What is Our Project?
We are developing modular bacterial regulators by integrating structural biochemistry with bioinformatics approaches. These include structure predictions, molecular dynamics, and evolutionary information-based analyses such as Shannon entropy and direct coupling analysis.
Bacterial LuxR family regulators have been proven to be modular[1], with a typical architecture consisting of a signal receiver domain—such as a two-component system sensor or small molecule binding domain—and a LuxR family DNA binding domain.
Multiple studies have demonstrated that it is possible to swap DNA binding domains of these regulatory proteins under certain conditions, thereby achieving altered DNA binding specificity. This change leads to a modified regulatory output for a given signal. Our goal is to develop and apply metrics to guide the design of these "rewired" bacterial regulators.
Figure 1: 
Graphical Abstract
What are Two-Component Systems?
Two-component systems (TCS) are essential parts of bacterial signal transduction[3], regulating many vital functions. These systems consist of a histidine kinase and a response regulator. The histidine kinase detects specific signals, autophosphorylates, and activates the response regulator. The activated response regulator dimerizes, binds to DNA, and interacts with RNA polymerase to change gene expression. This transmits the signal further, allowing the cell to respond appropriately to the detected signal.
Why is That Interesting?
The most intriguing aspect of TCS is the conserved domain architecture of the response regulators. They are typically divided into a signal-receiving and signal-transmitting domain, separated by a linker. The signal-receiving domain interacts with the histidine kinase, allowing dimerization upon phosphorylation. This enables the DNA-binding domains to also dimerize, binding DNA and altering transcription by interacting with RNA polymerase. The independence of these domains makes them inherently modular[3], which can be exploited to reroute signaling pathways.
What is Our Idea?
Histidine kinases detect a wide variety of signals and transmit these to downstream promoters via the activation of their associated response regulators. Given the modularity of response regulators, it is possible to reroute signals from one sensory pathway to another by swapping the response regulator domains.
For example, an artificially activated sensor can be paired with a biosynthetic pathway, enabling controlled activation. Alternatively, bacterial biosensors can be designed to detect specific environmental factors, reacting as intended. This allows for the rapid construction of synthetic regulatory circuits, reducing the need for promoter engineering and enabling easier signal rerouting.
What Have We Achieved?
We identified the vast potential of TCS recombination, but a key challenge remains. Rewiring TCS components is not always straightforward, as poorly selected fusion points can render the regulators inactive. A recent review found that one of the biggest challenges is selecting optimal fusion points[4] as a significant challenge for rewiring bacterial regulators, necessitating labor-intensive screenings for active points.
Our project aims to model and identify optimal fusion points. The culmination of our research is Cluster Control, an online tool that predicts optimal rewiring points between signal-receiving and signal-transmitting domains. This tool will provide researchers with a convenient way to predict functional fusion points for their chosen domains. Additionally, Cluster Control includes a database and a wiki, which we aim to grow into an authoritative resource for bacterial two-component system domains, their compatibility, and their functions.
Validation of Our Tool
To validate our tool, we tested the rewiring of well-known bacterial regulatory systems. Specifically, we rewired the NarL response regulator from Escherichia coli[2], a nitrate sensor, to the pLux promoter, which is widely used in iGEM projects. Additionally, we incorporated EL222, a light-sensitive signal receiver domain from Erythrobacter litoralis.
References
- Mukherji, R., Zhang, S., Chowdhury, S., & Stallforth, P. (2020). Chimeric LuxR transcription factors rewire natural product regulation. Angewandte Chemie International Edition
- Schmidl, S. R., Ekness, F., Sofjan, K., Daeffler, K. N.-M., Brink, K. R., Landry, B. P., Gerhardt, K. P., Dyulgyarov, N., Sheth, R. U., & Tabor, J. J. (2019). Rewiring bacterial two-component systems by modular DNA-binding domain swapping. Nature Chemical Biology, 15(7), 690–698.
- Zschiedrich, C., Keidel, V, Szurmant, H. (2016). Molecular Mechanisms of Two-Component Signal Transduction. J Mol Biol 428, 3752-3775
- Chan, C.T.Y., Kennedy, V., Kinshuk, S. (2024). A domain swapping strategy to create modular transcriptional regulators for novel topology in genetic network., Biotechnology Advances