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

Abstract

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.

Regulation of cell reversal frequency in Myxococcus xanthus requires the balanced activity of CheY‐like domains inFrzEandFrzZ

“…FrzZ plays an important role in the regulation of cell reversal frequency because increasing amounts of phospho-FrzZ leads to an increase in cell reversals. In two-component systems of other species, accessory CheY-like regulators were described to act as phosphate sinks, that is, they solely accept phosphoryl-groups from a His-kinase to modulate the phosphorylation rate of the main CheY-effector of the system (Sourjik and Schmitt, 1998;Amin et al, 2014). Does a similar mechanism apply for Frz signaling?…”

What influences selection of native phosphorelay architectures?

“…Initially we compiled a list of physiological variables that are important for the function of these signal transduction circuits 9,10,[42][43][44][45][46][47]12,27,[36][37][38][39][40][41] . We compared the alternative architectures to understand how they differed with respect to each of those variables.…”

Analysis of substrate competition in regulatory network motifs: Stimulus–response curves, thresholds and ultrasensitivity

“…For example, in the chemotaxis network of E. coli the histidine kinase CheA can phosphorylate either of the two regulator proteins CheY and CheB: while CheY-P controls the sense of rotation of the flagellar motor CheB-P mediates feedback regulation at the receptor level (Sourjik, 2004). In a recent study Amin et al (2014) have shown that phosphorylation of two response regulators by a single histidine kinase may lead to thresholds and ultrasensitivity in a similar manner as for receptor-ligand binding and phosphorylation/dephosphorylation cycles. Hence, it seems likely that substrate competition in twocomponent systems can be described by similar equations as those derived in the present study.…”