In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter

Abstract

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.

FIG. 1.

Physical map of the pTn7SX transposon donor plasmid. This plasmid is derived from the pGPS2.1 donor plasmid from New England BioLabs (catalog no. N7121S) and contains the origin of replication from the transmissible plasmid R6K (which depends on the Π replication initiation protein, the product of the pir gene) and a tetracycline resistance gene. The transposon, flanked by the Tn7 left and right ends (Tn7L and Tn7R, respectively), contains a gram-positive SPEC resistance cassette and the outward-facing xylA promoter (P_xylA).

FIG. 2.

(A) Random insertion of mTn7SX across the B. subtilis chromosome. The insertion sites of 30 transposants were determined by arbitrary PCR and DNA sequencing, and their locations are shown as gray circles. (B) Insertions resulting in fosfomycin resistance. The positions of 10 insertions within the glpTQ genes and 2 upstream of the fosB gene were determined. Each insertion was at a different position within these genes and is indicated by a vertical arrow. (C) Insertions resulting in duramycin resistance. The positions of 10 insertions within the pssA-ybfM-psd operon were determined and are indicated by black arrows. Open arrows represent the open reading frames, and the −10 and −35 promoter regions are shown as gray boxes.