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Characterization of the cro-ori Region of the Streptococcus thermophilus Virulent Bacteriophage DT1

Geneviève Lamothe,^1,2, Céline Lévesque,^1,2, Frédéric Bissonnette,^1,2, Armelle Cochu,^1,2 Christian Vadeboncoeur,^1,2 Michel Frenette,^1,2 Martin Duplessis,^1,2 Denise Tremblay,^1,3 and Sylvain Moineau^1,2,3*

Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire,¹ Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie,² Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Québec City, Québec, Canada³

Received 17 December 2003/ Accepted 4 October 2004

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.

Present address: Centre de Recherche Fernand-Seguin, Hôpital Louis-H. Lafontaine, Montréal, Québec, Canada.

Present address: Oral Microbiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.

Present address: Health Canada, Antimicrobial and Fungicides Section, Ottawa, Ontario, Canada.

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