Lamothe Geneviève, Lévesque Céline, Bissonnette Frédéric, Cochu Armelle, Vadeboncoeur Christian, Frenette Michel, Duplessis Martin, Tremblay Denise, Moineau Sylvain
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P4.
Appl Environ Microbiol. 2005 Mar;71(3):1237-46. doi: 10.1128/AEM.71.3.1237-1246.2005.
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.
烈性cos型嗜热链球菌噬菌体DT1先前是从一份马苏里拉乳清样品中分离得到的，其完整基因组序列已公开。噬菌体DT1的推定ori的特征是位于orf36和orf37之间非编码区的三个反向重复序列和两个正向重复序列。由于无法确定推定ori及其侧翼基因的复制能力，因此对其赋予噬菌体抗性的能力进行了测试。当ori克隆到高拷贝数质粒上时，它能在牛奶发酵过程中为嗜热链球菌菌株提供针对噬菌体感染的保护。这种保护具有噬菌体特异性且依赖于菌株。然后，针对位于类cro基因（orf29）和ori之间的区域构建了详细的转录图谱。Northern印迹结果表明，该区域的转录在噬菌体感染开始后5分钟启动。对三种嗜热链球菌cos型噬菌体DT1、Sfi19（烈性）和Sfi21（温和性）中cro - ori区域表达的比较分析揭示了转录本数量和大小的显著差异。通过引物延伸分析进一步研究了orf29上游的启动子，并通过氯霉素乙酰转移酶测定法证实了其活性，结果表明噬菌体启动子比嗜热链球菌编码磷酸烯醇丙酮酸：糖磷酸转移酶系统通用蛋白的操纵子的组成型细菌启动子更有效。然而，噬菌体启动子在乳酸乳球菌和大肠杆菌中比pts启动子效率更低。
