Personne : Magadán, Alfonso H.
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Département de biochimie, de microbiologie et de bioinformatique, Faculté des sciences et de génie, Université Laval
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- PublicationRestreintLactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA(Tandfonline, 2018-10-02) Millen, Anne M.; Tremblay, Denise; Moineau, Sylvain; Samson, Julie; Magadán, Alfonso H.; Rousseau, Geneviève M.; Romero, Dennis A.CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcriptiondependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.
- 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.
- PublicationAccès libreThe CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA(Macmillan, 2010-11-03) Garneau, Josiane; Devillers, Rodolphe; Moineau, Sylvain; Romero, Dennis A.; Dupuis, Marie-Ève; Magadán, Alfonso H.; Boyaval, Patrick; Villion, Manuela; Fremaux, Christophe; Horvath, PhilippeBacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
- PublicationAccès librePhage-host interactions in Streptococcus thermophilus : genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array(Nature Publishing Group, 2017-03-06) Achigar, Rodrigo; Tremblay, Denise; Moineau, Sylvain; Magadán, Alfonso H.; Pianzzola, María JuliaThree cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5′-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array. Introductio
- PublicationAccès libreCRISPR-Cas and restriction–modification systems are compatible and increase phage resistance(Nature Publishing Group, 2013-07-02) Moineau, Sylvain; Dupuis, Marie-Ève; Magadán, Alfonso H.; Villion, ManuelaBacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction–modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.
- PublicationAccès libreCleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system(Public Library of Science, 2012-07-20) Moineau, Sylvain; Dupuis, Marie-Ève; Magadán, Alfonso H.; Villion, ManuelaStreptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.
- PublicationAccès libreThe CRISPR-Cas immune system and genetic transfers : reaching an equilibrium(ASM Press, 2015-02-20) Moineau, Sylvain; Samson, Julie; Magadán, Alfonso H.Horizontal gene transfer drives the evolution of bacterial genomes, including the adaptation to changing environmental conditions. Exogenous DNA can enter a bacterial cell through transformation (free DNA or plasmids) or through the transfer of mobile genetic elements by conjugation (plasmids) and transduction (bacteriophages). Favorable genes can be acquired, but undesirable traits can also be inadvertently acquired through these processes. Bacteria have systems, such as clustered regularly interspaced short palindromic repeat CRISPR–associated genes (CRISPR-Cas), that can cleave foreign nucleic acid molecules. In this review, we discuss recent advances in understanding CRISPR-Cas system activity against mobile genetic element transfer through transformation and conjugation. We also highlight how CRISPR-Cas systems influence bacterial evolution and how CRISPR-Cas components affect plasmid replication.
- PublicationRestreintStructure and function of phage p2 ORF34p2, a new type of single-stranded DNA binding protein(Blackwell Scientific Publications, 2009-09-14) Scaltriti, Erika; Tremblay, Denise; Tegoni, Mariella; Masson, Jean-Yves; Rivetti, Claudio; Launay, Hélène; Moineau, Sylvain; Magadán, Alfonso H.; Ramoni, Roberto; Lichière, Julie; Campanacci, Valérie; Cambillau, Christian; Ortiz-Lombardía, MiguelLactococcus lactis, a Gram‐positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single‐stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single‐strand annealing protein. Two‐dimensional gel electrophoresis showed that ORF34p2 is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel‐shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34p2 interacts with single‐strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34p2 and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding‐fold of SSBs. Finally, we found that ORF34p2 is able to stimulate Escherichia coli RecA‐mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34p2 from other SSB proteins are discussed.