Phosphate Sink Containing Two-Component Signaling Systems as Tunable Threshold Devices

Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications.Author Summary: Two-component signaling systems are found in bacteria, fungi and plants. Their modular structures make them ideal targets for de novo engineering through synthetic biology. Here, we explore the signal-response relationship arising from a common two-component system, where a single HK phosphotransfers reversibly to two separate output RRs. We show that under the experimentally observed parameters, this motif implements a sigmoidal signal-response relationship, whereby one of the RRs acts as a phosphate sink towards the other. We identify two mathematical conditions on the system parameters that are necessary for sigmoidality and define key parameters that control threshold levels and sensitivity. We confirm these findings experimentally by in vitro reconstitution of the “one HK-two RR” motif found in S. meliloti. Particularly, we show that the level of sigmoidality in this system can be experimentally controlled by the amount of sink RR and also through an auxiliary protein, CheS. These findings show that the one HK-two RR motif can open the way to the design of novel threshold systems in synthetic biology.