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Publications Département Microbiologie


  • F. Acosta, J. Agapito, A. M. Cabibbe, T. Cáceres, C. Sola, L. Pérez-Lago, E. Abascal, M. Herranz, E. Meza, B. Klotoe, P. Muñoz, G. M. Rossolini, A. Bartoloni, E. Tortoli, D. M. Cirillo, E. Gotuzzo, et D. García de Viedma, « Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru », Emerging Infectious Diseases, vol. 25, nᵒ 3, p. 596-598, mars 2019.
    Résumé : We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.
    Mots-clés : antimicrobial resistance, bacteria, Europe, exportation, Florence, IGEPE, Italy, Lima, Madrid, MDR, MICROBIO, migration, MIRU-VNTR, molecular epidemiology, mycobacterium, Mycobacterium tuberculosis, Peru, single-nucleotide polymorphism, Spain, spoligotype, transmission, tuberculosis, tuberculosis and other mycobacteria.

  • C. Aubry, J. - L. Pernodet, et S. Lautru, « A set of modular and integrative vectors for synthetic biology in Streptomyces », Applied and Environmental Microbiology, juin 2019.
    Résumé : With the development of synthetic biology in the field of (actinobacteria) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed.We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FRT sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains, when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalysed excision were all confirmed.To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the Biocrick assembly method. We also used the seamless Ligase Chain Reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain.IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites, or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacteria chromosome. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for protein production or genetic complementation, for example.
    Mots-clés : ACTINO, MICROBIO.

  • H. Barreteau, M. Vandervennet, L. Guedon, V. Point, S. Canaan, S. Rebuffat, J. Peduzzi, et A. Carre-Mlouka, « Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment », International Journal of Systematic and Evolutionary Microbiology, vol. 69, nᵒ 3, p. 732-738, mars 2019.
    Résumé : A halophilic organism, SWO25(T) , was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25(T) - grew optimally at 35-45 degrees C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25(T) to the genus Haloarcula. The DNA G+C content was 61.1 mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25(T) is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarcula sebkhae sp. nov. is proposed. The type strain is SWO25(T)(=CIP 110583(T) =JCM 19018(T)).
    Mots-clés : 16s ribosomal-rna, dna hybridization, ENVBAC, gen. nov., genera, haloarchaea, Haloarcula, halophilic archaeon, heterogeneity, hypersaline environments, MICROBIO, mukohataei, organic-solvent tolerance, rapid method, salt lake, sebkha, sequence.

  • P. Béguin, Y. Chekli, G. Sezonov, P. Forterre, et M. Krupovic, « Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei », Nucleic Acids Research, mai 2019.
    Résumé : Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.
    Mots-clés : ARCHEE, MICROBIO.

  • G. Briassoulis, P. Briassoulis, M. Miliaraki, S. Ilia, M. Parlato, F. Philippart, A. Rouquette, V. Moucadel, J. - M. Cavaillon, B. Misset, et Combined Approach for The eArly diagnosis of INfection in sepsis (CAPTAIN) study group, « Biomarker cruises in sepsis: who is the CAPTAIN? Discussion on "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, janv. 2019.

  • A. Briquet, R. Vong, J. - B. Roseau, E. Javelle, N. Cazes, F. Rivière, M. Aletti, M. - P. Otto, C. Ficko, S. Duron, M. Fabre, C. Pourcel, F. Simon, et C. Soler, « Clinical features of Mycobacterium canettii infection: a retrospective study of 20 cases among French soldiers and relatives », Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, févr. 2019.
    Résumé : Background: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. M. canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Our study aims to describe M. canettii infections among French military or their families between 1998 and 2015. Methods: This retrospective study relied on 3 sources of data: the reference centre for mycobacteria in the Biology Department at Percy military hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families. Results: 20 cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; one case was observed one month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6) and 3 atypical forms are described. All patients had stayed in Djibouti. Conclusions: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments were comparable to tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • E. C. Conceição, G. Refregier, H. M. Gomes, X. Olessa-Daragon, F. Coll, N. H. Ratovonirina, V. Rasolofo-Razanamparany, M. L. Lopes, D. van Soolingen, L. Rutaihwa, S. Gagneux, V. R. Bollela, P. N. Suffys, R. S. Duarte, K. V. B. Lima, et C. N. Sola, « Mycobacterium tuberculosis lineage 1 genetic diversity in Pará, Brazil, suggests common ancestry with east-African isolates potentially linked to historical slave trade », Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, vol. 73, p. 337-341, juin 2019.
    Résumé : Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sub lineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48, sp. 40) and L1/EAI6-BGD1 (SIT591, sp. 23) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil.
    Mots-clés : IGEPE, Lineage 1, MICROBIO, Molecular evolution, Mycobacterium tuberculosis complex, Single-nucleotide polymorphisms, Spoligotyping, Whole genome sequencing.

  • P. Dubois, I. Correia, F. Le Chevalier, S. Dubois, I. Jacques, N. Canu, M. Moutiez, R. Thai, M. Gondry, O. Lequin, et P. Belin, « Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids », Scientific Reports, vol. 9, nᵒ 1, p. 9208, juin 2019.
    Résumé : Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2-26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.
    Mots-clés : BIOSYN, 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, vol. 45, nᵒ 4, p. 508-511, avr. 2019.
    Mots-clés : chest radiography, ESHR, infants, MICROBIO, pneumonia, ultrasonography, ventilation.

  • C. Essoh, J. - P. Vernadet, G. Vergnaud, A. Coulibaly, A. Kakou-N'Douba, A. S. - P. N'Guetta, G. Resch, et C. Pourcel, « Complete Genome Sequences of Five Acinetobacter baumannii Phages from Abidjan, Côte d'Ivoire », Microbiology Resource Announcements, vol. 8, nᵒ 1, janv. 2019.
    Résumé : Five bacteriophages of Acinetobacter baumannii were isolated from sewage water in Abidjan, Côte d'Ivoire. Phages Aci01-1, Aci02-2, and Aci05 belong to an unclassified genus of the Myoviridae family, with double-stranded DNA (dsDNA) genomes, whereas Aci07 and Aci08 belong to the Fri1virus genus of the Podoviridae family of phages.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • A. Gonzalez-Mula, M. Torres, et D. Faure, « Integrative and deconvolution omics approaches to uncover the Agrobacterium tumefaciens lifestyle in plant tumors », Plant Signaling & Behavior, vol. 14, nᵒ 3, p. e1581562, mars 2019.
    Résumé : Agrobacterium tumefaciens is a plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in plant tumors: an integrative approach when omics were used on A. tumefaciens cells collected from plant tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in plant tumors.
    Mots-clés : Agrobacterium, MICROBIO, omics, PBI, plant pathogens, plant tumors.

  • A. Gorlas, R. Catchpole, E. Marguet, et P. Forterre, « Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation », Extremophiles, vol. 23, nᵒ 1, p. 141-149, janv. 2019.
    Résumé : Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.
    Mots-clés : ARCHEE, DBG, Double-strand breaks, MICROBIO, Plasmid, RBA, Topology, UV irradiation.

  • M. Hrasta, K. Rozman, I. Ogris, V. Skedelj, D. Patin, M. Sova, H. Barreteau, S. Gobec, S. G. Grdadolnik, et A. Zega, « H Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases », Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 34, nᵒ 1, p. 1010-1017, janv. 2019.
    Résumé : The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 mu M). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.
    Mots-clés : antibacterial agents, Bacterial Mur (MurC-MurF) ligases, Bacterial Mur (MurC–MurF) ligases, crystal-structure, cytoplasmic steps, d-glutamate ligase, design, discovery, drugs, efficiency, ENVBAC, enzymes, l-alanine, MICROBIO, NMR studies, published kinase inhibitor set, purification, steady-state kinetics measurements.

  • B. J. Klotoe, S. Kacimi, E. Costa-Conceicão, H. M. Gomes, R. B. Barcellos, S. Panaiotov, D. Haj Slimene, N. Sikhayeva, S. Sengstake, A. R. Schuitema, M. Akhalaia, A. Alenova, E. Zholdybayeva, P. Tarlykov, R. Anthony, G. Refrégier, et C. Sola, « Genomic characterization of MDR/XDR-TB in Kazakhstan by a combination of high-throughput methods predominantly shows the ongoing transmission of L2/Beijing 94-32 central Asian/Russian clusters », BMC infectious diseases, vol. 19, nᵒ 1, p. 553, juin 2019.
    Résumé : BACKGROUND: Kazakhstan remains a high-burden TB prevalence country with a concomitent high-burden of multi-drug resistant tuberculosis. For this reason, we performed an in depth genetic diversity and population structure characterization of Mycobacterium tuberculosis complex (MTC) genetic diversity in Kazakhstan with both patient and community benefit. METHODS: A convenience sample of 700 MTC DNA cultures extracts from 630 tuberculosis patients recruited from 12 out of 14 regions in Kazakhstan, between 2010 and 2015, was independently studied by high-throughput hybridization-based methods, TB-SPRINT (59-Plex, n = 700), TB-SNPID (50-Plex, n = 543). DNA from 391 clinical isolates was successfully typed by two methods. To resolve the population structure of drug-resistant clades in more detail two complementary assays were run on the L2 isolates: an IS6110-NTF insertion site typing assay and a SigE SNP polymorphism assay. RESULTS: Strains belonged to L2/Beijing and L4/Euro-American sublineages; L2/Beijing prevalence totaled almost 80%. 50% of all samples were resistant to RIF and to INH., Subtyping showed that: (1) all L2/Beijing were "modern" Beijing and (2) most of these belonged to the previously described 94-32 sublineage (Central Asian/Russian), (3) at least two populations of the Central Asian/Russian sublineages are circulating in Kazakhstan, with different evolutionary dynamics. CONCLUSIONS: For the first time, the global genetic diversity and population structure of M. tuberculosis genotypes circulating in Kazakhstan was obtained and compared to previous local studies. Results suggest a region-specific spread of a very limited number of L2/Beijing clonal complexes in Kazakhstan many strongly associated with an MDR phenotype.
    Mots-clés : Genomics, High-throughput diagnostics methods, IGEPE, Kazakhstan, MDR-TB, MICROBIO, Molecular evolution, Public health, Tuberculosis, XDR-TB.

  • D. Knierim, Q. Barrière, I. Grigoras, S. Winter, H. - J. Vetten, M. Schwinghamer, J. Thomas, P. Chu, B. Gronenborn, et T. Timchenko, « Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components », Viruses, vol. 11, nᵒ 2, p. 138, févr. 2019.
    Résumé : Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.
    Mots-clés : circular ssdna components, DBG, dna, dwarf virus, high-throughput sequencing, high-throughput sequencing, identification, MICROBIO, nanovirus, nanovirus-alphasatellite complex, necrotic yellows virus, PBI, pea, PROMTI, proteins, recombination, replication, rolling circle replication, virus evolution.

  • M. Krupovic, K. S. Makarova, Y. I. Wolf, S. Medvedeva, D. Prangishvili, P. Forterre, et E. V. Koonin, « Integrated mobile genetic elements in Thaumarchaeota », Environmental Microbiology, vol. 21, nᵒ 6, p. 2056-2078, juin 2019.
    Résumé : To explore the diversity of mobile genetic elements (MGE) associated with archaea of the phylum Thaumarchaeota, we exploited the property of most MGE to integrate into the genomes of their hosts. Integrated MGE (iMGE) were identified in 20 thaumarchaeal genomes amounting to 2 Mbp of mobile thaumarchaeal DNA. These iMGE group into five major classes: (i) proviruses, (ii) casposons, (iii) insertion sequence-like transposons, (iv) integrative-conjugative elements and (v) cryptic integrated elements. The majority of the iMGE belong to the latter category and might represent novel families of viruses or plasmids. The identified proviruses are related to tailed viruses of the order Caudovirales and to tailless icosahedral viruses with the double jelly-roll capsid proteins. The thaumarchaeal iMGE are all connected within a gene sharing network, highlighting pervasive gene exchange between MGE occupying the same ecological niche. The thaumarchaeal mobilome carries multiple auxiliary metabolic genes, including multicopper oxidases and ammonia monooxygenase subunit C (AmoC), and stress response genes, such as those for universal stress response proteins (UspA). Thus, iMGE might make important contributions to the fitness and adaptation of their hosts. We identified several iMGE carrying type I-B CRISPR-Cas systems and spacers matching other thaumarchaeal iMGE, suggesting antagonistic interactions between coexisting MGE and symbiotic relationships with the ir archaeal hosts.
    Mots-clés : ammonia-oxidizing archaeon, ARCHEE, crispr-cas systems, diversity, evolution, genome sequence, insights, MICROBIO, photosystem-i, protein, tailed viruses, transfer agents.

  • F. Laddomada, M. M. Miyachiro, M. Jessop, D. Patin, V. Job, D. Mengin-Lecreulx, A. Le Roy, C. Ebel, C. Breyton, I. Gutsche, et A. Dessen, « The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis », Scientific Reports, vol. 9, nᵒ 1, p. 4656, mars 2019.
    Résumé : Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.
    Mots-clés : complexes, crystal-structure, ENVBAC, escherichia-coli murg, macromolecules, MICROBIO, precursors, proteins, purification, reveals, small-angle scattering, ultracentrifugation.

  • F. Lamouche, N. Bonadé-Bottino, P. Mergaert, et B. Alunni, « Symbiotic Efficiency of Spherical and Elongated Bacteroids in the Aeschynomene-Bradyrhizobium Symbiosis », Frontiers in Plant Science, vol. 10, p. 377, 2019.
    Résumé : The legume-rhizobium symbiosis is a major supplier of fixed nitrogen in the biosphere and constitutes a key step of the nitrogen biogeochemical cycle. In some legume species belonging to the Inverted Repeat Lacking Clade (IRLC) and the Dalbergioids, the differentiation of rhizobia into intracellular nitrogen-fixing bacteroids is terminal and involves pronounced cell enlargement and genome endoreduplication, in addition to a strong loss of viability. In the Medicago truncatula-Sinorhizobium spp. system, the extent of bacteroid differentiation correlates with the level of symbiotic efficiency. Here, we used different physiological measurements to compare the symbiotic efficiency of photosynthetic bradyrhizobia in different Aeschynomene spp. (Dalbergioids) hosts inducing different bacteroid morphotypes associated with increasing ploidy levels. The strongly differentiated spherical bacteroids were more efficient than the less strongly differentiated elongated ones, providing a higher mass gain to their hosts. However, symbiotic efficiency is not solely correlated with the extent of bacteroid differentiation especially in spherical bacteroid-inducing plants, suggesting the existence of other factors controlling symbiotic efficiency.
    Mots-clés : accessions, Aeschynomene, bacteroid differentiation, Bradyrhizobium, cell-cycle, differentiation, evolution, family, flow cytometry, genes, legume-rhizobium symbiosis, legumes, medicago-truncatula, MICROBIO, PBI, peptide, plant, symbiotic efficiency, symbiotic efficiency.

  • F. Lamouche, A. Chaumeret, I. Guefrachi, Q. Barrière, O. Pierre, F. Guérard, F. Gilard, E. Giraud, Y. Dessaux, B. Gakière, T. Timchenko, A. Kereszt, P. Mergaert, et B. Alunni, « From intracellular bacteria to differentiated bacteroids: transcriptome and metabolome analysis in Aeschynomene nodules using the Bradyrhizobium sp. ORS285 bclA mutant », Journal of Bacteriology, juin 2019.
    Résumé : Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells where they differentiate into nitrogen-fixing bacteroids. Several legume lineages enforce their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. ORS285, a symbiont of Aeschynomene spp.. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells but they cannot differentiate into enlarged polyploid and functional bacteroids. The bclA nodule bacteria constitute thus an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied with a first transcriptome switch involving several hundreds of upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving less genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a non-symbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.ImportanceThe legume-rhizobium symbiosis is a major ecological process fueling the biogeochemical nitrogen cycle with reduced nitrogen. It represents also a promising strategy to cut down the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.
    Mots-clés : MICROBIO, PBI.

  • L. Latino, D. Patin, D. Cherier, T. Touze, C. Pourcel, H. Barreteau, et D. Mengin-Lecreulx, « Impact of FiuA Outer Membrane Receptor Polymorphism on the Resistance of Pseudomonas aeruginosa toward Peptidoglycan Lipid II-Targeting PaeM Pyocins », Journal of Bacteriology, vol. 201, nᵒ 13, p. e00164, juill. 2019.
    Résumé : Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination. IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.
    Mots-clés : acquisition, bacteriocins, cell wall, colicin M, colicin-m, crystal-structure, DBG, emergence, ENVBAC, escherichia-coli, FiuA, lipid II, locus variable-number, mechanism, MICROBIO, PaeM pyocins, peptidoglycan, protein, Pseudomonas aeruginosa, receptor structure-function, sequence, SSFA, TonB, tonb gene.

  • J. Lossouarn, A. Briet, E. Moncaut, S. Furlan, A. Bouteau, O. Son, M. Leroy, M. S. DuBow, F. Lecointe, P. Serror, et M. - A. Petit, « Enterococcus faecalis Countermeasures Defeat a Virulent Picovirinae Bacteriophage », Viruses, vol. 11, nᵒ 1, p. 48, janv. 2019.
    Résumé : Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested—including V583—were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abiα gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10−4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
    Mots-clés : <i>Enterococcus</i>, abortive infection, adsorption, bacterial, encodes, Enterococcus, escherichia-coli, family, gene, identification, LGBMB, MICROBIO, phage abortive infection, prophage, protein, resistance mechanism, rhamnopolysaccharide, system.
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  • J. Maire, C. Vincent-Monegat, S. Balmand, A. Vallier, M. Herve, F. Masson, N. Parisot, A. Vigneron, C. Anselme, J. Perrin, J. Orlans, I. Rahioui, P. Da Silva, M. - O. Fauvarque, D. Mengin-Lecreulx, A. Zaidman-Remy, et A. Heddi, « Weevil pgrp-lb prevents endosymbiont TCT dissemination and chronic host systemic immune activation », Proceedings of the National Academy of Sciences of the United States of America, vol. 116, nᵒ 12, p. 5623-5632, mars 2019.
    Résumé : Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil Sitophilus zeamais's association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC-independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. pgrp-lb silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas pgrp-lb was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.
    Mots-clés : bacteria, coevolution, drosophila, endosymbiosis, ENVBAC, infection, innate immunity, insect, MICROBIO, mitochondrial, mutualist wigglesworthia, neisseria-gonorrhoeae, nod1, peptidoglycan recognition proteins, PGRP.

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

  • A. Massarweh, M. Bosco, I. Chantret, T. Léger, L. Jamal, D. I. Roper, C. G. Dowson, P. Busca, A. Bouhss, C. Gravier-Pelletier, et S. E. H. Moore, « Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities », Molecules (Basel, Switzerland), vol. 24, nᵒ 11, p. 2135, juin 2019.
    Résumé : Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.
    Mots-clés : alanyl-d-alanine, congenital disorders of glycosylation, congenital disorders of glycosylation, endoplasmic reticulum, ENVBAC, glycosylation, lipid-linked oligosaccharide, MICROBIO, peptidoglycan biosynthesis, peptidoglycan biosynthesis, precursors, protein N-glycosylation.

  • F. Migliardo, Y. Bourdreux, M. Buchotte, G. Doisneau, J. - M. Beau, et N. Bayan, « Study of the conformational behaviour of trehalose mycolates by FT-IR spectroscopy », Chemistry and Physics of Lipids, p. 104789, juin 2019.
    Résumé : Mycolic acids are fundamental cell wall components, found in the outer membrane barrier (mycomembrane) of Mycobacterium related genera, that have shown antigenic, murine innate immunity inducting and inflammatory activity triggering action. The mycolic acid derivatives, such as the lipid extractable trehalose monomycolates (TMM) and dimycolates (TDM), have been extensively investigated by several biochemical and biological methods and, more recently, we have performed the first neutron scattering measurements on these molecules in order to characterize their dynamical behavior as well as their rigidity properties. In the present paper, we show the first systematic FT-IR study on TMM, TDM and glucose monomycolate (GMM). It includes the analysis of individual lipids but also mixtures of TMM/TDM (ratio of 1:1) or TMM/GMM (ratio of 1:2). The present work is aimed to the first characterization of the vibrational behavior of mycolates and their mixtures enabling us to elucidate the molecular mechanisms responsible for the capability of mycolic acids to affect the flexibility and permeability properties of the mycomembrane. As a whole, the present FT-IR findings provide information that have relevant biological implications, allowing to demonstrate that the membrane fluidity is not only linked to the chain length, but also to the specific conformational behavior adopted by mycolates, which in the mixtures is strongly affected by their mutual interactions. In addition, the capability of trehalose to drive the mycolate conformational behavior and then the chain order and packing is emphasized; due to the TDM relevant evidences shown by our data, this trehalose effect could be related to the TDM toxicity and inflammation action.
    Mots-clés : chain length, chain order, CORYNE, fluidity, Infrared spectroscopy, MICROBIO, mycomembrane, trehalose mycolates.

  • L. Morin, M. Kneyber, N. J. G. Jansen, M. J. Peters, E. Javouhey, S. Nadel, G. Maclaren, L. J. Schlapbach, P. Tissieres, et ESPNIC Refractory Septic Shock Definition taskforce and the Infection, Systemic Inflammation and Sepsis ESPNIC section, « Translational gap in pediatric septic shock management: an ESPNIC perspective », Annals of Intensive Care, vol. 9, nᵒ 1, p. 73, juin 2019.
    Résumé : BACKGROUND: The Surviving Sepsis Campaign and the American College of Critical Care Medicine guidelines have provided recommendations for the management of pediatric septic shock patients. We conducted a survey among the European Society of Pediatric and Neonatal Intensive Care (ESPNIC) members to assess variations to these recommendations. METHODS: A total of 114 pediatric intensive care physicians completed an electronic survey. The survey consisted of four standardized clinical cases exploring seven clinical scenarios. RESULTS: Among the seven different clinical scenarios, the types of fluids were preferentially non-synthetic colloids (albumin) and crystalloids (isotonic saline) and volume expansion was not limited to 60 ml/kg. Early intubation for mechanical ventilation was used by 70% of the participants. Norepinephrine was stated to be used in 94% of the PICU physicians surveyed, although dopamine or epinephrine is recommended as first-line vasopressors in pediatric septic shock. When norepinephrine was used, the addition of another inotrope was frequent. Specific drugs such as vasopressin or enoximone were used in < 20%. Extracorporeal life support was used or considered by 91% of the physicians audited in certain specific situations, whereas the use of high-flow hemofiltration was considered for 44%. CONCLUSIONS: This pediatric septic shock management survey outlined variability in the current clinician-reported practice of pediatric septic shock management. As most recommendations are not supported by evidence, these findings outline some limitation of existing pediatric guidelines in regard to context and patient's specificity.
    Mots-clés : ESHR, MICROBIO.

  • S. Najah, C. Saulnier, J. - L. Pernodet, et S. Bury-Moné, « Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci », BMC biotechnology, vol. 19, nᵒ 1, p. 18, mars 2019.
    Résumé : BACKGROUND: The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci. RESULTS: We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy. CONCLUSION: By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.
    Mots-clés : ACTINO, Bait DNA, CRISPR-Cas9, deletions, dna, Essential gene, Foreign DNA, Generic tool, Lsr2, MICROBIO, Multicopy gene, Nucleoid-associated proteins, Streptomyces, Xenogeneic silencers.

  • A. Novikov, N. Marr, et M. Caroff, « A comparative study of the complete lipopolysaccharide structures and biosynthesis loci of Bordetella avium, B. hinzii, and B. trematum », Biochimie, vol. 159, p. 81-92, avr. 2019.
    Résumé : A dozen species of human and animal pathogens have been described to date in the Bordetella genus, with the majority being respiratory tract pathogens. Bordetella avium lipopolysaccharides have been shown to be important virulence factors for this bird pathogen. B. hinzii is closely related to the B. avium species, but has also been isolated from humans. B. trematum is associated to ear and blood infections in humans. Its lipid A structure, the biological active moiety of LPS, was found to be closely related to those of B. avium and B. hinzii. It is important to unveil the subtle structural modifications orchestrated during the LPS biosynthetic pathway to better understand host adaptation. The present data are also important in the context of deciphering the virulence pathways of this important genus containing the major pathogens B. pertussis and B. parapertussis, responsible for whooping cough. We recently reported the isolated lipid A structures of the three presented species, following the previously identified O-chain structures. In the present study, we provide details on the free and O-chain-linked core oligosaccharides which were required to characterize the complete LPS structures. Data are presented here in relation to relevant biosynthesis genes. The present characterization of the three species is well illustrated by Matrix Assisted Laser Desorption Mass Spectrometry experiments, and data were obtained mainly on native LPS molecules for the first time. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
    Mots-clés : antigen, B. hinzii, B. trematum, B. hinzii, B. trematum, Bordetella avium, bronchiseptica, chain linkage region, core, desorption mass-spectrometry, Endotoxin, ESHR, Genomics, Lipopolysaccharide MALDI MS, MICROBIO, o-specific polysaccharide, parapertussis, pertussis, sp nov., takamatsuzuka tumulus.

  • T. Ohbayashi, R. Futahashi, M. Terashima, Q. Barrière, F. Lamouche, K. Takeshita, X. - Y. Meng, Y. Mitani, T. Sone, S. Shigenobu, T. Fukatsu, P. Mergaert, et Y. Kikuchi, « Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture », The ISME journal, vol. 13, nᵒ 6, p. 1469-1483, juin 2019.
    Résumé : In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.
    Mots-clés : antimicrobial peptides, bacteria, blattabacterium, colonization, diversity, endosymbiont, genome sequence, gut symbiont, MICROBIO, outer-membrane vesicles, PBI, transmission.

  • T. Ohbayashi, H. Itoh, J. Lachat, Y. Kikuchi, et P. Mergaert, « Burkholderia Gut Symbionts Associated with European and Japanese Populations of the Dock Bug Coreus marginatus (Coreoidea: Coreidae) », Microbes and Environments, juin 2019.
    Résumé : Insects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern.
    Mots-clés : Burkholderia, MICROBIO, obligate gut symbiosis, PBI, region-dependent symbionts, stinkbug.

  • J. R. Osman, C. Regeard, C. Badel, G. Fernandes, et M. S. DuBow, « Variation of bacterial biodiversity from saline soils and estuary sediments present near the Mediterranean Sea coast of Camargue (France) », Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, vol. 112, nᵒ 3, p. 351-365, mars 2019.
    Résumé : Salinity is an important environmental factor influencing microbial community composition. To better understand this influence, we determined the bacterial communities present in 17 different sites of brackish sediment (underwater) and soil (surface) samples from the Camargue region (Rhone river delta) in southern France during the fall of 2013 and 2014 using pyrosequencing of the V3-V4 regions of the 16S rRNA genes amplified by PCR. This region is known for abundant flora and fauna and, though saline, 30% of rice consumed in France is grown here. We found that bacterial abundance in 1g of soil or sediment, calculated by qPCR, was higher in sediments than in surface soil samples. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes phyla dominated the bacterial communities of sediment samples, while members belonging to the Proteobacteria, Bacteroidetes, Gemmatimonadetes, Actinobacteria, Firmicutes and Acidobacteria phyla dominated the bacterial communities of the soil samples. The most abundant bacterial genera present in the saline sediments and soils from the Camargue belonged mostly to halophilic and sulphate reducing bacteria, suggesting that the Camargue may be a valuable system to investigate saline, yet agriculturally productive, sediment and soil microbial ecosystem.
    Mots-clés : ARCHEE, Bacterial biodiversity, community structure, de-giraud, diversity, gen. nov., Halophilic bacteria, hypersaline microbial mat, LGBMB, MICROBIO, molecular analysis, Pyrosequencing, rhone river delta, rice fields, rna, Salinity, temporal variation.

  • S. Oulghazi, J. Cigna, Y. Y. Lau, M. Moumni, K. G. Chan, et D. Faure, « Transfer of the waterfall source isolate Pectobacterium carotovorum M022 to Pectobacterium fontis sp. nov., a deep-branching species within the genus Pectobacterium », International Journal of Systematic and Evolutionary Microbiology, vol. 69, nᵒ 2, p. 470-475, févr. 2019.
    Résumé : Pectobacterium carotovorum M022(T) has been isolated from a waterfall source in Selangor district (Malaysia). Using genomic and phenotypic tests, we re-examined the taxonomical position of this strain. Based on 14 concatenated housekeeping genes (fusA, rpoD, rpoS, acnA, purA, gyrB, recA, mdh, mtID, groEL, secY, glyA, gapA and rpIB), multi-locus sequence analysis revealed that strain M022(T) falls into a novel Glade separated from the other Pectobacterium species. The in silico DNA-DNA hybridization and average nucleotide identity values were lower than the 70 and 95% threshold values, respectively. In addition, by combining genomic and phenotypic tests, strain M022(T) may be distinguished from the other Pectobacterium isolates by its incapacity to grow on D(+)-xylose, L-rhamnose, cellobiose and lactose. Strain M022(T) (=CFBP 8629(T)=LMG 30744(T)) is proposed as the type strain of the Pectobacterium fontis sp. nov.
    Mots-clés : 1st report, atrosepticum, bacterial soft-rot, brasiliense, Dickeya, MICROBIO, pathogen, PBI, pectinolytic, Pectobacterium, potato, stem rot, subsp carotovorum, taxonomy, wasabiae, water, waterfall.

  • S. Oulghazi, J. Pédron, J. Cigna, Y. Y. Lau, M. Moumni, F. Van Gijsegem, K. - G. Chan, et D. Faure, « Dickeya undicola sp. nov., a novel species for pectinolytic isolates from surface waters in Europe and Asia », International Journal of Systematic and Evolutionary Microbiology, juin 2019.
    Résumé : Strains 2B12T, FVG1-MFV-O17 and FVG10-MFV-A16 were isolated from fresh water samples collected in Asia and Europe. The nucleotide sequences of the gapA barcodes revealed that all three strains belonged to the same cluster within the genus Dickeya. Using 13 housekeeping genes (fusA, rpoD, rpoS, glyA, purA, groEL, gapA, rplB, leuS, recA, gyrB, infB and secY), multilocus sequence analysis confirmed the existence of a new clade. When the genome sequences of these three isolates and other Dickeya species were compared, the in silico DNA-DNA hybridization and average nucleotide identity values were found to be no more than 45.50 and 91.22 %, respectively. The closest relative species was Dickeya fangzhongdai. Genome comparisons also highlighted genetic traits differentiating the new strains from D. fangzhongdai strains DSM 101947T (=CFBP 8607T) and B16. Phenotypical tests were performed to distinguish the three strains from D. fangzhongdai and other Dickeya species. The name Dickeya undicola sp. nov. is proposed with strain 2B12T (=CFBP 8650T=LMG 30903T) as the type strain.
    Mots-clés : MICROBIO, PBI.

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

  • J. C. Reina, M. Torres, et I. Llamas, « Stenotrophomonas maltophilia AHL-Degrading Strains Isolated from Marine Invertebrate Microbiota Attenuate the Virulence of Pectobacterium carotovorum and Vibrio coralliilyticus », Marine Biotechnology (New York, N.Y.), févr. 2019.
    Résumé : Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.
    Mots-clés : Acylase, MICROBIO, N-Acylhomoserine lactones, PBI, Quorum quenching, Stenotrophomonas.

  • M. Rondeau, Q. Esmaeel, J. Crouzet, P. Blin, I. Gosselin, C. Sarazin, M. Pernes, J. Beaugrand, F. Wisniewski-Dyé, L. Vial, D. Faure, C. Clément, E. Ait Barka, C. Jacquard, et L. Sanchez, « Biofilm constructing variants of Paraburkholderia phytofirmans PsJN outcompete the wild-type form in free-living and static conditions but not in planta », Applied and Environmental Microbiology, vol. 85, nᵒ 11, p. UNSP e02670-18, mars 2019.
    Résumé : Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several Microbe Associated Molecular Pattern (MAMPs) including exopolysaccharides (EPS), flagellin and GroEL. Interestingly, the variants induce a low PR1 and PDF1.2 expression compared to the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an Iron-Sulfur Cluster: hscA (encoding a co-chaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and co-express a variant that outcompete the wild-type form in free-living and static conditions but not in plantaIMPORTANCEParaburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneaous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneaous mutations in an Iron-Sulfur Cluster. Overall, the data provided here allowed a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in heterogeneous environment.
    Mots-clés : antigenic variation, arabidopsis-thaliana, biofilm, burkholderia-cepacia complex, colony morphology, competition, defense responses, growth promotion, iron-sulfur cluster, MICROBIO, necrotrophic pathogens, Paraburkholderia phytofirmans, PBI, phase variation, phenotypic variation, plant defense, root-colonization, static cultures.

  • V. Škedelj, U. P. Fonović, P. Molek, S. Magnet, J. - L. Mainardi, D. Blanot, S. Gobec, J. Stojan, et A. Zega, « Kinetic mechanism of Enterococcus faecium D-aspartate ligase », Biochimie, vol. 158, p. 217-223, janv. 2019.
    Résumé : Enterococcus faecium D-aspartate ligase (Aslfm) is a peptide bond-forming enzyme that is involved in the peptidoglycan assembly pathway. It catalyzes the ATP-dependent ligation of the β-carboxylate of D-Asp to the ε-amino group of L-Lys in the nucleotide precursor UDP- MurNAc-pentapeptide. The enzyme is of interest as a target of new, potential, narrow-spectrum antibiotics directed against multiresistant E. faecium. The kinetic mechanism of Aslfm has not been fully characterized. To determine it, a progress curve analysis of Aslfm catalytic process using pyruvate kinase/lactate dehydrogenase ATPase detection assay was performed. With an inspection of the shape of measured progress curves and the results of specific qualitative experiments, the Aslfm reaction mechanism was singled out. The proposed Aslfm kinetics reaction scheme was evaluated by fitting the parameters of the corresponding differential equations to progress curves using the computer program ENZO. The complete kinetic analysis result is consistent with the substrate binding order 1) ATP, 2) D-Asp, and 3) UDP-MurNAc-pentapeptide. The analysis suggests that slowly establishing non-productive equilibria between the free and ATP-bound enzyme with the participating pentapeptide are responsible for initial reaction burst followed by a steady-state period before the complete depletion of the reactant added in the lowest concentration.
    Mots-clés : acid, bacterial cell-walls, biosynthesis, D-aspartate ligase (Asl(fm)), Enterococcus faecium, ENVBAC, enzyme, Kinetic mechanism, MICROBIO, Progress curve analysis.

  • O. Soutourina, « Type I Toxin-Antitoxin Systems in Clostridia », Toxins, vol. 11, nᵒ 5, mai 2019.
    Résumé : Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.
    Mots-clés : ARNCLO, Clostridium difficile, MICROBIO, prophages, RNA antitoxins, toxin-antitoxin systems, type I.

  • M. Torres, Y. Dessaux, et I. Llamas, « Saline Environments as a Source of Potential Quorum Sensing Disruptors to Control Bacterial Infections: A Review », Marine Drugs, vol. 17, nᵒ 3, mars 2019.
    Résumé : Saline environments, such as marine and hypersaline habitats, are widely distributed around the world. They include sea waters, saline lakes, solar salterns, or hypersaline soils. The bacteria that live in these habitats produce and develop unique bioactive molecules and physiological pathways to cope with the stress conditions generated by these environments. They have been described to produce compounds with properties that differ from those found in non-saline habitats. In the last decades, the ability to disrupt quorum-sensing (QS) intercellular communication systems has been identified in many marine organisms, including bacteria. The two main mechanisms of QS interference, i.e., quorum sensing inhibition (QSI) and quorum quenching (QQ), appear to be a more frequent phenomenon in marine aquatic environments than in soils. However, data concerning bacteria from hypersaline habitats is scarce. Salt-tolerant QSI compounds and QQ enzymes may be of interest to interfere with QS-regulated bacterial functions, including virulence, in sectors such as aquaculture or agriculture where salinity is a serious environmental issue. This review provides a global overview of the main works related to QS interruption in saline environments as well as the derived biotechnological applications.
    Mots-clés : hypersaline habitat, marine habitat, marine pathogens, MICROBIO, PBI, plant pathogens, QQ, QSI, quorum quenching, quorum sensing, saline environment, Vibrio.

  • M. Torres, K. - W. Hong, T. - M. Chong, J. C. Reina, K. - G. Chan, Y. Dessaux, et I. Llamas, « Genomic analyses of two Alteromonas stellipolaris strains reveal traits with potential biotechnological applications », Scientific Reports, vol. 9, nᵒ 1, p. 1215, févr. 2019.
    Résumé : The Alteromonas stellipolaris strains PQQ-42 and PQQ-44, previously isolated from a fish hatchery, have been selected on the basis of their strong quorum quenching (QQ) activity, as well as their ability to reduce Vibrio-induced mortality on the coral Oculina patagonica. In this study, the genome sequences of both strains were determined and analyzed in order to identify the mechanism responsible for QQ activity. Both PQQ-42 and PQQ-44 were found to degrade a wide range of N-acylhomoserine lactone (AHL) QS signals, possibly due to the presence of an aac gene which encodes an AHL amidohydrolase. In addition, the different colony morphologies exhibited by the strains could be related to the differences observed in genes encoding cell wall biosynthesis and exopolysaccharide (EPS) production. The PQQ-42 strain produces more EPS (0.36 g l-1) than the PQQ-44 strain (0.15 g l-1), whose chemical compositions also differ. Remarkably, PQQ-44 EPS contains large amounts of fucose, a sugar used in high-value biotechnological applications. Furthermore, the genome of strain PQQ-42 contained a large non-ribosomal peptide synthase (NRPS) cluster with a previously unknown genetic structure. The synthesis of enzymes and other bioactive compounds were also identified, indicating that PQQ-42 and PQQ-44 could have biotechnological applications.
    Mots-clés : acylhomoserine lactone-acylase, alginate lyase, cloning, exopolysaccharide production, identification, l-fucose, marine bacterium, MICROBIO, PBI, protein, quorum, sp nov..

  • T. Tosi, F. Hoshiga, C. Millership, R. Singh, C. Eldrid, D. Patin, D. Mengin-Lecreulx, K. Thalassinos, P. Freemont, et A. Gründling, « Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM », PLoS pathogens, vol. 15, nᵒ 1, p. e1007537, janv. 2019.
    Résumé : c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
    Mots-clés : cyclic dinucleotide, degradation, domain, ENVBAC, enzyme, functional-analysis, growth, integrity, MICROBIO, mobility mass-spectrometry, resistance, scanning protein-a.

  • S. Werten, N. H. Rustmeier, M. Gemmer, M. - J. Virolle, et W. Hinrichs, « Structural and Biochemical Analysis of a Phosin from Streptomyces chartreusis Reveals a Combined Polyphosphate- and Metal-Binding Fold », FEBS letters, juin 2019.
    Résumé : X-ray crystallographic analysis of a phosin (PptA) from Steptomyces chartreusis reveals a metal-associated, lozenge-shaped fold featuring a 5-10 Å wide, positively charged tunnel that traverses the protein core. Two distinct metal-binding sites were identified in which the predominant metal ion was Cu2+ . In solution, PptA forms stable homodimers that bind with nanomolar affinity to polyphosphate, a stress-related biopolymer acting as a phosphate and energy reserve in conditions of nutrient depletion. A single protein dimer interacts with 14-15 consecutive phosphate moieties within the polymer. Our observations suggest that PptA plays a role in polyphosphate metabolism, mobilisation or sensing, possibly by acting in concert with polyphosphate kinase (Ppk). Like Ppk, phosins may inuence antibiotic synthesis by streptomycetes. This article is protected by copyright. All rights reserved.
    Mots-clés : antibiotics, Conserved histidine α-helical domain (CHAD), MESMIC, MICROBIO, nutritional stress, phosphate metabolism, secondary metabolism, signalling.

  • W. Zhang-Sun, F. Terce, R. Burcelin, A. Novikov, M. Serino, et M. Caroff, « Structure function relationships in three lipids A from the Ralstonia genus rising in obese patients », Biochimie, vol. 159, p. 72-80, avr. 2019.
    Résumé : The identification of a functional molecular moiety relating the lipopolysaccharides (LPSs) to their capacity to induce inflammation-mediated metabolic diseases needed to be performed. We previously described a proportional increase in the relative abundance of the 16 SrDNA bacterial gene from the genus Ralstonia, within the microbiota from the adipose tissue stroma vascular fraction of obese patients, suggesting a causal role of the bacteria. Therefore, we first characterized the structures of the lipids A, the inflammatory inducing moieties of LPSs, of three Ralstonia species: Ralstonia eutropha, R. mannitolilytica and R. pickettii, and then compared each, in terms of in vitro inflammatory capacities. R. pickettii lipid A displaying only 5 Fatty Acids (FA) was a weaker inducer of inflammation, compared to the two other species harboring hexa-acylated lipids A, despite the presence of 2 AraN substituents on the phosphate groups. With regard to in vitro pro-inflammatory activities, TNF-alpha and IL-6 inducing capacities were compared on THP-1 cells treated with LPSs isolated from the three Ralstonia. R. pickettii, with low inflammatory capacities, and recently involved in nosocomial outcomes, could explain the low inflammatory level reported in previous studies on diabetic patients and animals. In addition, transmission electron microscopy was performed on the three Ralstonia species. It showed that the R. pickettii under-acylated LPSs, with a higher level of phosphate substitution had the capacity of producing more outer membrane vesicles (OMVs). The latter could facilitate transfer of LPSs to the blood and explain the increased low-grade inflammation observed in obese/diabetic patients. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
    Mots-clés : bacteria, Cytokines, endotoxemia, ESHR, Lipopolysaccharide, lipopolysaccharides, lps, Mass spectrometry, Metabolic diseases, MICROBIO, microextraction, onset, phosphate groups, Ralstonia, strains, Structure.


  • P. Abranyi-Balogh, L. Petri, T. Imre, P. Szijj, A. Scarpino, M. Hrast, A. Mitrovic, U. P. Fonovic, K. Nemeth, H. Barreteau, D. I. Roper, K. Horvati, G. G. Ferenczy, J. Kos, J. Ilas, S. Gobec, et G. M. Keseru, « A road map for prioritizing warheads for cysteine targeting covalent inhibitors », European Journal of Medicinal Chemistry, vol. 160, p. 94-107, déc. 2018.
    Résumé : Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.
    Mots-clés : Cathepsin B, Cathepsin X, cathepsin-b, Covalent inhibitors, design, drug discovery, electrophiles, Electrophilic warheads, ENVBAC, fragments, GSH reactivity assay, kinase inhibitors, MICROBIO, MurA, Oligopeptide specificity assay.
  • G. Agbota, M. Accrombessi, S. Ezinmegnon, U. Ahouayito, B. Agossou, A. Massougbodji, L. Ganee, J. Marcos, A. Pachot, P. Tissieres, N. Fievet, M. Cot, et V. Briand, « Malaria in the First Trimester of Pregnancy Is Associated with Non-Malaria Fever During the First Three Months of Life in a Beninese Infant Population », American Journal of Tropical Medicine and Hygiene, vol. 99, nᵒ 4, p. 235-235, 2018.

  • 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.

  • W. Briand, O. Dao, G. Garnier, R. Guegan, B. Marta, C. Maupu, J. Miesch, K. Papadopoulo, A. Radoux, J. Rojahn, Y. Zhu, C. Aubry, P. Bouloc, S. Bury-Moné, A. Ferré, S. Lautru, O. Namy, et M. Sabeti-Azad, « Dégradation d’un anticancéreux dans les eaux usées - Une médaille d’or pour l’équipe GO Paris-Saclay », médecine/sciences, vol. 34, nᵒ 12, p. 1111-1114, déc. 2018.
    Résumé : iGEM (pour <i>international genetically engineered machine)<i/> est un concours international autour de la biologie synthétique réunissant des étudiants de toutes disciplines (mathématiques, physique, biologie, arts, etc.). « L’objectif est de construire un système biologique fonctionnel complexe, en assemblant des composants individuels moléculaires simples et standardisés (fragments d’ADN), appelés « briques biologiques » (biobriques), sorte de « legos » moléculaires, entreposés au MIT (<i>Massachusetts Institute of Technology<i/>) (le <i>registry of standard biological parts<i/> contient environ 20 000 biobriques). C’est une démarche proche de celle de l’ingénieur qui assemble des circuits électroniques ». En 2004, lors de sa création par le MIT (<b>→<b/>), la compétition iGEM regroupait une quarantaine de projets ; 14 ans plus tard, elle accueille 350 équipes (6 000 étudiants, avec leurs instructeurs) issues des universités du monde entier. Elle culmine en un <i>Giant Jamboree<i/> de quatre jours à Boston en novembre, au cours duquel les équipes présentent leur projet. Le « wiki » de la compétition ( présente l’ensemble des projets ainsi que le palmarès. Cette année, ont été décernées 114 médailles d’or, 68 d’argent et 107 de bronze. Neuf équipes françaises étaient engagées.<b>(→) Voir l’article de J. Peccoud et L. Coulombel, dont certains passages sont repris dans ce « chapo », m/s n° 5, mai 2007, page 551<b/>
    Pièce jointe Full Text PDF 1.5 Mo (source)
    Pièce jointe Full Text PDF 1.5 Mo (source)

  • 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.

  • C. Cassier‐Chauvat et F. Chauvat, « Cyanobacteria: Wonderful Microorganisms for Basic and Applied Research », in eLS, American Cancer Society, 2018, p. 1-11.
    Résumé : Cyanobacteria, the only prokaryotes to perform photosynthesis, are fascinating organisms. They are regarded as the oldest phylum of bacteria that shaped the oxygenic atmosphere of our planet, and the progenitor of the plant-chloroplast. Contemporary cyanobacteria colonise most water (fresh, brackish and marine) and soils of our planet, as free-living or symbiotic organisms, and generate a large part of the oxygen and the biomass for the biosphere. Hence, a few edible cyanobacteria are being tested as a way to replenish O2, provide food and recycle wastes (CO2 and urea) during long-term space missions. Furthermore, cyanobacteria also synthesise a vast array of biologically active metabolites with great potential for human health and industries. Thus, cyanobacteria constitute promising microbial factories for the production of chemicals from highly abundant natural resources: solar energy, CO2, minerals and waters (even polluted). Key Concepts Cyanobacteria are ancient organisms. Cyanobacteria are environmentally important organisms. Cyanobacteria produce a huge biomass for the food chain. Cyanobacteria are widely diverse organisms. Cyanobacteria have a versatile metabolism of great biotechnological interest.
    Mots-clés : B2CYA, biodiversity, biofuels, bioplastics, cell division, cell morphology, cyanobacteria, MICROBIO, nitrogen fixation, photosynthesis, respiration, therapeutics.
    Pièce jointe Full Text PDF 1.8 Mo (source)

  • 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, DBG, Gene cloning and expression, Genetics of extremophiles, Hyperthermophiles, MICROBIO, Molecular biology, Molecular biology of archaea, RBA.

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