Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage phi 105

Abstract

A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this "promoter probe" vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage phi 105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage phi 105 inserted into the temperature-sensitive replicon pE194. A repressor-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the phi 105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against phi 105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage lambda, the phi 105 repressor interacts with an operator sequence mapping very close to its own gene.