Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis

Abstract

The Escherichia coli lacUV5 promoter is used inefficiently by the major vegetative form of Bacillus subtilis RNA polymerase, despite very close adherence in the -35 and -10 regions to consensus sequences for promoters recognized by this enzyme. To select derivatives of this promoter with increased activity in B. subtilis, the lacUV5 promoter was fused to a promoter-less chloramphenicol acetyltransferase gene and mutagenized by passage through an E. coli mutD5 mutator strain. Derivatives that conferred resistance to chloramphenicol in B. subtilis were isolated. Twenty-three independent isolates each contained single mutations in the 207 bp lac fragment. These mutations, which were in ten different positions, fell in two clusters. One set of mutations, located between positions -18 and -14, resulted in greater homology to a consensus sequence previously noted for this region in B. subtilis vegetative promoters. The remaining mutations were located near the transcription initiation site. The effects of these mutations and additional mutations constructed by oligonucleotide-directed mutagenesis on expression in B. subtilis and E. coli was determined by measurements of chloramphenicol acetyltransferase activities directed by these promoters. While most mutations had little effect on expression in E. coli, the increase in activity in B. subtilis was as much as 28-fold.