CONSTRUCTION OF TN917AC1, A TRANSPOSON USEFUL FOR MUTAGENESIS AND CLONING OF BACILLUS-SUBTILIS GENES

A Tn917 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 Tn917, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the erm-tnpR -tnpA gene cluster of Tn917 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 conc entration of erythromycin. Cells with a Tn917ac1 insertion in the chro mosome were selected on agar containing chloramphenicol after heat tre atment to eliminate the plasmidic form of pD917. DNA fragments adjacen t to Tn917ac1 could be cloned by restriction digestion of the chromoso mal DNA and by transforming the self-ligated restriction fragments int o Escherichia coli. Sequence analysis revealed that Tn987ac1 was integ rated into the chromosome of B. subtilis by transposition in a recE st rain and by transposition or integration of pD917 in a wild-type strai n. Tn917ac1 has been demonstrated to be useful for mutating and clonin g of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.