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 global regulators that play a major role in gene expression control in Escherichia coli, and usually, multiple cis-regulatory elements for these proteins are present 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 analyze 42 synthetic promoter variants harboring consensus cis-elements for Fis and IHF at different distances from the core −35/−10 region and in various 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 to recognize Fis binding sites. However, changing the arrangement of the cis-elements (i.e., the position or number of sites) can completely abolish the non-specific binding of both TFs. More remarkably, we demonstrated 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.

Background: Transcription factors (TFs) are proteins controlling the flow of genetic information by regulating cellular gene expression. A better understanding of TFs in a bacterial community context may open novel revenues for exploring gene regulation in ecosystems where bacteria play a key role. Here we describe PredicTF, a platform supporting the prediction and classification of novel bacterial TF in single species and complex microbial communities. PredicTF is based on a deep learning algorithm. Results: To train PredicTF, we created a TF database (BacTFDB) by manually curating a total of 11,961 TF distributed in 99 TF families. Five model organisms were used to test the performance and the accuracy of PredicTF. PredicTF was able to identify 24–62% of the known TFs with an average precision of 88% in our five model organisms. We demonstrated PredicTF using pure cultures and a complex microbial community. In these demonstrations, we used (meta)genomes for TF prediction and (meta)transcriptomes for determining the expression of putative TFs. Conclusion: PredicTF demonstrated high accuracy in predicting transcription factors in model organisms. We prepared the pipeline to be easily implemented in studies profiling TFs using (meta)genomes and (meta)transcriptomes. PredicTF is an open-source software available at https://github.com/mdsufz/PredicTF.

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.

Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.