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Accueil > Publications

Publications Département Microbiologie

2017


  • S. Al Dahouk, S. Köhler, A. Occhialini, M. P. Jiménez de Bagüés, J. A. Hammerl, T. Eisenberg, G. Vergnaud, A. Cloeckaert, M. S. Zygmunt, A. M. Whatmore, F. Melzer, K. P. Drees, J. T. Foster, A. R. Wattam, et H. C. Scholz, « Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts », Scientific Reports, vol. 7, p. 44420, 2017.
    Résumé : Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.
    Mots-clés : LGBMB, MICROBIO.

  • A. K. Alame-Emane, C. Pierre-Audigier, O. C. Aboumegone-Biyogo, A. Nzoghe-Mveang, V. Cadet-Daniel, C. Sola, J. F. Djoba-Siawaya, B. Gicquel, et H. E. Takiff, « The use of GeneXpert remnants for drug resistance profiling and molecular epidemiology of tuberculosis in Libreville, Gabon », Journal of Clinical Microbiology, 2017.
    Résumé : Multidrug (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis are major problems in global health. The GeneXpertMTB/RIF (Xpert) rapidly detects resistance to rifampicin (RIF-R), but detection of the additional resistance that defines MDR and XDR-TB, and for molecular epidemiology, specimen cultures and biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for Xpert could be used directly to find mutations associated with drug resistance and for molecular epidemiology, and thus provide a precise characterization of MDR-TB cases in countries lacking BSL3 facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for Xpert were used for PCR amplification and sequencing or line probe assay to detect mutations associated with resistance to additional drugs, and for molecular epidemiology with spoligotyping and selective MIRU-VNTR. Of 130 sputum samples from Gabon tested with Xpert, 124 yielded interpretable results, of which 21 were determined to be RIF-R (17%). Amplification and sequencing or line probe assay of the Xpert remnants confirmed 18/21 as MDR: 11/116 (9.5%) new and 7/8 (87%) previously treated TB patients. Spoligotyping and MIRU with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for Xpert in PCR reactions can be used to find mutations associated with the resistance to the additional drugs that define MDR and XDR-TB, and to study molecular epidemiology without the need for culturing or biosafe infrastructure.
    Mots-clés : IGEPE, MICROBIO.

  • M. Bosco, A. Massarweh, S. Iatmanen-Harbi, A. Bouhss, I. Chantret, P. Busca, S. E. H. Moore, et C. Gravier-Pelletier, « Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP) », European Journal of Medicinal Chemistry, vol. 125, p. 952-964, 2017.
    Résumé : Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [(3)H]OSP from [(3)H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.
    Mots-clés : Animals, Biological evaluation, CDG, Diphosphatase, Disubstituted diphosphates, Dolichol, Dolichol Phosphates, ENVBAC, Glycochemistry, Humans, liver, MICROBIO, Monoterpenes, Oligosaccharides, Phosphoric Diester Hydrolases, Phosphoric Monoester Hydrolases, Phosphosugars, Polyisoprenyl Phosphate Sugars, Polyisoprenyl Phosphates, Substrate Specificity.

  • A. Breton, A. Novikov, R. Martin, P. Tissieres, et M. Caroff, « Structural and biological characteristics of different forms of V. filiformis lipid A: use of MS to highlight structural discrepancies », Journal of Lipid Research, vol. 58, nᵒ 3, p. 543-552, 2017.
    Résumé : Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. We characterized the detailed structure of its lipopolysaccharide (LPS) lipid A moiety, an active component of the bacterium that contributes to the observed skin activation properties. Two different batches differing in postculture cell recovery were tested. Chemical analyses and mass spectra, obtained before and after mild-alkali treatments, revealed that these lipids A share the common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxydecanoic acid in an amide linkage. Short-chain FAs, hydroxydecanoic and dodecanoic acid, were found in a 2:1 ratio. The two lipid A structures differed by the relative amount of the hexa-acyl molecular species and phosphoethanolamine substitution of the phosphate groups. The two V. filiformis LPS batches induced variable interleukin-6 and TNF-α secretion by stimulated myelomonocytic THP-1 cells, without any difference in reactive oxygen species production or activation of caspase 3/7. Other different well-known highly purified LPS samples were characterized structurally and used as standards. The structural data obtained in this work explain the low inflammatory response observed for V. filiformis LPS and the previously demonstrated beneficial effects on the skin.
    Mots-clés : cytokines, ESHR, lipid biochemistry, lipopolysaccharide, Mass Spectrometry, MICROBIO, skin, Toll-like receptor, V. filiformis.

  • S. Bury-Moné et B. Sclavi, « Stochasticity of gene expression as a motor of epigenetics in bacteria: from individual to collective behavior », Research in Microbiology, 2017.
    Résumé : Measuring gene expression at the single cell and single molecule level has recently made possible the quantitative measurement of stochasticity of gene expression. This enables identification of the probable sources and roles of noise. Stochastic gene expression can result in bacterial population heterogeneity, offering specific advantages for fitness and survival in various environments. This trait is therefore selected during the evolution of the species, and is consequently regulated by specific genetic network architecture. Examples exist in stress-response mechanisms, as well as in infection and pathogenicity strategies, pointing to advantages for multicellularity of bacterial populations.
    Mots-clés : ACTINO, Behavior, Bi-stability, Epigenetics, gene expression, MICROBIO, Multicellularity, regulatory networks, Stochasticity.

  • C. Cassier-Chauvat, V. Dive, et F. Chauvat, « Cyanobacteria: photosynthetic factories combining biodiversity, radiation resistance, and genetics to facilitate drug discovery », Applied Microbiology and Biotechnology, vol. 101, nᵒ 4, p. 1359-1364, 2017.
    Résumé : Cyanobacteria are ancient, abundant, and widely diverse photosynthetic prokaryotes, which are viewed as promising cell factories for the ecologically responsible production of chemicals. Natural cyanobacteria synthesize a vast array of biologically active (secondary) metabolites with great potential for human health, while a few genetic models can be engineered for the (low level) production of biofuels. Recently, genome sequencing and mining has revealed that natural cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The corresponding panoply of enzymes (polyketide synthases and non-ribosomal peptide synthases) of interest for synthetic biology can still be increased through gene manipulations with the tools available for the few genetically manipulable strains. In this review, we propose to exploit the metabolic diversity and radiation resistance of cyanobacteria, and when required the genetics of model strains, for the production and radioactive ((14)C) labeling of bioactive products, in order to facilitate the screening for new drugs.
    Mots-clés : B2CYA, Biodiversity, Cyanobacteria, MICROBIO, Peptide Synthases, photosynthesis, Radioactive labeling, Secondary metabolites, Toxins.

  • T. M. Chong, J. - W. Chen, W. - S. See-Too, C. - Y. Yu, G. - Y. Ang, Y. L. Lim, W. - F. Yin, C. Grandclément, D. Faure, Y. Dessaux, et K. - G. Chan, « Phenotypic and genomic survey on organic acid utilization profile of Pseudomonas mendocina strain S5.2, a vineyard soil isolate », AMB Express, vol. 7, nᵒ 1, p. 138, 2017.
    Résumé : Root exudates are chemical compounds that are released from living plant roots and provide significant energy, carbon, nitrogen and phosphorus sources for microbes inhabiting the rhizosphere. The exudates shape the microflora associated with the plant, as well as influences the plant health and productivity. Therefore, a better understanding of the trophic link that is established between the plant and the associated bacteria is necessary. In this study, a comprehensive survey on the utilization of grapevine and rootstock related organic acids were conducted on a vineyard soil isolate which is Pseudomonas mendocina strain S5.2. Phenotype microarray analysis has demonstrated that this strain can utilize several organic acids including lactic acid, succinic acid, malic acid, citric acid and fumaric acid as sole growth substrates. Complete genome analysis using single molecule real-time technology revealed that the genome consists of a 5,120,146 bp circular chromosome and a 252,328 bp megaplasmid. A series of genetic determinants associated with the carbon utilization signature of the strain were subsequently identified in the chromosome. Of note, the coexistence of genes encoding several iron-sulfur cluster independent isoenzymes in the genome indicated the importance of these enzymes in the events of iron deficiency. Synteny and comparative analysis have also unraveled the unique features of D-lactate dehydrogenase of strain S5.2 in the study. Collective information of this work has provided insights on the metabolic role of this strain in vineyard soil rhizosphere.
    Mots-clés : Carbon utilization enzymes, Grapevine exudates, MICROBIO, Organic acids, PBI, Pseudomonas mendocina, Single molecule real-time (SMRT) sequencing, Vineyard soil.

  • R. Chouari, M. Leonard, M. Bouali, S. Guermazi, N. Rahli, I. Zrafi, L. Morin, et A. Sghir, « Eukaryotic molecular diversity at different steps of the wastewater treatment plant process reveals more phylogenetic novel lineages », World Journal of Microbiology & Biotechnology, vol. 33, nᵒ 3, p. 44, 2017.
    Résumé : Wastewater microbiota represents important actors of organic depollution. Nowadays, some species used as bioindicators of the effluent quality are still identified by microscopy. In the present study, we investigated eukaryotic diversity at the different steps of the treatment process of a wastewater treatment plant (aerobic, anaerobic, clarifier basins and anaerobic digester) using the 18S rRNA gene sequencing approach. Of the 1519 analysed sequences, we identified 160 operational taxonomic units. Interestingly, 56.9% of the phylotypes were assigned to novel phylogenetic molecular species since they show <97% sequence identity with their nearest affiliated representative within public databases. Peritrichia ciliates were the most predominant group, with Epistylis as the most common genus. Although anaerobic, the digester appears to harbor many unclassified phylotypes of protozoa species. Novel lineages such as LKM11 and LKM118 were widely represented in the digester. Diversity values given by Shannon indexes show that the clarifier is the most diversified. This work will help designing molecular tools that are fast, reliable, and reproducible for monitoring wastewater depollution and studying phylogenetic relationships among the wonderful world of protists within this anthropogenic ecosystem.
    Mots-clés : 18S rRNA gene, Activated sludge, Ciliates, Cryptomycota, LGBMB, LKM118, MICROBIO, Wastewater microbiota.

  • M. Cossu, C. Badel, R. Catchpole, D. Gadelle, E. Marguet, V. Barbe, P. Forterre, et J. Oberto, « Flipping chromosomes in deep-sea archaea », PLoS genetics, vol. 13, nᵒ 6, p. e1006847, 2017.
    Résumé : One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element.
    Mots-clés : ARCHEE, MICROBIO.

  • N. Dautin, C. de Sousa-d'Auria, F. Constantinesco-Becker, C. Labarre, J. Oberto, I. L. de la Sierra-Gallay, C. Dietrich, H. Issa, C. Houssin, et N. Bayan, « Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales », Biochimica et Biophysica Acta (BBA) - General Subjects, vol. 1861, nᵒ 1 Pt B, p. 3581-3592, 2017.
    Résumé : Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.
    Mots-clés : Antigen 85, ARCHEE, CORYNE, Esterase, Fibronectin-binding protein, MICROBIO, Mycobacterium, Mycolyltransferases, Mycomembrane.

  • D. De Luca, A. H. van Kaam, D. G. Tingay, S. E. Courtney, O. Danhaive, V. P. Carnielli, L. J. Zimmermann, M. C. J. Kneyber, P. Tissieres, J. Brierley, G. Conti, J. J. Pillow, et P. C. Rimensberger, « The Montreux definition of neonatal ARDS: biological and clinical background behind the description of a new entity », The Lancet. Respiratory Medicine, 2017.
    Résumé : Acute respiratory distress syndrome (ARDS) is undefined in neonates, despite the long-standing existing formal recognition of ARDS syndrome in later life. We describe the Neonatal ARDS Project: an international, collaborative, multicentre, and multidisciplinary project which aimed to produce an ARDS consensus definition for neonates that is applicable from the perinatal period. The definition was created through discussions between five expert members of the European Society for Paediatric and Neonatal Intensive Care; four experts of the European Society for Paediatric Research; two independent experts from the USA and two from Australia. This Position Paper provides the first consensus definition for neonatal ARDS (called the Montreux definition). We also provide expert consensus that mechanisms causing ARDS in adults and older children-namely complex surfactant dysfunction, lung tissue inflammation, loss of lung volume, increased shunt, and diffuse alveolar damage-are also present in several critical neonatal respiratory disorders.
    Mots-clés : ESHR, MICROBIO.

  • S. Di Gregorio, S. Fernandez, A. Cuirolo, O. Verlaine, A. Amoroso, D. Mengin-Lecreulx, A. Famiglietti, B. Joris, et M. Mollerach, « Different Vancomycin-Intermediate Staphylococcus aureus Phenotypes Selected from the Same ST100-hVISA Parental Strain », Microbial Drug Resistance (Larchmont, N.Y.), vol. 23, nᵒ 1, p. 44-50, 2017.
    Résumé : The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.
    Mots-clés : ENVBAC, hVISA, MICROBIO, MRSA, ST100, Staphylococcus aureus, vancomycin, VISA.


  • C. Esnault, T. Dulermo, A. Smirnov, A. Askora, M. David, A. Deniset-Besseau, I. - B. Holland, et M. - J. Virolle, « Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145 », Scientific Reports, vol. 7, nᵒ 1, 2017.
    Mots-clés : DIR, MESMIC, MICROBIO, SWARM.

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

  • M. E. Ghachi, N. Howe, R. Auger, A. Lambion, A. Guiseppi, F. Delbrassine, G. Manat, S. Roure, S. Peslier, E. Sauvage, L. Vogeley, J. - C. Rengifo-Gonzalez, P. Charlier, D. Mengin-Lecreulx, M. Foglino, T. Touzé, M. Caffrey, et F. Kerff, « Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis », Cellular and molecular life sciences: CMLS, 2017.
    Résumé : Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.
    Mots-clés : Bacterial lipids metabolism, ENVBAC, Membrane protein structure, MICROBIO, Peptidoglycan-related lipid, Undecaprenyl phosphate.

  • K. Gloux, M. Guillemet, C. Soler, C. Morvan, D. Halpern, C. Pourcel, H. Vu Thien, G. Lamberet, et A. Gruss, « Clinical relevance of FASII bypass in Staphylococcus aureus », Antimicrobial Agents and Chemotherapy, 2017.
    Résumé : The need for new antimicrobials to treat bacterial infections has led to the use of fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host, and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, non-culturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of non-culturable variants.
    Mots-clés : LGBMB, MICROBIO.

  • M. Hrast, K. Rožman, M. Jukič, D. Patin, S. Gobec, et M. Sova, « Synthesis and structure-activity relationship study of novel quinazolinone-based inhibitors of MurA », Bioorganic & Medicinal Chemistry Letters, 2017.
    Résumé : MurA is an intracellular bacterial enzyme that is essential for peptidoglycan biosynthesis, and is therefore an important target for antibacterial drug discovery. We report the synthesis, in silico studies and extensive structure-activity relationships of a series of quinazolinone-based inhibitors of MurA from Escherichia coli. 3-Benzyloxyphenylquinazolinones showed promising inhibitory potencies against MurA, in the low micromolar range, with an IC50 of 8µM for the most potent derivative (58). Furthermore, furan-substituted quinazolinones (38, 46) showed promising antibacterial activities, with MICs from 1µg/mL to 8µg/mL, concomitant with their MurA inhibitory potencies. These data represent an important step towards the development of novel antimicrobial agents to combat increasing bacterial resistance.
    Mots-clés : Antibacterial agents, ENVBAC, MICROBIO, MurA enzyme, Peptidoglycan, Quinazolinones.

  • H. Issa, E. Huc-Claustre, T. Reddad, N. Bonadé Bottino, M. Tropis, C. Houssin, M. Daffé, N. Bayan, et N. Dautin, « Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum », PloS One, vol. 12, nᵒ 2, p. e0171955, 2017.
    Résumé : Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.
    Mots-clés : CORYNE, MICROBIO.


  • T. Kazmierczak, M. Nagymihaly, F. Lamouche, Q. Barrière, I. Guefrachi, B. Alunni, M. Ouadghiri, J. Ibijbijen, É. Kondorosi, P. Mergaert, et V. Gruber, « Specific host-responsive associations between Medicago truncatula accessions and Sinorhizobium strains », Molecular Plant-Microbe Interactions, mars 2017.

  • N. Kint, C. Janoir, M. Monot, S. Hoys, O. Soutourina, B. Dupuy, et I. Martin-Verstraete, « The alternative sigma factor σ(B) plays a crucial role in adaptive strategies of Clostridium difficile during gut infection », Environmental Microbiology, 2017.
    Résumé : Clostridium difficile is a major cause of diarrhoea associated with antibiotherapy. Exposed to stresses in the gut, C. difficile can survive by inducing protection, detoxification and repair systems. In several firmicutes, most of these systems are controlled by the general stress response involving σ(B) . In this work, we studied the role of σ(B) in the physiopathology of C. difficile. We showed that the survival of the sigB mutant during the stationary phase was reduced. Using a transcriptome analysis, we showed that σ(B) controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σ(B) does not control toxin gene expression. In agreement with the up-regulation of sporulation genes, the sporulation efficiency is higher in the sigB mutant than in the wild-type strain. sigB inactivation also led to increased sensitivity to acidification, cationic antimicrobial peptides, nitric oxide and ROS. In addition, we showed for the first time that σ(B) also plays a crucial role in oxygen tolerance in this strict anaerobe. Finally, we demonstrated that the fitness of colonisation by the sigB mutant is greatly affected in a dixenic mouse model of colonisation when compared to the wild-type strain.
    Mots-clés : ARNCLO, MICROBIO.


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

  • L. Latino, M. Caroff, et C. Pourcel, « Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants », Microbiology (Reading, England), vol. 163, nᵒ 6, p. 848-855, 2017.
    Résumé : Pseudomonas aeruginosa lipopolysaccharides (LPS) serve as primary receptors for many bacteriophages and, consequently, their biosynthesis is frequently affected in phage-resistant mutants. We previously isolated phage-resistant PAO1 mutants using three different phages, and showed that they were affected in the synthesis of LPS. Here we have investigated in detail the effect of mutations in seven genes involved in different steps of the production of core and oligosaccharide chains. The band profile of purified LPS was analysed by PAGE, and we further characterized the O-chains and core structures by MALDI mass spectrometry (MS). Mild LPS extraction conditions and native LPS MS analyses helped unveil lipid A molecular species with three phosphate residues in the close vicinity of the already highly charged inner-core region. No other MS direct analysis has allowed this peculiarity to be demonstrated for native lipid A high-molecular-weight molecular species, in normal growth conditions and without involving separation techniques. The present results shed light on the possible interactions between the phages and the LPS structures in the early phase of infection.
    Mots-clés : CAROFF, DIR, LGBMB, MICROBIO.

  • U. Lausten-Thomsen, Z. Merchaoui, C. Dubois, S. Eleni Dit Trolli, N. Le Saché, M. Mokhtari, et P. Tissières, « Ultrasound-Guided Subclavian Vein Cannulation in Low Birth Weight Neonates », Pediatric Critical Care Medicine: A Journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies, vol. 18, nᵒ 2, p. 172-175, 2017.
    Résumé : OBJECTIVES: Central venous access in critically ill, small infants remains technically challenging even in experienced hands. Several vascular accesses exist, but the subclavian vein is often preferred for central venous catheter insertion in infants where abdominal malformation and/or closure of the vein preclude the use of umbilical venous catheters, as catheterization of the subclavian vein is easier in very short necks than the internal jugular vein for age-related anatomical reasons. The subclavian vein approach is yet relatively undescribed in low birth weight infants (i.e., < 2,500 g), and this study aims to explore the feasibility of this technique in very small infants. DESIGN: Retrospective data collection of prospectively registered data on central venous catheter insertion in infants. SETTING: Neonatal ICU and PICU at a university hospital. PATIENTS: One hundred and five newborn children hospitalized in at the ICU. INTERVENTIONS: An ultrasound-guided supraclavicular approach was applied on all infants who had an subclavian vein catheterization during a 30-month period from January 2013 to July 2015. MEASUREMENTS AND MAIN RESULTS: One hundred seven supraclavicular subclavian vein catheters were placed in 105 children weighing less than 5,000 g. Among those, 40 patients weighed less than 2,500 g and 10 patients weighed less than 1,500 g. Successful central venous catheter insertion, defined as the correct placement of a functional double-lumen catheter (3F or 4F), was obtained in 97.3%. All three registered failed attempts were due to hematomas from venous bleeding and occurred in infants weighing greater than 2,500 g. No case of accidental arterial puncture or pleural puncture was registered. CONCLUSIONS: This large series of subclavian vein catheterizations in small infants demonstrates the feasibility of subclavian vein catheterizations even in very small neonates weighing less than 1,500 g.
    Mots-clés : ESHR, MICROBIO.


  • M. Levy, N. Le Sache, M. Mokhtari, G. Fagherazzi, G. Cuzon, B. Bueno, V. Fouquet, A. Benachi, S. Eleni Dit Trolli, et P. Tissieres, « Sepsis risk factors in infants with congenital diaphragmatic hernia », Annals of Intensive Care, vol. 7, nᵒ 1, 2017.

  • P. Mergaert, Y. Kikuchi, S. Shigenobu, et E. C. M. Nowack, « Metabolic Integration of Bacterial Endosymbionts through Antimicrobial Peptides », Trends in Microbiology, 2017.
    Résumé : Antimicrobial peptides (AMPs) are massively produced by eukaryotic hosts during symbiotic interactions with bacteria. Among other roles, these symbiotic AMPs have the capacity to permeabilize symbiont membranes and facilitate metabolite flow across the host-symbiont interface. We propose that an ancestral role of these peptides is to facilitate metabolic exchange between the symbiotic partners through membrane permeabilization. This function may be particularly critical for integration of endosymbiont and host metabolism in interactions involving bacteria with strongly reduced genomes lacking most small metabolite transporters. Moreover, AMPs could have acted in a similar way at the onset of plastid and mitochondrion evolution, after a host cell took up a bacterium and needed to extract nutrients from it in the absence of dedicated solute transporters.
    Mots-clés : antimicrobial peptide, endosymbiosis, genome reduction, metabolic integration, MICROBIO, PBI, solute transport.

  • A. Millan-Oropeza, C. Henry, M. Blein-Nicolas, A. Aubert-Frambourg, F. Moussa, J. Bleton, et M. - J. Virolle, « Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans », Journal of Proteome Research, 2017.
    Résumé : Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.
    Mots-clés : dormancy regulon, glycolytic metabolism, Mass Spectrometry, MESMIC, MICROBIO, oxidative metabolism, oxidative stress, Secondary metabolites, shotgun label-free quantitative proteomics, Streptomyces, tellurium resistance proteins.

  • A. Millan-Oropeza, R. Rebois, M. David, F. Moussa, A. Dazzi, J. Bleton, M. - J. Virolle, et A. Deniset-Besseau, « Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) for Rapid Determination of Microbial Cell Lipid Content: Correlation with Gas Chromatography-Mass Spectrometry (GC-MS) », Applied Spectroscopy, p. 3702817709459, 2017.
    Résumé : There is a growing interest worldwide for the production of renewable oil without mobilizing agriculture lands; fast and reliable methods are needed to identify highly oleaginous microorganisms of potential industrial interest. The aim of this study was to demonstrate the relevance of attenuated total reflection (ATR) spectroscopy to achieve this goal. To do so, the total lipid content of lyophilized samples of five Streptomyces strains with varying lipid content was assessed with two classical quantitative but time-consuming methods, gas chromatography-mass spectrometry (GC-MS) and ATR Fourier transform infrared (ATR FT-IR) spectroscopy in transmission mode with KBr pellets and the fast ATR method, often questioned for its lack of reliability. A linear correlation between these three methods was demonstrated allowing the establishment of equations to convert ATR values expressed as CO/amide I ratio, into micrograms of lipid per milligram of biomass. The ATR method proved to be as reliable and quantitative as the classical GC-MS and FT-IR in transmission mode methods but faster and more reproducible than the latter since it involves far less manipulation for sample preparation than the two others. Attenuated total reflection could be regarded as an efficient fast screening method to identify natural or genetically modified oleaginous microorganisms by the scientific community working in the field of bio-lipids.
    Mots-clés : ATR FT-IR, attenuated total reflection Fourier transform, Biofuel, gas chromatography–mass spectrometry, GC-MS, infrared spectroscopy, lipid quantification, MESMIC, MICROBIO, Streptomyces, triacylglycerol.

  • J. Moh-Klaren, G. Bodivit, M. Jugie, P. Chadebech, L. Chevret, M. Mokhtari, X. Chamillard, P. Gallon, P. Tissières, P. Bierling, R. Djoudi, F. Pirenne, et N. Burin-des-Roziers, « Severe hemolysis after plasma transfusion in a neonate with necrotizing enterocolitis, Clostridium perfringens infection, and red blood cell T-polyagglutination », Transfusion, 2017.
    Résumé : BACKGROUND: Red blood cell (RBC) Thomsen-Friedenreich antigen exposure (T activation) in infants with necrotizing enterocolitis (NEC) has occasionally been associated with posttransfusional intravascular hemolysis thought to be due to anti-T antibodies in the donor plasma. STUDY DESIGN AND METHODS: We describe an infant with NEC and Clostridium perfringens infection complicated by severe hemolysis after plasma transfusion. After this case, infants with confirmed NEC were prospectively evaluated for T activation. We checked for hemolysis in patients with T activation receiving plasma-containing blood products. RESULTS: The infant had received 80 mL of fresh-frozen plasma (FFP). His RBCs displayed strong T activation, and agglutination was observed with four of six ABO-compatible FFP units. A direct antiglobulin test was negative. IgM-class anti-T antibodies were present in small amounts (titer of 8) in the transfused FFP. Anti-T antibodies from the blood donor were not hemolytic in vitro. In the prospective study, T activation was observed in three of 28 infants with NEC (11%). One infant presented moderate T activation and two infants presented very strong T activation but only moderate decreases in sialic acid expression on the RBC membrane. These three infants presented no signs of hemolysis after transfusion with unwashed blood products or FFP. CONCLUSION: Anti-T antibodies are unlikely to be the etiologic factor for the hemolytic reactions observed in infants with NEC and T activation. Massive RBC desialylation and the direct action of bacterial toxins are more probable causes. Strict avoidance of plasma-containing blood products does not seem justified in these infants.
    Mots-clés : ESHR, MICROBIO.

  • B. Molina-Moya, M. K. Gomgnimbou, C. Lafoz, A. Lacoma, C. Prat, G. Refrégier, S. Samper, J. Dominguez, et C. Sola, « Molecular Characterization of Mycobacterium tuberculosis Strains with TB-SPRINT », The American Journal of Tropical Medicine and Hygiene, 2017.
    Résumé : We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.
    Mots-clés : IGEPE, MICROBIO.

  • J. Montiel, J. A. Downie, A. Farkas, P. Bihari, R. Herczeg, B. Bálint, P. Mergaert, A. Kereszt, et É. Kondorosi, « Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides », Proceedings of the National Academy of Sciences of the United States of America, 2017.
    Résumé : In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.
    Mots-clés : legume, MICROBIO, nitrogen-fixing bacteroids, nodule, nodule-specific cysteine-rich peptides, PBI, Symbiosis.

  • M. Moutiez, P. Belin, et M. Gondry, « Aminoacyl-tRNA-Utilizing Enzymes in Natural Product Biosynthesis », Chemical Reviews, 2017.
    Résumé : Aminoacyl-tRNAs were long thought to be involved solely in ribosome-dependent protein synthesis and essential primary metabolism processes, such as targeted protein degradation and peptidoglycan synthesis. About 10 years ago, an aminoacyl-tRNA-dependent enzyme involved in the biosynthesis of the antibiotic valanimycin was discovered in a Streptomyces strain. Far from being an isolated case, this discovery has been followed by the description of an increasing number of aminoacyl-tRNA-dependent enzymes involved in secondary metabolism. This review describes the three groups of aminoacyl-tRNA-dependent enzymes involved in the synthesis of natural products. Each group is characterized by a particular chemical reaction, and its members are predicted to share a specific fold. The three groups are cyclodipeptide synthases involved in diketopiperazine synthesis, LanB-like dehydratases involved in the posttranslational modification of ribosomal peptides, and transferases from various biosynthesis pathways.
    Mots-clés : BIOSYN, MICROBIO.

  • M. Nagymihály, A. Veluchamy, Z. Györgypál, F. Ariel, T. Jégu, M. Benhamed, A. Szűcs, A. Kereszt, P. Mergaert, et É. Kondorosi, « Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression », Proceedings of the National Academy of Sciences of the United States of America, 2017.
    Résumé : The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.
    Mots-clés : chromatin structure, DNA Methylation, Medicago truncatula, MICROBIO, nodule-specific cysteine-rich peptides, PBI, Symbiosis.

  • A. Novikov, A. Breton, et M. Caroff, « Micromethods for Isolation and Structural Characterization of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides », Methods in Molecular Biology (Clifton, N.J.), vol. 1600, p. 167-186, 2017.
    Résumé : Lipopolysaccharides (LPS) are major components of the external membrane of most Gram-negative bacteria, providing them with an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region (PS) linked to a hydrophobic one, termed lipid A. The LPS polysaccharide moiety is divided into the core oligosaccharide (OS) and O-chain repetitive elements. Depending on their individual variable fine structures, LPS may be potent immunomodulators. The lipid A structure is a key determinant for LPS activity. However, the presence of the core region, or at least of the highly charged 3-deoxy-d-manno-oct-2-ulosonic acid molecules, is also important for preserving the native lipid A conformation within individual LPS molecules. We describe herein four rapid and practical micromethods for LPS, lipid A, and core OS structural analyses. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids enabling the characterization of their substitution position in the lipid A backbone, to be determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The third one is a microscale procedure for the mass spectra screening of LPS, lipid A, and PS using triethylamine and citric acid. The fourth method is a chromatography procedure for Rough-type LPS on thin-layer-chromatography. These methods were developed to be coupled to mass-spectrometry (e.g., MALDI-MS) but can also be used with other analytical techniques (e.g., chromatography). Examples are given with reference to two major human pathogens: Bordetella pertussis and Pseudomonas aeruginosa; to one porcine pathogen: Actinobacillus pleuropneumoniae; and to commercial samples of Salmonella Minnesota Re595 LPS.
    Mots-clés : 3-Deoxy-d-manno-oct-2-ulosonic acid, ESHR, Gas chromatography, Lipooligosaccharide, lipopolysaccharide, Matrix-assisted laser desorption/ionization mass spectrometry, MICROBIO, Polyacrylamide gel electrophoresis, Sodium dodecyl sulfate, Thin-layer chromatography, Triethylamine.


  • J. Pédron, E. Chapelle, B. Alunni, et F. Van Gijsegem, « Transcriptome analysis of the Dickeya dadantii PecS regulon during early stages of interaction with Arabidopsis thaliana : D. dadantii in planta PecS regulon », Molecular Plant Pathology, 2017.
    Mots-clés : Arabidopsis, Dickeya dadantii, epiphytic colonisation, gene expression, in planta transcriptome, MICROBIO, PBI, regulatory networks, soft rot, T6SS, Virulence.


  • M. J. Peters, A. Argent, M. Festa, S. Leteurtre, J. Piva, A. Thompson, D. Willson, P. Tissières, M. Tucci, et J. Lacroix, « The intensive care medicine clinical research agenda in paediatrics », Intensive Care Medicine, mars 2017.

  • C. Pourcel, C. Midoux, Y. Hauck, G. Vergnaud, et L. Latino, « Large Preferred Region for Packaging of Bacterial DNA by phiC725A, a Novel Pseudomonas aeruginosa F116-Like Bacteriophage », PloS One, vol. 12, nᵒ 1, p. e0169684, 2017.
    Résumé : Bacteriophage vB_PaeP_PAO1_phiC725A (short name phiC725A) was isolated following mitomycin C induction of C7-25, a clinical Pseudomonas aeruginosa strain carrying phiC725A as a prophage. The phiC725A genome sequence shows similarity to F116, a P. aeruginosa podovirus capable of generalized transduction. Likewise, phiC725A is a podovirus with long tail fibers. PhiC725A was able to lysogenize two additional P. aeruginosa strains in which it was maintained both as a prophage and in an episomal state. Investigation by deep sequencing showed that bacterial DNA carried inside phage particles originated predominantly from a 700-800kb region, immediately flanking the attL prophage insertion site, whether the phages were induced from a lysogen or recovered after infection. This indicates that during productive replication, recombination of phage genomes with the bacterial chromosome at the att site occurs occasionally, allowing packaging of adjacent bacterial DNA.
    Mots-clés : LGBMB, MICROBIO.

  • C. W. Ribeiro, F. Baldacci-Cresp, O. Pierre, M. Larousse, S. Benyamina, A. Lambert, J. Hopkins, C. Castella, J. Cazareth, G. Alloing, E. Boncompagni, J. Couturier, P. Mergaert, P. Gamas, N. Rouhier, F. Montrichard, et P. Frendo, « Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1 », Current biology: CB, vol. 27, nᵒ 2, p. 250-256, 2017.
    Résumé : Legumes associate with rhizobia to form nitrogen (N2)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N2-fixing bacteroids by producing defensin-like nodule-specific cysteine-rich peptides (NCRs) [3, 4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules [9]. Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N2-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S. meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S. meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions.
    Mots-clés : bacteroids, Differentiation, disulfide bond reduction, Medicago truncatula, MICROBIO, nitrogen-fixing symbiosis, nodule cysteine-rich peptides, PBI, redox state, Sinorhizobium meliloti, thiol modifications, thioredoxins.

  • K. Rožman, S. Lešnik, B. Brus, M. Hrast, M. Sova, D. Patin, H. Barreteau, J. Konc, D. Janežič, et S. Gobec, « Discovery of new MurA inhibitors using induced-fit simulation and docking », Bioorganic & Medicinal Chemistry Letters, vol. 27, nᵒ 4, p. 944-949, 2017.
    Résumé : We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1μM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.
    Mots-clés : Antibacterial agents, ENVBAC, induced fit, MICROBIO, MurA inhibitors, ProBiS-CHARMMing web server, Small-molecule inhibitors.

  • O. Soutourina, « RNA-based control mechanisms of Clostridium difficile », Current Opinion in Microbiology, vol. 36, p. 62-68, 2017.
    Résumé : Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD. More than 200 regulatory RNAs have been identified by deep sequencing and targeted approaches, including Hfq-dependent trans riboregulators, cis-antisense RNAs, CRISPR RNAs, and c-di-GMP-responsive riboswitches. These regulatory RNAs are involved in the control of major processes in the CD infection cycle, for example motility, biofilm formation, adhesion, sporulation, stress response, and defence against bacteriophages. We will discuss recent advances in elucidation of the original features of RNA-based mechanisms in this important enteropathogen. This knowledge may pave the way for further discoveries in this emergent field.
    Mots-clés : ARNCLO, MICROBIO.

  • M. Tannières, J. Lang, C. Barnier, J. A. Shykoff, et D. Faure, « Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders », Scientific Reports, vol. 7, p. 40126, 2017.
    Résumé : Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QS-disruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants.
    Mots-clés : MICROBIO, PBI.


  • L. Yan, Q. Zhang, M. - J. Virolle, et D. Xu, « In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24 », PLOS ONE, vol. 12, nᵒ 3, p. e0174781, mars 2017.

  • D. Zhivaki, S. Lemoine, A. Lim, A. Morva, P. - O. Vidalain, L. Schandene, N. Casartelli, M. - A. Rameix-Welti, P. - L. Hervé, E. Dériaud, B. Beitz, M. Ripaux-Lefevre, J. Miatello, B. Lemercier, V. Lorin, D. Descamps, J. Fix, J. - F. Eléouët, S. Riffault, O. Schwartz, F. Porcheray, F. Mascart, H. Mouquet, X. Zhang, P. Tissières, et R. Lo-Man, « Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity », Immunity, vol. 46, nᵒ 2, p. 301-314, 2017.
    Résumé : Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.
    Mots-clés : Breg cell, bronchiolitis, ESHR, MICROBIO, Newborn, respiratory syncytial virus.

2016


  • B. Al-Dabbagh, S. Olatunji, M. Crouvoisier, M. El Ghachi, D. Blanot, D. Mengin-Lecreulx, et A. Bouhss, « Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily », Biochimie, vol. 127, p. 249-257, 2016.
    Résumé : The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the β-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed.
    Mots-clés : Amines, Bacillus subtilis, Biocatalysis, catalytic mechanism, ENVBAC, Lipid Metabolism, MICROBIO, MraY, Peptidoglycan, Sequence Homology, Amino Acid, Substrate Specificity, Thermotoga maritima, Transferases, WecA.

  • B. Alunni et B. Gourion, « Terminal bacteroid differentiation in the legume-rhizobium symbiosis: nodule-specific cysteine-rich peptides and beyond », The New Phytologist, vol. 211, nᵒ 2, p. 411-417, 2016.
    Résumé : Contents 411 I. 411 II. 412 III. 412 IV. 413 V. 414 VI. 414 VII. 415 VIII. 415 416 References 416 SUMMARY: Terminal bacteroid differentiation (TBD) is a remarkable case of bacterial cell differentiation that occurs after rhizobia are released intracellularly within plant cells of symbiotic legume organs called nodules. The hallmarks of TBD are cell enlargement, genome amplification and membrane permeabilization. This plant-driven process is governed by a large family of bacteroid-targeted nodule-specific cysteine-rich (NCR) peptides that were until recently thought to be restricted to a specific lineage of the legume family, including the model plant Medicago truncatula. Recently, new plant and bacterial factors involved in TBD have been identified, challenging our view of this phenomenon at mechanistic and evolutionary levels. Here, we review the recent literature and discuss emerging questions about the mechanisms and the role(s) of TBD.
    Mots-clés : antimicrobial peptides, endoreduplication, host-symbiont coevolution, legume-rhizobium symbiosis, MICROBIO, Nitrogen Fixation, nodule-specific cysteine-rich (NCR) peptides, PBI, terminal bacteroid differentiation.

  • S. M. Basheer, V. Bouchez, A. Novikov, L. A. Augusto, N. Guiso, et M. Caroff, « Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates », Biochimie, vol. 120, p. 87-95, 2016.
    Résumé : Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography-mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3' in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.
    Mots-clés : Bordetella, Bordetella petrii, Cell Line, Tumor, Endotoxin, ESHR, Female, Humans, Interleukin-6, Lipid A, Male, Mass Spectrometry, MICROBIO, Monocytes, Structure-Activity Relationship, Structure–activity, Tumor Necrosis Factor-alpha.

  • P. Béguin, N. Charpin, E. V. Koonin, P. Forterre, et M. Krupovic, « Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems », Nucleic Acids Research, vol. 44, nᵒ 21, p. 10367-10376, 2016.
    Résumé : Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.
    Mots-clés : ARCHEE, MICROBIO.


  • M. Benincasa, Q. Barrière, G. Runti, O. Pierre, M. Bourge, M. Scocchi, et P. Mergaert, « Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria », BIO-PROTOCOL, vol. 6, nᵒ 23, 2016.
    Mots-clés : CYTO, MICROBIO, PBI, PF.

  • I. Boukhris, T. Dulermo, H. Chouayekh, et M. - J. Virolle, « Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor », Journal of Basic Microbiology, vol. 56, nᵒ 1, p. 59-66, 2016.
    Résumé : Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.
    Mots-clés : 6-Phytase, Bacterial Proteins, Base Sequence, Beta-glucuronidase reporter gene, Gene Expression Regulation, Bacterial, Glucuronidase, MESMIC, MICROBIO, Negative, Operon, PHO box, PhoP, PhoR, Phosphate limitation, Phytic Acid, Positive regulation, Promoter Regions, Genetic, Protein Binding, Repetitive Sequences, Nucleic Acid, Sequence Deletion, Soil Microbiology, Streptomyces coelicolor, Streptomyces lividans, Streptomyces phytase.

  • I. Boukhris, A. Farhat-Khemakhem, K. Bouchaala, M. - J. Virolle, et H. Chouayekh, « Cloning and characterization of the first actinomycete β-propeller phytase from Streptomyces sp. US42 », Journal of Basic Microbiology, vol. 56, nᵒ 10, p. 1080-1089, 2016.
    Résumé : A gene encoding an extracellular phytase was cloned for the first time from an Actinomycete, Streptomyces sp. US42 and sequenced. The sequence of this gene revealed an encoded polypeptide (PHY US42) exhibiting one and six residues difference with the putative phytases of Streptomyces lividans TK24 and Streptomyces coelicolor A3(2), respectively. The molecular modeling of PHY US42 indicated that this phytase belongs to the group of β-propeller phytases that are usually calcium-dependent. PHY US42 was purified and characterized. Its activity was calcium-dependent and maximal at pH 7 and 65 °C. The enzyme was perfectly stable at pH ranging from 5 to 10 and its thermostability was greatly enhanced in the presence of calcium. Indeed, PHY US42 maintained 80% of activity after 10 min of incubation at 75 °C in the presence of 5 mM CaCl2 . PHY US42 was also found to exhibit high stability after incubation at 37 °C for 1 h in the presence of bovine bile and digestive proteases like of pepsin, trypsin, and chymotrypsin. Considering its biochemical properties, PHY US42 could be used as feed additive in combination with an acid phytase for monogastric animals.
    Mots-clés : 6-Phytase, Amino Acid Sequence, Base Sequence, Calcium, Calcium-dependent, Cloning, Molecular, DNA, Fungal, Enzyme Stability, Feed additive, MESMIC, MICROBIO, Sequence Analysis, DNA, Streptomyces, Substrate Specificity, β-propeller phytase.

  • C. Cassier-Chauvat, T. Veaudor, et F. Chauvat, « Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications », Frontiers in Microbiology, vol. 7, p. 1809, 2016.
    Résumé : Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings indicate that not all cyanobacteria have an E. coli-type SOS system. Also interestingly, several cyanobacteria possess multiple copies of E. coli-like DNA repair genes, such as Acaryochloris marina MBIC11017 (2 alkB, 3 ogt, 7 recA, 3 recD, 2 ssb, 3 umuC, 4 umuD, and 8 xerC), Cyanothece ATCC51142 (2 lexA and 4 ruvC), and Nostoc PCC7120 (2 ssb and 3 xerC).
    Mots-clés : B2CYA, Cyanobacteria, DNA recombination, DNA Repair, genetic instability, insertion sequences, MICROBIO, natural transformation, photoproduction, radiation resistance.

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