Germany), mai 2017.
    Résumé : αRep is a new family of artificial proteins based on a thermostable alpha-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to receive metal complexes and thus appears as suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen and changed independently into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine of each protein variant and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was bound specifically by the two biohybrids with two different binding modes and, in addition, the holo biohybrid A3F119NPH was found to be able to catalyze enantioselectively the Diels-Alder (D-A) cycloaddition with up to 62% ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a new promising route for the design and production of new enantioselective biohybrids based on entirely artificial proteins issued from a highly diverse library.
    Mots-clés : artificial repeat proteins, B3S, Diels-Alder reaction, Enantioselective Catalysis, MIP, PF, PIM.


  • R. El Helou, G. Pinna, O. Cabaud, J. Wicinski, R. Bhajun, L. Guyon, C. Rioualen, P. Finetti, A. Gros, B. Mari, P. Barbry, F. Bertucci, G. Bidaut, A. Harel-Bellan, D. Birnbaum, E. Charafe-Jauffret, et C. Ginestier, « miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling », Cell Reports, vol. 18, nᵒ 9, p. 2256-2268, 2017.

  • C. Esnault, D. Leiber, C. Toffano-Nioche, Z. Tanfin, et M. - J. Virolle, « Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity », Applied Microbiology and Biotechnology, vol. 101, nᵒ 1, p. 139-145, janv. 2017.
    Résumé : Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [(3)H]phosphatidic acid (PA) released from [(3)H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.
    Mots-clés : Amino Acid Motifs, Cell Membrane, Choline, DBG, eBio, Hydrolysis, Lipid droplets, MESMIC, MICROBIO, PF, Phosphatidic Acids, Phosphatidylcholines, Phospholipase D, Phosphotransferases (Phosphate Group Acceptor), Polyphosphate kinase, Promiscuous enzyme, Protein Conformation, SSFA, Streptomyces lividans.


  • P. P. Weil, Y. Jaszczyszyn, A. Baroin-Tourancheau, J. Postberg, et L. Amar, « Holistic and Affordable Analyses of MicroRNA Expression Profiles Using Tagged cDNA Libraries and a Multiplex Sequencing Strategy », in Functional Genomics, vol. 1654, M. Kaufmann, C. Klinger, et A. Savelsbergh, Éd. New York, NY: Springer New York, 2017, p. 179-196.


  • J. Lang, A. Vigouroux, A. El Sahili, A. Kwasiborski, M. Aumont-Nicaise, Y. Dessaux, J. A. Shykoff, S. Moréra, et D. Faure, « Fitness costs restrict niche expansion by generalist niche-constructing pathogens », The ISME Journal, vol. 11, nᵒ 2, p. 374-385, 2017.
    Mots-clés : B3S, MESB3S, MICROBIO, PBI, PF, PIM.

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