Modulating Fis and IHF binding specificity, crosstalk and regulatory logic through the engineering of complex promoters

Bacterial promoters are usually formed by multiple cis-regulatory elements recognized by a plethora of transcriptional factors (TFs). From those, global regulators are key elements since these TFs are responsible for the regulation of hundreds of genes in the bacterial genome. For instance, Fis and IHF are two global regulators which play a major role in gene expression control in Escherichia coli and usually multiple cis-regulatory elements for these proteins co-occur at target promoters. Here, we investigated the relationship between the architecture of the cis-regulatory elements for Fis and IHF in E. coli. For this, we constructed 42 synthetic promoter variants harboring consensus cis-elements for Fis and IHF at different distances from a core -35/-10 region and in different numbers and combinations. We first demonstrated that although Fis preferentially recognizes its consensus cis-element , it can also recognize, to some extent, the consensus binding site for IHF, and the same was true for IHF, which was also able of recognizing Fis binding sites. However, changing the arrangement of the cis-elements (i.e., the position or the number of sites) can completely abolish unspecific binding of both TFs. More remarkably, we demonstrate that combining cis-elements for both TFs could result in Fis and IHF repressed or activated promoters depending on the final architecture of the promoters in an unpredictable way. Taken together, the data presented here demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications. Importance The understanding of the regulatory complex in bacteria is a key issue in modern microbiology. Here, we constructed synthetic bacterial promoters in order to investigate how binding of transcriptional factors to multiple target sites at the promoters can influence gene expression. Our results demonstrate in a systematic way that the arrangement and number of these cis-regulatory elements are crucial for the final expression dynamics of the target promoters. In particular, we show that TF binding specificity or promiscuity can be modulated using different promoter architectures based on consensus cis-regulatory elements, and that transcriptional repression and activation can also be affected by promoter architecture. These results are relevant both for the understanding of natural systems and for the construction of synthetic circuits for biotechnological applications.