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Accueil > Publications

Publications Département Microbiologie

2018


  • P. Abranyi-Balogh, L. Petri, T. Imre, P. Szijj, A. Scarpino, M. Hrast, A. Mitrovic, U. P. Fonovic, K. Nemeth, H. Barreteau, D. I. Roper, K. Horvati, G. G. Ferenczy, J. Kos, J. Ilas, S. Gobec, et G. M. Keseru, « A road map for prioritizing warheads for cysteine targeting covalent inhibitors », European Journal of Medicinal Chemistry, vol. 160, p. 94-107, déc. 2018.
    Résumé : Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.
    Mots-clés : Cathepsin B, Cathepsin X, cathepsin-b, Covalent inhibitors, design, drug discovery, electrophiles, Electrophilic warheads, ENVBAC, fragments, GSH reactivity assay, kinase inhibitors, MICROBIO, MurA, Oligopeptide specificity assay.


  • G. Bourgeois, J. Seguin, M. Babin, P. Belin, M. Moutiez, Y. Mechulam, M. Gondry, et E. Schmitt, « Structural basis for partition of the cyclodipeptide synthases into two subfamilies », Journal of Structural Biology, vol. 203, nᵒ 1, p. 17-26, juill. 2018.
    Résumé : Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
    Mots-clés : Aminoacyl-tRNA synthetases, BIOSYN, Cyclodipeptides, Diketopiperazines, MICROBIO, Non-ribosomal peptide synthesis, Rossmann fold, Transfer RNA.

  • N. Canu, P. Belin, R. Thai, I. Correia, O. Lequin, J. Seguin, M. Moutiez, et M. Gondry, « Incorporation of Non-canonical Amino Acids into 2,5-Diketopiperazines by Cyclodipeptide Synthases », Angewandte Chemie (International Ed. in English), vol. 57, nᵒ 12, p. 3118-3122, mars 2018.
    Résumé : The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5-diketopiperazines (2,5-DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an in vivo method for incorporating non-canonical amino acids (ncAAs) into 2,5-DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5-DKP biosynthetic pathways. CDPSs use aminoacyl-tRNAs as substrates. We exploited the natural ability of aminoacyl-tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non-canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5-DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide-tailoring enzymes found in 2,5-DKP biosynthetic pathways.
    Mots-clés : biocatalysis, BIOSYN, biosynthesis, Cyclodipeptide synthases, cyclodipeptides, Diketopiperazines, MICROBIO, Natural product engineering, Non-canonical amino acid.

  • R. Catchpole, A. Gorlas, J. Oberto, et P. Forterre, « A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors », Extremophiles: Life Under Extreme Conditions, vol. 22, nᵒ 4, p. 591-598, juill. 2018.
    Résumé : Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli-T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli-T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.
    Mots-clés : Archaea, ARCHEE, Cloning, Gene cloning and expression, Genetics of extremophiles, Hyperthermophiles, MICROBIO, Molecular biology, Molecular biology of archaea.


  • F. Coll, J. Phelan, G. A. Hill-Cawthorne, M. B. Nair, K. Mallard, S. Ali, A. M. Abdallah, S. Alghamdi, M. Alsomali, A. O. Ahmed, S. Portelli, Y. Oppong, A. Alves, T. B. Bessa, S. Campino, M. Caws, A. Chatterjee, A. C. Crampin, K. Dheda, N. Furnham, J. R. Glynn, L. Grandjean, D. Minh Ha, R. Hasan, Z. Hasan, M. L. Hibberd, M. Joloba, E. C. Jones-López, T. Matsumoto, A. Miranda, D. J. Moore, N. Mocillo, S. Panaiotov, J. Parkhill, C. Penha, J. Perdigão, I. Portugal, Z. Rchiad, J. Robledo, P. Sheen, N. T. Shesha, F. A. Sirgel, C. Sola, E. Oliveira Sousa, E. M. Streicher, P. V. Helden, M. Viveiros, R. M. Warren, R. McNerney, A. Pain, et T. G. Clark, « Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis », Nature Genetics, janv. 2018.

  • D. Couvin, A. Bernheim, C. Toffano-Nioche, M. Touchon, J. Michalik, B. Néron, E. P. C Rocha, G. Vergnaud, D. Gautheret, et C. Pourcel, « CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins », Nucleic Acids Research, mai 2018.
    Résumé : CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.
    Mots-clés : BDG, LGBMB, MICROBIO, SSFA.

  • M. Crüsemann, R. Reher, I. Schamari, A. O. Brachmann, T. Ohbayashi, M. Kuschak, D. Malfacini, A. Seidinger, M. Pinto-Carbó, R. Richarz, T. Reuter, S. Kehraus, A. Hallab, M. Attwood, H. B. Schiöth, P. Mergaert, Y. Kikuchi, T. F. Schäberle, E. Kostenis, D. Wenzel, C. E. Müller, J. Piel, A. Carlier, L. Eberl, et G. M. König, « Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359 », Angewandte Chemie (International Ed. in English), vol. 57, nᵒ 3, p. 836-840, janv. 2018.
    Résumé : The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.
    Mots-clés : biosynthesis, FR900359, G proteins, G proteins, Heterologous expression, MICROBIO, natural products, PBI.


  • V. Da Cunha, M. Gaia, A. Nasir, et P. Forterre, « Asgard archaea do not close the debate about the universal tree of life topology », PLOS Genetics, vol. 14, nᵒ 3, p. e1007215, mars 2018.

  • L. Debarbieux, P. Forterre, M. Krupovic, M. Kutateladze, et D. Prangishvili, « Centennial celebration of the bacteriophage research », Research in Microbiology, vol. 169, nᵒ 9, p. 479-480, nov. 2018.
    Mots-clés : ARCHEE, MICROBIO, viruses.

  • Y. Dessaux et D. Faure, « Niche Construction and Exploitation by Agrobacterium: How to Survive and Face Competition in Soil and Plant Habitats », Berlin, Heidelberg: Springer Berlin Heidelberg, 2018.

  • Y. Dessaux et D. Faure, « Quorum Sensing and Quorum Quenching in Agrobacterium: A "Go/No Go System"? », Genes, vol. 9, nᵒ 4, avr. 2018.
    Résumé : The pathogen Agrobacterium induces gall formation on a wide range of dicotyledonous plants. In this bacteria, most pathogenicity determinants are borne on the tumour inducing (Ti) plasmid. The conjugative transfer of this plasmid between agrobacteria is regulated by quorum sensing (QS). However, processes involved in the disturbance of QS also occur in this bacteria under the molecular form of a protein, TraM, inhibiting the sensing of the QS signals, and two lactonases BlcC (AttM) and AiiB that degrade the acylhomoserine lactone (AHL) QS signal. In the model Agrobacteriumfabrum strain C58, several data, once integrated, strongly suggest that the QS regulation may not be reacting only to cell concentration. Rather, these QS elements in association with the quorum quenching (QQ) activities may constitute an integrated and complex “go/no go system” that finely controls the biologically costly transfer of the Ti plasmid in response to multiple environmental cues. This decision mechanism permits the bacteria to sense whether it is in a gall or not, in a living or decaying tumor, in stressed plant tissues, etc. In this scheme, the role of the lactonases selected and maintained in the course of Ti plasmid and agrobacterial evolution appears to be pivotal.
    Mots-clés : (p)ppGpp, Agrobacterium, GABA, lactonase, MICROBIO, PBI, proline, quorum quenching, quorum sensing, Ti plasmid.

  • A. Durand, M. - L. Bourbon, A. - S. Steunou, B. Khalfaoui-Hassani, C. Legrand, A. Guitton, C. Astier, et S. Ouchane, « Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model », The Journal of Biological Chemistry, vol. 293, nᵒ 3, p. 808-818, janv. 2018.
    Résumé : The cbb3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus.
    Mots-clés : BACADA, bacteria, cbb3 cytochrome c oxidase biogenesis, cytochrome oxidase, Membrane protein, MICROBIO.

  • P. Durand, D. De Luca, et P. Tissieres, « What's new in lung ultrasound in the critically ill or injured child », Intensive Care Medicine, sept. 2018.


  • M. El Ghachi, N. Howe, C. - Y. Huang, V. Olieric, R. Warshamanage, T. Touzé, D. Weichert, P. J. Stansfeld, M. Wang, F. Kerff, et M. Caffrey, « Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis », Nature Communications, vol. 9, nᵒ 1, 2018.

  • D. Faure, J. - C. Simon, et T. Heulin, « Holobiont: a conceptual framework to explore the eco-evolutionary and functional implications of host-microbiota interactions in all ecosystems », The New Phytologist, vol. 218, nᵒ 4, p. 1321-1324, juin 2018.
    Mots-clés : evolution, holobiont, hologenome, MICROBIO, microbiome, microbiota, PBI, plant-microbiota interactions.


  • M. J. Fer, L. L. Corre, N. Pietrancosta, N. Evrard-Todeschi, S. Olatunji, A. Bouhss, S. Calvet-Vitale, et C. Gravier-Pelletier, « Bacterial Transferase MraY, a Source of Inspiration towards New Antibiotics », Current Medicinal Chemistry, vol. 25, mars 2018.

  • M. Fonvielle, A. Bouhss, C. Hoareau, D. Patin, D. Mengin-Lecreulx, L. Iannazzo, N. Sakkas, A. El Sagheer, T. Brown, M. Etheve-Quelquejeu, et M. Arthur, « Synthesis of Lipid-Carbohydrate-Peptidyl-RNA Conjugates to Explore the Limits Imposed by the Substrate Specificity of Cell Wall Enzymes on the Acquisition of Drug Resistance », Chemistry-a European Journal, vol. 24, nᵒ 56, p. 14911-14915, oct. 2018.
    Résumé : Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNA(Gly) and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 = 56 nm). The synthetic route was exploited to generate substrates and inhibitors containing D-lactate residue (D-Lac) instead of D-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.
    Mots-clés : complex, ENVBAC, Fem, mechanism, MICROBIO, mrsa, oligonucleotide, proteins, RNA conjugates, small interfering rna, Staphylococcus aureus, staphylococcus-aureus, targeted delivery, VanA, vancomycin.
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  • R. Fujikane, I. Behm-Ansmant, A. - S. Tillault, C. Loegler, V. Igel-Bourguignon, E. Marguet, P. Forterre, C. Branlant, Y. Motorin, et B. Charpentier, « Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein », Scientific Reports, vol. 8, nᵒ 1, p. 13815, sept. 2018.
    Résumé : Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.
    Mots-clés : ARCHEE, conserved pseudouridine, crystal-structure, escherichia-coli, haloferax-volcanii, hyperthermophilic archaeon, in-vivo, MICROBIO, pseudouridine synthase, substrate rna, sulfolobus-solfataricus, thermococcus-kodakaraensis kod1.

  • S. Gill, R. Catchpole, et P. Forterre, « Extracellular membrane vesicles (EVs) in the three domains of life and beyond », FEMS microbiology reviews, nov. 2018.
    Résumé : Cells from all three domains of life, Archaea, Bacteria and Eukarya, produce extracellular vesicles (EVs) which are sometimes associated to filamentous structures known as nanopods or nanotubes. The mechanisms of EV biogenesis in the three domains remain poorly understood, although studies in Bacteria and Eukarya indicate that the regulation of lipid composition plays a major role in initiating membrane curvature. EVs are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. They have been implicated in many aspects of cell physiology such as stress response, inter-cellular competition, lateral gene transfer (via RNA or DNA), pathogenicity, and detoxification. Their role in various human pathologies and aging has aroused much interest in recent years. EVs can be used as decoys against viral attack but virus infected cells also produce EVs that boost viral infection. Here, we review current knowledge on EVs in the three domains of life and their interactions with the viral world.
    Mots-clés : ARCHEE, MICROBIO.

  • M. Gondry, I. B. Jacques, R. Thai, M. Babin, N. Canu, J. Seguin, P. Belin, J. - L. Pernodet, et M. Moutiez, « A Comprehensive Overview of the Cyclodipeptide Synthase Family Enriched with the Characterization of 32 New Enzymes », Frontiers in Microbiology, vol. 9, p. 46, 2018.
    Résumé : Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored. Here, we describe the activity of 32 new CDPSs, bringing the number of experimentally characterized CDPSs to about 100. We detect 16 new cyclodipeptides, one of which containing an arginine which has never been observed previously. This brings to 75 the number of cyclodipeptides formed by CDPSs out of the possible 210 natural ones. We also identify several consensus sequences related to the synthesis of a specific cyclodipeptide, improving the predictive model of CDPS specificity. The improved prediction method enables to propose the main product synthesized for about 80% of the CDPS sequences available in databases and opens the way for the deciphering of CDPS-dependent pathways. Analysis of phylum distribution and predicted activity for all CDPSs identified in databases shows that the experimentally characterized set is representative of the whole family. Our work also demonstrates that some cyclodipeptides, precursors of diketopiperazines with interesting pharmacological properties and previously described as being synthesized by fungal non-ribosomal peptide synthetases, can also be produced by CDPSs in bacteria.
    Mots-clés : ACTINO, activity prediction, BIOSYN, Biosynthetic Pathways, cyclodipeptide MS/MS, cyclodipeptide synthase, diketopiperazine, MICROBIO, Secondary metabolites, tRNA-dependent enzymes.

  • 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, 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 : biotroph, ecological niche, fitness, MICROBIO, NGS, PBI, PF, plant pathogen, transcriptomics, transposon-sequencing (Tn-seq).

  • 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.

  • A. Gorlas, R. Catchpole, E. Marguet, et P. Forterre, « Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation », Extremophiles: Life Under Extreme Conditions, nov. 2018.
    Résumé : Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.
    Mots-clés : ARCHEE, Double-strand breaks, MICROBIO, Plasmid, Topology, UV irradiation.

  • A. Gorlas, P. Jacquemot, J. - M. Guigner, S. Gill, P. Forterre, et F. Guyot, « Greigite nanocrystals produced by hyperthermophilic archaea of Thermococcales order », Plos One, vol. 13, nᵒ 8, p. e0201549, août 2018.
    Résumé : Interactions between hyperthermophilic archaea and minerals occur in hydrothermal deep-ea vents, one of the most extreme environments for life on Earth. These interactions occur in the internal pores and at surfaces of active hydrothermal chimneys. In this study, we show that, at 85 degrees C, Thermococcales, the predominant hyperthermophilic microorganisms inhabiting hot parts of hydrothermal deep-sea vents, produce greigite nanocrystals (Fe3S4) on extracellular polymeric substances, and that an amorphous iron phosphate acts as a precursor phase. Greigite, although a minor component of chimneys, is a recognized catalyst for CO2 reduction thus implying that Thermococcales may influence the balance of CO2 in hydrothermal ecosystems. We propose that observation of greigite nanocrystals on extracellular polymeric substances could provide a signature of hyperthermophilic life in hydrothermal deep-sea vents.
    Mots-clés : ARCHEE, biomineralization, growth, iron sulfides, life, mechanisms, MICROBIO, mineralization, minerals, oxidizing bacteria, sequence, vesicles.

  • B. Gourion et B. Alunni, « Strain-Specific Symbiotic Genes: A New Level of Control in the Intracellular Accommodation of Rhizobia Within Legume Nodule Cells », Molecular plant-microbe interactions: MPMI, vol. 31, nᵒ 3, p. 287-288, mars 2018.
    Résumé : This is a short commentary on the article by Wang et al. published in MPMI Vol. 31, No. 2, pages 240-248.
    Mots-clés : MICROBIO, PBI.


  • 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, 2018.
    Mots-clés : MICROBIO, PBI, PF, SICAPS.


  • M. Hrast, M. Jukič, D. Patin, J. Tod, C. G. Dowson, D. I. Roper, H. Barreteau, et S. Gobec, « In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB », Chemical Biology & Drug Design, vol. 91, nᵒ 6, p. 1101-1112, 2018.

  • Y. Ishino, M. Krupovic, et P. Forterre, « History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology », Journal of Bacteriology, vol. 200, nᵒ 7, avr. 2018.
    Résumé : Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in theEscherichia coligenome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence inE. colito genome editing in humans.
    Mots-clés : archaea, ARCHEE, casposon, genome editing, MICROBIO, repeated sequence.

  • A. Kereszt, P. Mergaert, J. Montiel, G. Endre, et É. Kondorosi, « Impact of Plant Peptides on Symbiotic Nodule Development and Functioning », Frontiers in Plant Science, vol. 9, p. 1026, 2018.
    Résumé : Ribosomally synthesized peptides have wide ranges of functions in plants being, for example, signal molecules, transporters, alkaloids, or antimicrobial agents. Legumes are an unprecedented rich source of peptides, which are used to control the symbiosis of these plants with the nitrogen-fixing Rhizobium bacteria. Here, we discuss the function and the evolution of these peptides playing an important role in the formation or functioning of the symbiotic organs, the root nodules. We distinguish peptides that can be either cell-autonomous or secreted short-range or long-range signals, carrying messages in or between plant cells or that can act as effectors interacting with the symbiotic bacteria. Peptides are further classified according to the stage of the symbiotic process where they act. Several peptide classes, including RALF, DLV, ENOD40, and others, control Rhizobium infection and the initiation of cell divisions and the formation of nodule primordia. CLE and CEP peptides are implicated in systemic and local control of nodule initiation during autoregulation of nodulation and in response to the nutritional demands of the plant. Still other peptides act at later stages of the symbiosis. The PSK peptide is thought to be involved in the suppression of immunity in nodules and the nodule-specific cysteine-rich, GRP, and SNARP (LEED..PEED) peptide families are essential in the functioning of the nitrogen fixing root nodules. The NCRs and possibly also the GRP and SNARPs are targeted to the endosymbionts and play essential roles in the terminal differentiation of these bacteria.
    Mots-clés : CEP, CLE, GRP, legume-rhizobium symbiosis, MICROBIO, NCR, nodule development, PBI, signaling peptides.

  • S. Khayi, P. Blin, T. M. Chong, K. - G. Chan, et D. Faure, « Complete Chromosome and Plasmid Sequences of Two Plant Pathogens, Dickeya solani Strains D s0432-1 and PPO 9019 », Genome Announcements, vol. 6, nᵒ 17, avr. 2018.
    Résumé : Dickeya solani species are emerging bacterial pathogens of Solanum tuberosum Here, we announce the complete genome sequences of two strains, Dickeya solani D s0432-1 and PPO 9019. Strain PPO 9019 represents the first described member of the genus Dickeya with an extrachromosomal genetic element.
    Mots-clés : MICROBIO, PBI.

  • N. Kieffer, P. Nordmann, A. M. Moreno, L. Z. Moreno, R. Chaby, A. Breton, P. Tissières, et L. Poirel, « Genetic and functional characterization of an MCR-3-like producing Escherichia coli recovered from swine, Brazil », Antimicrobial Agents and Chemotherapy, avr. 2018.
    Résumé : A collection of 126 pigs were screened for carriage of colistin-resistant Enterobacteriaceae in a farm in Minas Gerais, Brazil. Out of this collection, eigth colistin-resistant Escherichia coli isolates were recovered, including one from Minas Gerais State, producing a new MCR-3 variant (MCR-3.12). Analysis of the lipopolysaccharide revealed that MCR-3.12 had a similar function as MCR-1 and MCR-2 by adding a phosphoethanolamine group to the lipid A. Genetic analysis showed that the mcr-3.12 gene was carried by an IncA/C2 plasmid and was embedded in an original genetic environment. This study reports the occurrence of the MCR-3-like determinant in South America and firstly demonstrates the functionality of this group of enzymes as a phosphoethanolamine transferase.
    Mots-clés : ESHR, MICROBIO.

  • B. J. Klotoe, B. Molina-Moya, H. M. Gomes, M. K. Gomgnimbou, L. O. Suzarte, M. H. Féres Saad, S. Ali, J. Dominguez, E. Pimkina, E. Zholdybayeva, C. Sola, et G. Refrégier, « TB-EFI, a novel 18-Plex microbead-based method for prediction of second-line drugs and ethambutol resistance in Mycobacterium tuberculosis complex », Journal of Microbiological Methods, vol. 152, p. 10-17, sept. 2018.
    Résumé : Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus. Basic analysis can be performed manually. An upgraded interpretation is made available in Excel 2016®. Using a reference set of 61 DNA extracts, we show that TB-EFI provides perfect concordance with pyrosequencing. Concordance between genotypic resistance and phenotypic DST was relatively good (72 to 98% concordance), with lower efficiency for fluoroquinolones and ethambutol due to some untargeted mutations. When compared to phenotypical resistance, performances were similar to those obtained with Hain MTBDRsl assay, possibly thanks to the use of automatized processing of data although some mutations involved in fluoroquinolone resistance could not be included. When applied on three uncharacterized sets, phenotype could be predicted for 51% to 98% depending on the setting and the drug investigated, detecting one extensively drug-resistant isolate in each of a Pakistan and a Brazilian set of 91 samples, and 9 XDR among 43 multi-resistant Kazakhstan samples. By allowing high-throughput detection of second-line drugs resistance and of resistance to ethambutol that is often combined to second-line treatments, TB-EFI is a cost-effective assay for large-scale worldwide surveillance of resistant tuberculosis and XDR-TB control.
    Mots-clés : Allele call, assay, Automatized analysis, diagnosis, fluoroquinolone, genotype mtbdrplus, IGEPE, MICROBIO, microsphere-based platform, molecular characterization, mutations, NAAT, Second-line drug resistance, TB, XDR-TB.

  • A. - S. L'Honneur, H. Leh, F. Laurent-Tchenio, U. Hazan, F. Rozenberg, et S. Bury-Moné, « Exploring the role of NCCR variation on JC polyomavirus expression from dual reporter minicircles », PloS One, vol. 13, nᵒ 6, p. e0199171, 2018.
    Résumé : JC virus (JCV), a ubiquitous human polyomavirus, can cause fatal progressive multifocal leukoencephalopathy (PML) in immune compromised patients. The viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR). We analyzed the NCCR sequence from 10 PML JCV strains and found new mutations. Remarkably, the NCCR f section was mutated in most cases. We therefore explored the importance of this section in JCV expression in renal (HEK293H) and glioblastoma (U-87MG) cell lines, by adapting the emerging technology of DNA minicircles. Using bidirectional fluorescent reporters, we revealed that impaired NCCR-driven late expression in glioblastoma cells was restored by a short deletion overlapping e and f sections. This study evidenced a relevant link between JCV NCCR polymorphism and cell-type dependent expression. The use of DNA minicircles opens new insights for monitoring the impact of NCCR variation.
    Mots-clés : ACTINO, MICROBIO.

  • F. Lamouche, D. Gully, A. Chaumeret, N. Nouwen, C. Verly, O. Pierre, C. Sciallano, J. Fardoux, C. Jeudy, A. Szücs, S. Mondy, C. Salon, I. Nagy, A. Kereszt, Y. Dessaux, E. Giraud, P. Mergaert, et B. Alunni, « Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency », Environmental Microbiology, juin 2018.
    Résumé : To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E>U and S>U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids. This article is protected by copyright. All rights reserved.
    Mots-clés : MICROBIO, PBI.

  • L. Latino et C. Pourcel, « Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage », Methods in Molecular Biology (Clifton, N.J.), vol. 1693, p. 85-98, 2018.
    Résumé : Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations.
    Mots-clés : Bacterial phenotype, Bacteriophages, Complementation, Genome sequencing, LGBMB, MICROBIO.


  • H. Liu, Y. Zhang, Z. Liu, J. Liu, Y. Hauck, J. Liu, H. Dong, J. Liu, X. Zhao, B. Lu, Y. Jiang, G. Vergnaud, C. Pourcel, et K. Wan, « Associations between Mycobacterium tuberculosis Beijing genotype and drug resistance to four first-line drugs: a survey in China », Frontiers of Medicine, vol. 12, nᵒ 1, p. 92-97, 2018.
    Mots-clés : CHERDIR, LGBMB, MICROBIO.

  • X. Liu, N. Y. Meiresonne, A. Bouhss, et T. den Blaauwen, « FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli », Molecular Microbiology, vol. 109, nᵒ 6, sept. 2018.
    Résumé : Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in E. coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of MurJ recruitment during cell division. This article is protected by copyright. All rights reserved.
    Mots-clés : 1st membrane step, bacillus-subtilis, beta-lactam antibiotics, divisome, E. coli, ENVBAC, flippase, flippase activity, lipid II, MICROBIO, penicillin-binding proteins, peptidoglycan, peptidoglycan synthesis, terminal domain, udp-n-acetylglucosamine, wall synthesis.

  • A. Maikova, J. Peltier, P. Boudry, E. Hajnsdorf, N. Kint, M. Monot, I. Poquet, I. Martin-Verstraete, B. Dupuy, et O. Soutourina, « Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile », Nucleic Acids Research, vol. 46, nᵒ 9, p. 4733-4751, mai 2018.
    Résumé : Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.
    Mots-clés : ARNCLO, MICROBIO.


  • A. Maikova, K. Severinov, et O. Soutourina, « New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System », Frontiers in Microbiology, vol. 9, p. 1740, 2018.
    Résumé : Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) - has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.
    Mots-clés : antimicrobials, ARNCLO, C. difficile, CRISPR, CRISPR regulation, Genome editing, I-B subtype CRISPR-Cas system, MICROBIO.
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  • M. Mardirossian, Q. Barriere, T. Timchenko, C. Mueller, S. Pacor, P. Mergaert, M. Scocchi, et D. N. Wilson, « Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis », Antimicrobial Agents and Chemotherapy, vol. 62, nᵒ 8, août 2018.
    Résumé : Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coil and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo. Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coil ribosomes but a poor inhibitor on Thermus thermophiius ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of inter-action is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.
    Mots-clés : antibacterial peptides, Bac5, bactenecins, bovine neutrophils, coli, ef-p, gram-negative bacteria, macrolide antibiotics, mechanism, MICROBIO, PBI, PrAMPs, proline-rich antimicrobial agents, proline-rich antimicrobial peptide, protein synthesis inhibitor, ribosomal-proteins, ribosome, translation.


  • M. Mardirossian, Q. Barrière, T. Timchenko, C. Müller, S. Pacor, P. Mergaert, M. Scocchi, et D. N. Wilson, « Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis », Antimicrobial Agents and Chemotherapy, vol. 62, nᵒ 8, p. e00534-18, août 2018.
    Résumé : Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coli and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo. Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coli ribosomes but a poor inhibitor on Thermus thermophilus ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.
    Mots-clés : MICROBIO, PBI.

  • P. Mergaert, « Role of antimicrobial peptides in controlling symbiotic bacterial populations », Natural Product Reports, vol. 35, nᵒ 4, p. 336-356, avr. 2018.
    Résumé : Covering: up to 2018 Antimicrobial peptides (AMPs) have been known for well over three decades as crucial mediators of the innate immune response in animals and plants, where they are involved in the killing of infecting microbes. However, AMPs have now also been found to be produced by eukaryotic hosts during symbiotic interactions with bacteria. These symbiotic AMPs target the symbionts and therefore have a more subtle biological role: not eliminating the microbial symbiont population but rather keeping it in check. The arsenal of AMPs and the symbionts' adaptations to resist them are in a careful balance, which contributes to the establishment of the host-microbe homeostasis. Although in many cases the biological roles of symbiotic AMPs remain elusive, for a number of symbiotic interactions, precise functions have been assigned or proposed to the AMPs, which are discussed here. The microbiota living on epithelia in animals, from the most primitive ones to the mammals, are challenged by a cocktail of AMPs that determine the specific composition of the bacterial community as well as its spatial organization. In the symbiosis of legume plants with nitrogen-fixing rhizobium bacteria, the host deploys an extremely large panel of AMPs - called nodule-specific cysteine-rich (NCR) peptides - that drive the bacteria into a terminally differentiated state and manipulate the symbiont physiology to maximize the benefit for the host. The NCR peptides are used as tools to enslave the bacterial symbionts, limiting their reproduction but keeping them metabolically active for nitrogen fixation. In the nutritional symbiotic interactions of insects and protists that have vertically transmitted bacterial symbionts with reduced genomes, symbiotic AMPs could facilitate the integration of the endosymbiont and host metabolism by favouring the flow of metabolites across the symbiont membrane through membrane permeabilization.
    Mots-clés : MICROBIO, PBI.

  • L. Meyer, G. Coste, S. Sommer, J. Oberto, F. Confalonieri, P. Servant, et C. Pasternak, « DdrI, a CRP family member, acts as a major regulator for adaptation of Deinococcus radiodurans to various stresses », Journal of Bacteriology, avr. 2018.
    Résumé : The DNA damage response gene ddrI encodes a transcription regulator belonging to the CRP (cAMP Receptor Protein) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects, sensitivity to DNA damaging agents and to oxidative stress. Here, we show that the absence of DdrI protein also confers sensitivity to heat shock treatment and several genes involved in heat shock response were shown to be up-regulated in a DdrI dependent manner. Interestingly, expression of the E. coli CRP protein partially compensates the absence of the DdrI protein. Microscopic observations of ΔddrI mutant cells revealed an increased proportion of two-tetrads and anucleated cells in the population as compared to the wild-type strain, indicating that DdrI is crucial for completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and acts as a dimer. All these data underline the major role of DdrI for D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, as well as diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights in the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.
    Mots-clés : ARCHEE, BDG, MICROBIO, RBA.

  • N. Mirouze, C. Ferret, C. Cornilleau, et R. Carballido-López, « Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis », Nature Communications, vol. 9, nᵒ 1, p. 5072, 2018.
    Résumé : Despite decades of investigation of genetic transformation in the model Gram-positive bacterium Bacillus subtilis, the factors responsible for exogenous DNA binding at the surface of competent cells remain to be identified. Here, we report that wall teichoic acids (WTAs), cell wall-anchored anionic glycopolymers associated to numerous critical functions in Gram-positive bacteria, are involved in this initial step of transformation. Using a combination of cell wall-targeting antibiotics and fluorescence microscopy, we show that competence-specific WTAs are produced and specifically localized in the competent cells to mediate DNA binding at the proximity of the transformation apparatus. Furthermore, we propose that TuaH, a putative glycosyl transferase induced during competence, modifies competence-induced WTAs in order to promote (directly or indirectly) DNA binding. On the basis of our results and previous knowledge in the field, we propose a model for DNA binding and transport during genetic transformation in B. subtilis.
    Mots-clés : adsorption, architecture, bacterial-cell, biosynthesis, cell-wall, concanavalin-a, deoxyribonucleic-acid, expression, MICROBIO, THG, transcription factor, transforming dna.

  • B. Molina-Moya, S. T. Abdurrahman, L. Madukaji, M. K. Gomgnimbou, L. Spinasse, M. Gomes-Fernandes, H. M. Gomes, S. Kacimi, R. Dacombe, J. S. Bimba, L. Lawson, C. Sola, L. E. Cuevas, et J. Dominguez, « Genetic characterization of Mycobacterium tuberculosis complex isolates circulating in Abuja, Nigeria », Infection and Drug Resistance, vol. 11, p. 1617-1625, 2018.
    Résumé : Objective: Nigeria ranks fourth among the high tuberculosis (TB) burden countries. This study describes the prevalence of drug resistance and the genetic diversity of Mycobacterium tuberculosis in Abuja's Federal Capital Territory. Materials and methods: Two hundred and seventy-eight consecutive sputum samples were collected from adults with presumptive TB during 2013-2014. DNA was extracted from Lowenstein-Jensen cultures and analyzed for the identification of nontuberculous mycobacteria species, detection of drug resistance with line probe assays, and high-throughput spacer oligonucleotide typing (spoligotyping) using microbead-based hybridization. Results: Two hundred and two cultures were positive for M. tuberculosis complex, 24 negative, 38 contaminated, and 15 positive for nontuberculous mycobacteria. Five (2.5%)M. tuberculosis complex isolates were resistant to rifampicin (RIF) and isoniazid (multidrug resistant), nine (4.5%) to RIF alone, and 15 (7.4%) to isoniazid alone; two RIF-resistant isolates were also resistant to fluoroquinolones and ethambutol, and one multidrug resistant isolate was also resistant to ethambutol. Among the 180 isolates with spoligotyping results, 164 (91.1%) were classified as lineage 4 (Euro-American), 13 (7.2%) as lineage 5 (West African 1), two (1.1%) as lineage 2 (East Asia), and one (0.6%) as lineage 6 (West African 2). One hundred and fifty-six (86.7%) isolates were grouped in 17 clusters (2-108 isolates/cluster), of which 108 (60.0%) were grouped as L4.6.2/Cameroon (spoligotype international type 61). Conclusion: The description of drug resistance prevalence and genetic diversity of M tuberculosis in this study may be useful for improving TB control in Nigeria.
    Mots-clés : clinical-samples, cross river state, drug-resistant tuberculosis, ethambutol resistance, hybridization assay, IGEPE, isoniazid, isoniazid resistance, line probe assay, microbeads, MICROBIO, molecular epidemiology, rapid detection, rifampicin, spoligotyping, strains, tuberculosis.


  • B. Molina-Moya, M. Agonafir, S. Blanco, R. Dacombe, M. K. Gomgnimbou, L. Spinasse, M. Gomes-Fernandes, D. G. Datiko, T. Edwards, L. E. Cuevas, J. Dominguez, et C. Sola, « Microbead-based spoligotyping of Mycobacterium tuberculosis from Ziehl-Neelsen-stained microscopy preparations in Ethiopia », Scientific Reports, vol. 8, nᵒ 1, 2018.

  • B. Molina-Moya, M. K. Gomgnimbou, L. Spinasse, J. Obasanya, O. Oladimeji, R. Dacombe, T. Edwards, X. - O. Daragon, L. Lawson, S. T. Abdurrahman, L. E. Cuevas, J. Dominguez, et C. Sola, « Mycobacterium tuberculosis complex genotypes circulating in Nigeria based on spoligotyping obtained from Ziehl-Neelsen stained slides extracted DNA », PLoS neglected tropical diseases, vol. 12, nᵒ 2, p. e0006242, févr. 2018.
    Résumé : METHODS: All State TB control programmes in Nigeria were requested to submit 25-50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013-2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays. RESULTS: Spoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations. CONCLUSION/SIGNIFICANCE: We present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB.
    Mots-clés : IGEPE, MICROBIO.

  • F. Ngadjeua, E. Braud, S. Saidjalolov, L. Iannazzo, D. Schnappinger, S. Ehrt, J. - E. Hugonnet, D. Mengin-Lecreulx, D. P. Patin, M. Ethève-Quelquejeu, M. Fonvielle, et M. Arthur, « Critical impact of peptidoglycan precursor amidation on the activity of L,D-transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis », Chemistry (Weinheim an Der Bergstrasse, Germany), févr. 2018.
    Résumé : The bacterial cell wall peptidoglycan contains unusual L and D amino acids assembled in branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by L,D-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the L,D-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essentiality of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.
    Mots-clés : Amidation, Amidotransferase, ENVBAC, MICROBIO, Mycobacterium tuberculosis, Peptidoglycan, Transpeptidase.


  • J. R. Osman, G. Fernandes, C. Regeard, C. Jaubert, et M. S. DuBow, « Examination of the Bacterial Biodiversity of Coastal Eroded Surface Soils from the Padza de Dapani (Mayotte Island) », Geomicrobiology Journal, vol. 35, nᵒ 5, p. 355-365, 2018.
    Résumé : To better understand microbial populations present in atypical soil environments, and to discern any relations between these environments and their bacterial communities, we examined the “Padza de Dapani” on the island of Mayotte off the east coast of Africa. This area is not a true (hot) desert, but resembles one in many places due to extensive soil erosion. We collected surface soil samples from five different sites of the Padza de Dapani in Mayotte. We examined bacterial biodiversity using pyrosequencing of PCR-amplified 16S V1–V3 rDNA sequences from total extracted DNA. Our results show that in the acidic (pH 4.6–6), oligotrophic (organic carbon; 0.1–0.7 g/kg of soil) and mineralized (Fe: 18 g/100 g; Al: 12 g/100 g) Dapani Padza soil samples, members of the Actinobacteria and Proteobacteria phyla dominated the bacterial communities, as was also observed in samples from Saudi Arabia hot desert sands.Interestingly, members belonging to the genera Acinetobacter, Arthrobacter and Bacillus were also found to be very abundant in our samples. These were also seen in hot Asian deserts sand samples, such as those from the Gobi (Mongolia) and Taklamaken (China) deserts, thus possibly pointing to microbial populations characteristic of denuded soils.
    Mots-clés : 16S rRNA, Bacteria, biodiversity, LGBMB, MICROBIO, pyrosequencing, soil erosion.

  • J. R. Osman, C. Regeard, C. Badel, G. Fernandes, et M. S. DuBow, « Variation of bacterial biodiversity from saline soils and estuary sediments present near the Mediterranean Sea coast of Camargue (France) », Antonie Van Leeuwenhoek, sept. 2018.
    Résumé : Salinity is an important environmental factor influencing microbial community composition. To better understand this influence, we determined the bacterial communities present in 17 different sites of brackish sediment (underwater) and soil (surface) samples from the Camargue region (Rhône river delta) in southern France during the fall of 2013 and 2014 using pyrosequencing of the V3-V4 regions of the 16S rRNA genes amplified by PCR. This region is known for abundant flora and fauna and, though saline, 30% of rice consumed in France is grown here. We found that bacterial abundance in 1 g of soil or sediment, calculated by qPCR, was higher in sediments than in surface soil samples. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes phyla dominated the bacterial communities of sediment samples, while members belonging to the Proteobacteria, Bacteroidetes, Gemmatimonadetes, Actinobacteria, Firmicutes and Acidobacteria phyla dominated the bacterial communities of the soil samples. The most abundant bacterial genera present in the saline sediments and soils from the Camargue belonged mostly to halophilic and sulphate reducing bacteria, suggesting that the Camargue may be a valuable system to investigate saline, yet agriculturally productive, sediment and soil microbial ecosystem.
    Mots-clés : ARCHEE, Bacterial biodiversity, Halophilic bacteria, LGBMB, microbio, Pyrosequencing, Salinity.

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