Bacterial two-component systems as sensors for synthetic biology applications

Abstract

Two-component systems (TCSs) are a ubiquitous family of signal transduction pathways that enable bacteria to sense and respond to diverse physical, chemical, and biological stimuli outside and inside the cell. Synthetic biologists have begun to repurpose TCSs for applications in optogenetics, materials science, gut microbiome engineering, and soil nutrient biosensing, among others. New engineering methods including genetic refactoring, DNA-binding domain swapping, detection threshold tuning, and phosphorylation cross-talk insulation are being used to increase the reliability of TCS sensor performance and tailor TCS signaling properties to the requirements of specific applications. There is now potential to combine these methods with large-scale gene synthesis and laboratory screening to discover the inputs sensed by many uncharacterized TCSs and develop a large new family of genetically-encoded sensors that respond to an unrivaled breadth of stimuli.

Figure 1.

Bacterial two-component systems and their function. Inputs are shown inside of the rounded box. Sensor histidine kinase (SHK): Black/dark blue. Phosphoryl group: black circle. Response regulator (RR): light blue. P_output: output promoter.

Figure 2.

Refactoring and rewiring TCSs. (A) Evolved TCSs can be computationally identified in bacterial genomes due to the conservation of SHK and RR domain architectures and the fact that the genes encoding interacting SHK-RR pairs often reside adjacent to one another. However, the promoters (dashed bent lines) and RBSs driving the expression of shk and rr genes can be difficult to predict and characterize from sequence information. This problem is exacerbated for output promoters, which may or may not reside adjacent to shk and rr genes. (B) TCSs can be engineered to function more reliably by replacing native promoters and RBSs with well-characterized synthetic versions. Promoters and RBSs of different strengths should be screened to achieve optimal SHK and RR expression levels. If an evolved output promoter is unknown or has undesirable features, it can be replaced by DBD swapping, wherein the native rr gene is replaced by a chimeric rec-DBD gene and the native output promoter is replaced by a well-characterized promoter that responds to the Rec-DBD protein (P_DBD). yfg: your favorite gene.