Regulation of the put operon in Salmonella typhimurium: characterization of promoter and operator mutations

Abstract

The two genes required for proline utilization by S. typhimurium form a divergent operon. Expression of the put operon is induced by proline and subject to catabolite repression. Genetic evidence suggests that putA protein autogenously represses transcription of the putA and putP genes. In order to establish the molecular mechanism of put operon regulation we isolated regulatory mutations in the put control region. These mutants were selected using two phenotypes: the ability to degrade a toxic proline analogue, dehydroproline, due to overexpression of putA enzyme activity, or overexpression of lacZ from put::Mud operon fusions. The effect of each mutation on transcription in both directions was determined by measuring lacZ expression from putA and putP operon fusions. These regulatory mutations were cis-dominant when the putA protein was provided in trans, and they map in a region between the two genes. The phenotypes of the mutants suggest that the put regulatory region has a single operator site where the putA protein binds to repress transcription in both directions, and the putA and putP promoters overlap.