SummaryThe evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerRmonovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3Ј and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerRmonovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.