Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t.

Abstract

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.