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Publications Département Microbiologie


  • 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
    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.

  • 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. Ethève-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 (Weinheim an Der Bergstrasse, Germany), juill. 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-tRNAGly 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-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 : ENVBAC, Fem, MICROBIO, RNA conjugates, Staphylococcus aureus, 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, 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. 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, 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, juin 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, Automatized analysis, IGEPE, MICROBIO, 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.

  • 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, août 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 : divisome, E. coli, ENVBAC, flippase, lipid II, MICROBIO, peptidoglycan.

  • 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 non-lytic proline-rich antimicrobial peptide Bac5 kill E. coli cells by inhibiting protein synthesis », Antimicrobial Agents and Chemotherapy, mai 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 could show that Bac5 (1-25) and Bac5 (1-31) retain excellent antimicrobial activity towards Escherichia coli and low toxicity towards 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 suggest 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.

  • 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.

  • M. Parlato, F. Philippart, A. Rouquette, V. Moucadel, V. Puchois, S. Blein, J. - P. Bedos, J. - L. Diehl, O. Hamzaoui, D. Annane, D. Journois, M. Ben Boutieb, L. Estève, C. Fitting, J. - M. Treluyer, A. Pachot, M. Adib-Conquy, J. - M. Cavaillon, B. Misset, et Captain Study Group, « Circulating biomarkers may be unable to detect infection at the early phase of sepsis in ICU patients: the CAPTAIN prospective multicenter cohort study », Intensive Care Medicine, juin 2018.
    Résumé : PURPOSE: Sepsis and non-septic systemic inflammatory response syndrome (SIRS) are the same syndromes, differing by their cause, sepsis being secondary to microbial infection. Microbiological tests are not enough to detect infection early. While more than 50 biomarkers have been proposed to detect infection, none have been repeatedly validated. AIM: To assess the accuracy of circulating biomarkers to discriminate between sepsis and non-septic SIRS. METHODS: The CAPTAIN study was a prospective observational multicenter cohort of 279 ICU patients with hypo- or hyperthermia and criteria of SIRS, included at the time the attending physician considered antimicrobial therapy. Investigators collected blood at inclusion to measure 29 plasma compounds and ten whole blood RNAs, and-for those patients included within working hours-14 leukocyte surface markers. Patients were classified as having sepsis or non-septic SIRS blindly to the biomarkers results. We used the LASSO method as the technique of multivariate analysis, because of the large number of biomarkers. RESULTS: During the study period, 363 patients with SIRS were screened, 84 having exclusion criteria. Ninety-one patients were classified as having non-septic SIRS and 188 as having sepsis. Eight biomarkers had an area under the receiver operating curve (ROC-AUC) over 0.6 with a 95% confidence interval over 0.5. LASSO regression identified CRP and HLA-DRA mRNA as being repeatedly associated with sepsis, and no model performed better than CRP alone (ROC-AUC 0.76 [0.68-0.84]). CONCLUSIONS: The circulating biomarkers tested were found to discriminate poorly between sepsis and non-septic SIRS, and no combination performed better than CRP alone.
    Mots-clés : Cohort, CRP, ESHR, HLA-DRA mRNA, MICROBIO, Sepsis.

  • A. Pichard-Kostuch, W. Zhang, D. Liger, M. - C. Daugeron, J. Létoquart, I. Li de la Sierra-Gallay, P. Forterre, B. Collinet, H. van Tilbeurgh, et T. Basta, « Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification », RNA (New York, N.Y.), vol. 24, nᵒ 7, p. 926-938, juill. 2018.
    Résumé : N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.
    Mots-clés : ARCHEE, B3S, FAAM, MICROBIO, Sua5, t6A37, threonylcarbamoyl adenosine, tRNA modification, TsaC.

  • F. Pompeo, D. Byrne, D. Mengin-Lecreulx, et A. Galinier, « Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth », Scientific Reports, vol. 8, nᵒ 1, 2018.

  • H. Salmi-Mani, G. Terreros, N. Barroca-Aubry, C. Aymes-Chodur, C. Regeard, et P. Roger, « Poly(ethylene terephthalate) films modified by UV-induced surface graft polymerization of vanillin derived monomer for antibacterial activity », European Polymer Journal, vol. 103, p. 51-58, juin 2018.
    Résumé : New antibacterial PET surfaces were developed from vanillin-derived biobased monomer. An easy one-step and high yielding synthesis of N-(4-hydroxy-3-methoxybenzyl)-acrylamide monomer was successfully achieved. PET was modified by a two-step procedure: Type II photoinitiator was first grafted through a PET aminolysis with N,N-diethylethylenediamine, then the photopolymerization of the biobased acrylamide monomer was performed according to a “grafting from” technique. PET surface modifications were characterized by XPS and UV–visible spectroscopies, as well as water contact angle measurements. Finally, antiadhesion biotests were conducted to evaluate the potential antibacterial performances of the modified surfaces against gram-positive (Rhodococcus wratislaviensis and Staphylococcus aureus) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains.
    Mots-clés : LGBMB, MICROBIO.

  • S. Sarfraz, K. Riaz, S. Oulghazi, J. Cigna, M. W. Alam, Y. Dessaux, et D. Faure, « First Report of Dickeya dianthicola Causing Blackleg Disease on Potato Plants in Pakistan », Plant Disease, p. PDIS04180551PDN, août 2018.

  • E. Schmitt, G. Bourgeois, M. Gondry, et A. Aleksandrov, « Cyclization Reaction Catalyzed by Cyclodipeptide Synthases Relies on a Conserved Tyrosine Residue », Scientific Reports, vol. 8, nᵒ 1, p. 7031, mai 2018.
    Résumé : Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs.
    Mots-clés : BIOSYN, MICROBIO.

  • T. Veaudor, M. Ortega-Ramos, T. Jittawuttipoka, H. Bottin, C. Cassier-Chauvat, et F. Chauvat, « Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment », PloS One, vol. 13, nᵒ 6, p. e0198836, 2018.
    Résumé : Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution).
    Mots-clés : B2CYA, MICROBIO.

  • G. Vergnaud, Y. Hauck, D. Christiany, B. Daoud, C. Pourcel, I. Jacques, A. Cloeckaert, et M. S. Zygmunt, « Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974-2006 », Frontiers in Microbiology, vol. 9, p. 1545, 2018.
    Résumé : Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.
    Mots-clés : animal, Brucella, genotyping, human, LGBMB, MICROBIO, MLVA, population structure.

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