Nauta A, van Sinderen D, Karsens H, Smit E, Venema G, Kok J
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Mol Microbiol. 1996 Mar;19(6):1331-41. doi: 10.1111/j.1365-2958.1996.tb02477.x.
A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized. It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec. Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences. The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators. Two of these repeats partially overlap the two promoter sequences. The distant third repeat is located within the tec coding sequence. Gel mobility shift assays demonstrated that Rro specifically binds to this sequence. To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed. Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low. Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle. In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion.
温和型乳酸乳球菌噬菌体r1t染色体的一个调控区域已被克隆并进行了表征。它包含两个方向相反的基因rro和tec，rro编码噬菌体阻遏物。这两个基因的转录起始位点已被定位，它们之前都有共有-35和-10启动子序列。该区域包含三个具有内部二重对称的21 bp直接重复序列，它们可能作为操纵子起作用。其中两个重复序列部分重叠两个启动子序列。距离较远的第三个重复序列位于tec编码序列内。凝胶迁移率变动分析表明，Rro特异性结合该序列。为了研究该区域可能的转录调控，构建了一个与tec下游开放阅读框的lacZ翻译融合体。在有利于r1t溶原性生活周期的条件下，β-半乳糖苷酶活性非常低。在促进r1t从溶原性转变为裂解性生活周期的丝裂霉素C浓度下，添加丝裂霉素C可使lacZ融合体的表达诱导70倍。在未诱导的细胞中，启动子活性被Rro抑制，因为rro中的移码突变导致lacZ基因融合体的组成型表达。
