Design and development of a novel genetic probe for the analysis of repressor-operator interactions

While the native diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion has allowed initial isolation and characterization of the diphtheria toxin repressor (DtxR), the low level of reporter gene expression has limited the detection and analysis of mutations affecting subtle changes in repressor-operator binding. In order to overcome this difficulty, we have constructed a novel hybrid promoter/operator-lacZ transcriptional fusion in which the "-35" and spacing of the tac promoter was fused to the "-10" and interrupted palindromic sequence of toxO. We show that the hybrid tacPtoxO is regulated by the transition metal ion-dependent DtxR and that lacZ expression is increased approximately 70-fold in the reporter strain Escherichia coli DH5alpha/lambdaRS45-tacPtoxO-lacZ relative to DH5alpha/lambdaRS45-toxPO-lacZ. In addition, we have constructed a transcriptional fusion between tacPtoxO and luc, pJL1. We have used pJL1 to program S30 extracts of E. coli in order to direct in vitro the coupled transcription and translation of luciferase. We demonstrate the utility of this in vitro system in providing a direct functional link between in vivo and in vitro observations with DtxR and mutants of DtxR, which display subtle changes in activity in a manner not previously possible.