Personne : Labrie, Simon
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Université Laval.Département de biochimie et de microbiologie
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- PublicationRestreintGenomic 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.
- PublicationRestreintIdentification 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, MarceloStreptococcus 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.
- PublicationRestreintThe 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, MurielVirulent 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 libreUse 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, ChristianThe 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.
- PublicationRestreintA new Microviridae phage isolated from a failed biotechnological process driven by Escherichia coli(American Society for Microbiology, 2014-10-17) Tremblay, Denise; Moineau, Sylvain; Dupuis, Marie-Ève; Labrie, Simon; Plante, Pier-Luc; Corbeil, JacquesBacteriophages are present in every environment that supports bacterial growth, including manmade ecological niches. Virulent phages may even slow or, in more severe cases, interrupt bioprocesses driven by bacteria. Escherichia coli is one of the most widely used bacteria for large-scale bioprocesses; however, literature describing phage-host interactions in this industrial context is sparse. Here, we describe phage MED1 isolated from a failed industrial process. Phage MED1 (Microviridae family, with a single-stranded DNA [ssDNA] genome) is highly similar to the archetypal phage phiX174, sharing >95% identity between their genomic sequences. Whole-genome phylogenetic analysis of 52 microvirus genomes from public databases revealed three genotypes (alpha3, G4, and phiX174). Phage MED1 belongs to the phiX174 group. We analyzed the distribution of single nucleotide variants in MED1 and 18 other phiX174-like genomes and found that there are more missense mutations in genes G, B, and E than in the other genes of these genomes. Gene G encodes the spike protein, involved in host attachment. The evolution of this protein likely results from the selective pressure on phages to rapidly adapt to the molecular diversity found at the surface of their hosts.
- PublicationAccès libreComplete genome sequence of Streptococcus thermophilus SMQ-301, a model strain for phage-host interactions(American Society for Microbiology, 2015-05-21) Tremblay, Denise; Moineau, Sylvain; Labrie, Simon; Wasserscheid, Jessica; Dewar, Ken; Plante, Pier-Luc; Corbeil, JacquesStreptococcus thermophilus is used by the dairy industry to manufacture yogurt and several cheeses. Using PacBio and Illumina platforms, we sequenced the genome of S. thermophilus SMQ-301, the host of several virulent phages. The genome is composed of 1,861,792 bp and contains 2,037 genes, 67 tRNAs, and 18 rRNAs.
- PublicationRestreintPhosphorylation, an altruistic bacterial trick to halt phages(Cell Press, 2016-10-12) Moineau, Sylvain; Labrie, SimonBacterial genomic islands contain hidden treasures. In this issue of Cell Host & Microbe, Depardieu et al. (2016) found, in one of those islands, an altruistic anti-phage system that is triggered by a phage component and induces a phosphorylation cascade leading to protection of the bacterial population.
- PublicationAccès libreProgramming native CRISPR arrays for the generation of targeted immunity(American Society for Microbiology, 2016-05-03) Moineau, Sylvain; Hynes, Alexander; Labrie, SimonABSTRACT : The adaptive immune system of prokaryotes, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes), results in specific cleavage of invading nucleic acid sequences recognized by the cell’s “memory” of past encounters. Here, we exploited the properties of native CRISPR-Cas systems to program the natural “memorization” process, efficiently generating immunity not only to a bacteriophage or plasmid but to any specifically chosen DNA sequence. IMPORTANCE : CRISPR-Cas systems have entered the public consciousness as genome editing tools due to their readily programmable nature. In industrial settings, natural CRISPR-Cas immunity is already exploited to generate strains resistant to potentially disruptive viruses. However, the natural process by which bacteria acquire new target specificities (adaptation) is difficult to study and manipulate. The target against which immunity is conferred is selected stochastically. By biasing the immunization process, we offer a means to generate customized immunity, as well as provide a new tool to study adaptation.
- PublicationRestreintInactivation of dairy bacteriophages by commercial sanitizers and disinfectants(Elsevier, 2013-11-19) Duchaine, Caroline; Moineau, Sylvain; Labrie, Simon; Campagna, Céline; Villion, ManuelaMany commercial sanitizers and disinfectants have been used over the years to control microbial contamination but their efficacy on phages is often unknown. Here, 23 commercial chemical products, including 21 food-grade sanitizers were tested against virulent dairy phages. These food-grade chemicals included oxidizing agents, halogenated agents, alcohols, quaternary ammonium compounds, anionic acids, iodine-based acids, and an amphoteric chemical. Phage P008 was first exposed to each sanitizer for 2 and 15 min at room temperature and at two different concentrations, namely the lowest and highest no-rinse sanitizing concentrations. Organic matter (whey or milk) was also added to the testing solutions. At the end of the exposure period, the test solution was neutralized and the number of infectious phages was determined by plaque assays. The five most efficient sanitizers against phage P008 (< 4 log of inactivation) were then tested against virulent lactococcal phages P008, CB13, AF6, P1532 of the 936 group, P001 (c2), Q54, and 1358 as well as Lactobacillus plantarum phage B1 and Streptococcus thermophilus phage 2972 using the same protocol. The oxidizing agents and the quaternary ammonium compounds were the most efficient against all phages although phages CB13 and P1532 were less sensitive to these chemicals than the other phages. This study may help in the selection of appropriate chemicals for controlling phage contamination in industrial factories and research laboratories.
- PublicationRestreintInvolvement 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, ChristianThe 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.
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