Construction of a derivative of Tn917 containing an outward-directed promoter and its use in Bacillus subtilis

Abstract

Summary: Engineered variants of the transposon Tn917 have been widely used to obtain insertion mutations and transcriptional fusions in Bacillus subtilis and other Gram-positive bacteria. We have developed a novel Tn917-based methodology useful for isolation and characterization of mutants resulting from gene over-expression. A Tn917 variant was constructed which contains a strong out-facing promoter near one end, able to promote transcription of genes in the vicinity of its insertion target. This transposon, designated Tn917PF1, was tested in model conditions. Three Tn917PF1 mutants of B. subtilis, with phenotypes presumed to result from gene over-expression, were analysed. Their phenotypes were shown to be due to transcription from the transposon promoter. In one mutant the promoter activated a deg gene, probably degQ. The other two contained different insertions decryptifying a B. subtilis gene encoding β-galactosidase.