Entangled adaptive landscapes facilitate the evolution of gene regulation by exaptation

Exaptation, the co-option of existing traits for new functions, is a central process in Darwinian evolution. However, the molecular changes leading to exaptations remain unclear. We investigated the potential of bacterial transcription factor binding sites (TFBSs) to evolve exaptively for the three global E. coli transcription factors (TFs) CRP, Fis, and IHF. Using a massively parallel reporter assay, we mapped three combinatorially complete adaptive landscapes, encompassing all intermediate sequences between three pairs of strong TFBSs for each TF. Our results revealed that these landscapes are smooth and navigable, with a monotonic relationship between mutations and their impact on gene regulation. Starting from a strong TFBS for one of our TFs, Darwinian evolution can create a strong TFBS for another TF through a small number of individually adaptive mutations. Notably, most intermediate genotypes are prone to transcriptional crosstalk – gene regulation mediated by both TFs. Because our landscapes are smooth, Darwinian evolution can also easily create TFBSs that show such crosstalk whenever it is adaptive. We also present evidence of exaptive evolution and crosstalk from an analysis of bacterial genomes. Our study presents the first in vivo evidence that new TFBSs can evolve exaptively through multiple small and adaptive mutational steps. It also highlights the importance of regulatory crosstalk for the diversification of gene regulation.