A precise deletion of escherichia coli transposable element IS1 from a plasmid during a competent cell transformation in Bacillus subtilis.

Abstract

Sakaguchi and Shishido (6) cloned in Escherichia coil a 4.5-kbp HindIII DNA fragment with the tetracycline-resistance (Tcr) determinant (named tetBS908) from Bacillus subtilis GSY908 chromosome using a shuttle plasmid vector pLS353 (4). In this isolation, a 5.2-kbp HindIII fragment was also obtained, which was an insertion derivative of the 4.5-kbp fragment (1). It carried E. coil transposable element IS1 (8) flanked by 8-bp duplications of the target sequence, AAAATTTG, just upstream from tetBS908 (Figs. l and 2) (1). The plasmid pLS353 with the 5.2-kbp HindIII fragment was named pTBS 1 ( Fig. 1) (1, 6). During a competent cell transformation of B. subtilis, IS1 was found to be deleted from pTBS 1 (see Figs. 1 and 2) with a high frequency.Two types of Tcr transformants were obtained by the competent cell transformation of B, subtilis RM 125 (9), which maintain either the pTBS 1 parent or particular deletion derivative. Nucleotide sequence analysis revealed that the deletion derivative lacks the IS1 element and one of the 8-bp target sequences for IS1 insertion (1) (Fig. 2). No other deletion occurred in the plasmid. We examined whether the IS1 element deleted from pTBS 1 had transposed into host chromosomal DNA. The Tcr transformant was treated with ethidium bromide to obtain plasmidless Tcs derivatives. The chromosomal DNAs were then isolated from the Tcs derivatives, digested with HindIII, and subjected to Southern blot hybridization (7) with a digoxigenin-labeled probe (the 0.6-kbp PvuII-Tth111I fragment of ISI element; part (a) of Fig. 2). No hybridization signal was obtained