Transcriptional control in the EcoRI-F immunity region of Bacillus subtilis phage phi 105. Identification and unusual structure of the operator

Abstract

We present the first evidence, in Bacillus subtilis, for gene regulation through the classical mechanism of repressor-operator interaction. The EcoRI-F immunity region (immF) of lysogenic phage phi 105 contains two promoters, PM and PR, in divergent orientations. PM initiates transcription of the phi 105 repressor (c phi 105) gene, whereas PR most probably signals the onset of the lytic pathway. Fusions between each of these promoters and the cat-86 gene were constructed, and in-vivo promoter activities were determined, in both the presence and absence of the functional c phi 105 product, using S1 nuclease analysis and chloramphenicol acetyl transferase assays. The results showed that transcription from PM is stimulated, whereas PR activity is negatively controlled by the repressor. This differential regulation appears to be mediated by recognition of a 14 base-pair (bp) sequence, 5' GACGGAAATACAAG 3', three identical copies of which are present as direct repeats. Two copies, OR1 and OR2, are located closely together in the non-transcribed region between PM and PR, but do not overlap with the -35 and -10 regions of these promoters. The third copy, OR3, is located some 250 bp downstream from PR, within the coding region (ORF3) of the proximal gene of the PR transcription unit. When a 231 bp restriction fragment containing only OR3 was inserted between a strong constitutive promoter (P138) and the cat-86 gene, the in-vivo expression of chloramphenicol resistance was considerably reduced in the presence, but not in the absence, of phi 105 repressor. This hybrid P138-OR-cat-86 construct was subsequently used to select in vivo for operator-constitutive (Oc) mutations. Of 25 Oc mutants analyzed, all showed base alterations or deletions affecting the 14 bp sequence. We show further that insertion of a chemically synthesized oligonucleotide, containing the 14 bp OR sequence, at a site more than 100 bp downstream from the constitutive P138 is sufficient for transcription to become negatively controlled by phi 105 repressor. In comparison with previously identified Gram-negative bacterial and phage operators, the most unusual aspect of the phi 105 OR sequence appears to be its complete lack of 2-fold rotational symmetry.