Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant

Abstract

The construction and characterization of a promoter expression vector, lambda RS205 , is described. lambda RS205 can be used for the in vitro construction of transcriptional (operon) fusions to the lacZ gene of Escherichia coli K-12. The level of beta-galactosidase activity in lysogens of lambda RS205 fusion phages provides a quantitative measure of promoter function under single-copy conditions. The regulation of the Tn10 tetracycline resistance gene ( tetA ) and the Tn10 tet repressor gene (tetR) was examined by inserting DNA fragments that span the tetR- tetA promoter-operator region into lambda RS205 . Levels of beta-galactosidase in tetA -lacZ and tetR-lacZ fusion strains indicate that the tetA and tetR promoters are strong promoters; the tetA promoter is fourfold more active than the tetR promoter. Introduction of tetR+ plasmids into tetA -lacZ and tetR-lacZ fusion strains represses beta-galactosidase synthesis 15- to 60-fold and 6- to 15-fold, respectively. The concentration of tetracycline required to induce half-maximal beta-galactosidase synthesis in these tetR+ tet-lac strains depends on both the tetracycline resistance phenotype and the level of tetR repressor in the fusion strain. However, the induction of beta-galactosidase in isogenic tetA -lacZ and tetR-lacZ strains is coordinate. The data presented here support the current model of Tn10 tet gene organization and regulation and provide quantitative information about the regulation of tetA and tetR in vivo.