Construction of Tn917ac1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes

A Tn977 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes. This transposon, Tn917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn977, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the ermtnpR-tnpA gene cluster of Tn977 and a temperature-sensitive ori of pE194. For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin. Cells with a Tn917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917. DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli. Sequence analysis revealed that Tn917ac1 was integrated into the chromosome of B. subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain. Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.