The highly rugged yet navigable regulatory landscape of the bacterial transcription factor TetR

Transcription factor binding sites ( TFBSs ) are important sources of evolutionary innovations. Understanding how evolution navigates the sequence space of such sites can be achieved by mapping TFBS adaptive landscapes. In such a landscape, an individual location corresponds to a TFBS bound by a transcription factor. The elevation at that location corresponds to the strength of transcriptional regulation conveyed by the sequence. We developed an in vivo massively parallel reporter assay to map the landscape of bacterial TFBSs. We applied this assay to the TetR repressor, for which few TFBSs are known. We quantify the strength of transcriptional repression for 17,765 TFBSs and show that the resulting landscape is highly rugged, with 2,092 peaks. Only a few peaks convey stronger repression than the wild type. Non-additive (epistatic) interactions between mutations are frequent. Despite these hallmarks of ruggedness, most high peaks are evolutionarily accessible. They have large basins of attraction and are reached by around 20% of populations evolving on the landscape. Which high peak is reached during evolution is unpredictable and contingent on the mutational path taken. This first in-depth analysis of a prokaryotic gene regulator reveals a landscape that is navigable but much more rugged than the landscapes of eukaryotic regulators. Significance Understanding how evolution explores the vast space of genotypic possibilities is a fundamental question in evolutionary biology. The mapping of genotypes to quantitative traits (such as phenotypes and fitness) allows us to delineate adaptive landscapes and their topological properties, shedding light on how evolution can navigate such vast spaces. In this study, we focused on mapping a transcription factor binding site (TFBS) landscape to gene expression levels, as changes in gene expression patterns play a crucial role in biological innovation. We developed a massively parallel reporter assay and mapped the first comprehensive in vivo gene regulatory landscape for a bacterial transcriptional regulator, TetR. Surprisingly, this landscape is way more rugged than those observed in eukaryotic regulators. Despite its ruggedness, the landscape remains highly navigable through adaptive evolution. Our study presents the first high-resolution landscape for a bacterial TFBS, offering valuable insights into the evolution of TFBS in vivo. Moreover, it holds promise as a framework for discovering new genetic components for synthetic biological systems. ### Competing Interest Statement The authors have declared no competing interest.