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Personne :
Duplessis, Martin

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Duplessis

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Martin

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Université Laval. Département de biochimie et de microbiologie

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ncf10569337

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Voici les éléments 1 - 5 sur 5
  • PublicationRestreint
    Global gene expression analysis of two Streptococcus thermophilus bacteriophages using DNA microarray
    (Elsevier, 2005-07-25) Moineau, Sylvain; Russell, W. Michael; Duplessis, Martin; Romero, Dennis A.
    A custom microarray was developed to study the temporal gene expression of the two groups of phages infecting the Gram-positive lactic acid bacterium Streptococcus thermophilus. The complete genomic sequence of the virulent cos-type phage DT1 (34,815 bp) and the pac-type phage 2972 (34,704 bp) were used for the construction of the microarray. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32 and 37 min) during phage infection and an expression curve was determined for each gene. Each phage gene was then classified into one of the three traditional transcription classes and these data were used to generate the complete transcriptional map of DT1 and 2972. Phage DT1 possesses 18 early genes, 12 middle genes and 12 late-expressed genes whereas 2972 has 16 early, 11 middle and 14 late genes. The trends of the phage gene expression profiles were also confirmed by slot blot hybridizations. Significant differences were observed when comparing the transcriptional maps of DT1 and 2972 with those already available for the S. thermophilus phages Sfi19 and Sfi21. To our knowledge, this report presents the first complete transcription analysis of bacteriophages infecting Gram-positive bacteria using the DNA microarray technology.
  • PublicationAccès libre
    Characterization of the cro-ori region of the Streptococcus thermophilus virulent bacteriophage DT1
    (American Society for Microbiology, 2005-03-03) Vadeboncoeur, Christian; Tremblay, Denise; Frenette, Michel; Cochu, Armelle; Moineau, Sylvain; Lamothe, Geneviève; Duplessis, Martin; Bissonnette, Frédéric.; Lévesque, Céline
    The virulent cos-type Streptococcus thermophilus phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative ori of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between orf36 and orf37. As the replication ability of the putative ori and flanking genes could not be established, its ability to confer phage resistance was tested. When ori is cloned on a high-copy-number plasmid, it provides protection to S. thermophilus strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the cro-like gene (orf29) and the ori. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the cro-ori region in the three S. thermophilus cos-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of orf29 was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the S. thermophilus operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the pts promoter in Lactococcus lactis and in Escherichia coli.
  • PublicationRestreint
    Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages
    (Wiley-Blackwell, 2001-12-21) Moineau, Sylvain; Duplessis, Martin
    Phage–host interactions remain poorly understood in lactic acid bacteria and essentially in all Grampositive bacteria. The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen-like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria.
  • PublicationRestreint
    Characterization of Streptococcus thermophilus host range phage mutants
    (American Society for Microbiology, 2006-04-05) Moineau, Sylvain; Duplessis, Martin; Lévesque, Céline
    To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10 6. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrixassisted laser desorption ionization–time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.
  • PublicationAccès libre
    Genomic organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing Streptococcus thermophilus strain
    (American Society for Microbiology, 2005-07-06) Tremblay, Denise; Labonté, Jessica.; Labrie, Steve; Moineau, Sylvain; Fremaux, Christophe; Duplessis, Martin; Lévesque, Céline
    The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.