Independent regulation of two genes in Escherichia coli by tetracyclines and Tet repressor variants

Abstract

We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

FIG. 1.

Chemical structures and designations of tetracyclines used in this study. The chemical structures of tetracycline and the antibiotically inactive 4-ddma-atc are shown.

FIG. 2.

Components of the dual regulation system. The host strain is WH207_λtet50. tetR(BD)-i1.9 [encoding the 4-DDMA-ATC-specific mutant TetR(BD) H64K L131I S135L] is constitutively expressed from plasmid pWH628 and binds tetO located upstream of lacZ on the chromosome. XynB, expressed from pWH628, is regulated via tetO-4C that is bound by TetR(B)-4C. TetR(B)-4C is constitutively expressed from pWH1925-tetR-4C. ori codes for origin of replication, cat codes for chloramphenicol acetyltransferase, bla codes for β-lactamase, xynB codes for β-Xyl, and lacZ codes for β-Gal.

FIG. 3.

Induction analysis of the dual regulation system. (A) Induction of β-Gal expression is shown in the presence of the respective inducers. β-Gal activity in the absence of tetR(BD)-i1.9 (pWH627) was set to 100% and represents about 2,500 Miller units. Open columns, no inducer; gray columns, 0.2 μM TET; black columns, 1.2 μM 4-DDMA-ATC; striped columns, 0.2 μM TET plus 2 μM 4-DDMA-ATC. (B) Induction of XynB expression in the presence of the inducers. XynB activity obtained in the absence of tetR(B)-4C (pWH1925-Δ) was set to 100% and represents about 10 Miller units.