Construction of a single-copy integration vector and its use to study gene expression in Bacillus licheniformis

Abstract

A versatile system consisting of an integrational vector and a bacitracin (Bt)-producing β-galactosidase (β-Gal)-negative (Lac-) Bacillus licheniformis TLH strain was constructed to quantify promoter activity and to study gene regulation in a single-copy set-up. The vector pTLH utilizes the promoterless Escherichia coli lacZ gene derived from pQF52 and contains the pBR322 origin of replication and a kanamycin-resistance gene for selection in both B. licheniformis and E. coli. The vector also contains an inner part of the first gene of the Bt synthetase (bts) operon which enables its integration into the bts of B. licheniformis by Campbell-type recombination. This recombination event can be easily tested on a Micrococcus flavus lawn where loss of Bt production, i.e. no clearing zone on the lawn, is indicative of the proper integration. The Lac- B. licheniformis TLH strain was developed by elimination of the natural β-Gal activity of B. licheniformis strain ATCC 10716 UM12 using NTG mutagenesis.