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Labrie, Simon

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Labrie

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Simon

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Université Laval.Département de biochimie et de microbiologie

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  • PublicationRestreint
    The tape measure protein is involved in the heat stability of Lactococcus lactis phages
    (American Society for Microbiology, 2018-01-17) Moineau, Sylvain; Geagea, Hany; Labrie, Simon; Subirade, Muriel
    Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group. IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.
  • PublicationAccès libre
    Use of an α-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus
    (American Dairy Science Association, 2010-03-18) Vadeboncoeur, Christian; Moineau, Sylvain; Labrie, Simon; Bart, Christian
    The α-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the α-galactosidase of L. raffinolactis is thermolabile and inoperative at 42°C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this α-galactosidase at 42°C was only 3% of that at 30°C, whereas the enzyme retained 23% of its activity at 37°C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37°C or below. The transformed cells possessed 6-fold more α-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.
  • PublicationRestreint
    Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2
    (American Society for Microbiology, 2003-09-19) Harel, Josée; Moineau, Sylvain; Martinez, Gabriela; Labrie, Simon; Nassar, Atef; Dezfulian, Hojabr; Brousseau, Roland; Gottschalk, Marcelo
    Streptococcus suis infection is considered to be a major problem in the swine industry worldwide. Most virulent Canadian isolates of S. suis serotype 2 do not produce the known virulence markers for this pathogen. PCR-based subtraction hybridization was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. Analysis of some subtracted DNA clones revealed significant homology with bacteriophages of gram-positive bacteria. An inducible phage (named Ss1) was observed in S. suis following the incubation of the virulent strain 89-999 with mitomycin C. Phage Ss1 has a long noncontractile tail and a small isometric nucleocapsid and is a member of the Siphoviridae family. Ss1 phage DNA appears to be present in most Canadian S. suis strains tested in this study, which were isolated from diseased pigs or had proven virulence in mouse or pig models. To our knowledge, this is the first report of the isolation of a phage in S. suis.
  • PublicationRestreint
    Genomic diversity of phages infecting probiotic strains of Lactobacillus paracasei
    (American Society for Microbiology, 2015-12-22) Mercanti, Diego J.; Tremblay, Denise; Moineau, Sylvain; Capra, María Luján; Labrie, Simon; Luján Quiberoni, Andrea del; Rousseau, Geneviève M.
    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.
  • PublicationRestreint
    Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism abiT
    (American Society for Microbiology, 2012-09-07) Tremblay, Denise; Moisan, Maxim; Moineau, Sylvain; Labrie, Simon; Magadán, Alfonso H.; Campanacci, Valérie; Villion, Manuela; Cambillau, Christian
    The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14bIL170]), orf41 (P008 [orf41P008]), and orf6 (p2 [orf6p2] and P008 [orf6P008]). The genes e14bIL170 and orf41P008 are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6p2 and ORF5p2, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.
  • PublicationAccès libre
    Caractérisation des phages et des mécanismes anti-phages chez Lactococcus lactis
    (2010) Labrie, Simon; Moineau, Sylvain
    Lactococcus lactis est une bactérie lactique très utilisée pour la fabrication de divers fromages. Cependant, cette bactérie est susceptible aux bactériophages qui peuvent causer sporadiquement des problèmes de fermentation. À cause de ces problèmes, les phages de L. lactis sont les plus étudiés après les entérophages. Néanmoins, bien des notions restent vagues au sujet de l’évolution et la biologie de ceux-ci, et ceci est particulièrement saillant pour les systèmes anti-phages. Au cours de ce projet, trois différents aspects de la biologie ces phages ont été abordés : leur diversité génétique, leur évolution, et les mécanismes anti-phages. Le séquençage du génome du phage P335 a permis de démontrer l’hétérogénéité des membres du groupe du même nom ainsi que de détecter l’émergence de sous-groupes à l’intérieur de celui-ci. De plus, un module de gènes morons a été identifié. Ces gènes possèdent de l’homologie avec des séquences provenant de prophages appartenant à d’autres taxons. Le second objectif a démontré la plasticité génétique des phages du groupe P335 et leur évolution en présence de deux mécanismes de résistance aux phages de type Abi. Un phage du groupe P335 a échangé jusqu’à 79% de son génome pour devenir résistant aux systèmes antiphages AbiK et AbiT. Les phages mutants ont acquis des nouveaux modules d’ADN provenant d’au moins deux prophages retrouvés dans le génome de leur hôte. Finalement, le mode d’action du mécanisme AbiT a été étudié. Plusieurs phages résistants à AbiT ont été isolés et caractérisés. Trois cibles ou activateurs d’AbiT ont été identifiés. Deux sont des gènes précoces dont la fonction est inconnue. Le troisième gène fait partie du module de morphogénèse et encode la protéine majeure de la capside. Lactococcus lactis et ses phages sont un modèle important pour la compréhension des interactions phage/hôte chez les bactéries lactiques. Ce projet a permis d’acquérir des connaissances sur la diversité génétique et l’évolution des phages du groupe P335. Ces phages possèdent une impressionnante plasticité génétique et leurs populations évoluent rapidement en présence d’une pression sélective, tels les systèmes anti-phages. Il s’est aussi avéré que le mode d’action mécanisme anti-phages AbiT est beaucoup plus complexe qu’initialement anticipé puisqu’il cible trois différentes composantes phagiques.
  • PublicationRestreint
  • PublicationRestreint
    Lactococcal abortive infection protein AbiV interacts directly with the phage protein SaV and prevents translation of phage proteins
    (American Society for Microbiology, 2010-10-25) Haaber, Jakob; Moineau, Sylvain; Labrie, Simon; Samson, Julie; Campanacci, Valérie; Cambillau, Christian; Hammer, Karin
    AbiV is an abortive infection protein that inhibits the lytic cycle of several virulent phages infecting Lactococcus lactis, while a mutation in the phage gene sav confers insensitivity to AbiV. In this study, we have further characterized the effects of the bacterial AbiV and its interaction with the phage p2 protein SaV. First, we showed that during phage infection of lactococcal AbiV+ cells, AbiV rapidly inhibited protein synthesis. Among early phage transcripts, sav gene transcription was slightly inhibited while the SaV protein could not be detected. Analyses of other phage p2 mRNAs and proteins suggested that AbiV blocks the activation of late gene transcription, probably by a general inhibition of translation. Using size exclusion chromatography coupled with on-line static light scattering and refractometry, as well as fluorescence quenching experiments, we also demonstrated that both AbiV and SaV formed homodimers and that they strongly and specifically interact with each other to form a stable protein complex.
  • PublicationRestreint
    Morphology, genome sequence, and structural Proteome of type phage P335 from Lactococcus lactis
    (American Society for Microbiology, 2008-08-01) Moineau, Sylvain; Josephsen, Jytte; Labrie, Simon; Neve, Hosrt; Vogensen, Finn K.
    Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.
  • PublicationAccès libre
    CRISPRStudio : A user-friendly software for rapid CRISPR array visualization
    (MDPI, 2018-11-01) Dion, Moïra; Moineau, Sylvain; Shah, Shiraz A.; Labrie, Simon
    The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.