You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Phosphate Sink Containing Two-Component Signaling Systems as Tunable Threshold Devices}
Article:
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.
Introduction:
Cells process external cues in order to produce appropriate responses that ensure survival and efficient proliferation. They achieve this goal through a myriad of signaling and gene regulatory networks, which implement specific signal processing capabilities such as switch-like threshold dynamics, logic gates, oscillations, and noise filtering[1]–[8]. Understanding the architecture and response dynamics of these systems is of fundamental value, providing us with a better insight into cell biology and allowing us to engineer de novo biological systems. The field of synthetic biology exploits the understanding and components from natural systems to rationally design synthetic systems that implement specific signaling dynamics. So far, this led to the development of oscillatory systems[9],[10], systems with threshold dynamics[1],[11]–[12] and logic gates[13]–[15]. In most cases, these studies use transcriptional regulation to implement the desired dynamics, while a few studies have explored the possibility of extending synthetic design approaches to signaling networks[16]–[18]. Bacterial systems are particularly attractive for attempting synthetic engineering of signaling networks. Most bacteria and certain eukaryotic microbes and plants utilize the so-called two-component signaling systems for signal transduction[19]–[21]. In their most simple implementation, these systems consist of a histidine protein kinase( HK) and a response regulator( RR). The activity of the HK is controlled in most cases by an environmental stimulus, which controls the rate of autophosphorylation. Once phosphorylated, the HK transfers its phosphoryl group to a cognate RR, which in its phosphorylated form mediates the output of the signaling pathway[21]. The phosphotransfer reaction is at the core of all two-component systems, and regulating its specificity could allow direct control over microbial( and to some extent plant) physiology, as well as creating synthetic signaling systems. Thus, several studies have attempted to decipher the coupling specificity of HK and RR proteins[22]–[26] and have generated chimeric HKs with specified and controllable inputs[27]–[31]. More recently, artificial molecular scaffolds have been used to increase the local concentrations of HK and RR proteins, allowing significant control over the phosphotransfer rate[17]. Generating synthetic systems with specified signal processing capabilities, however, requires a deeper understanding of system properties such as the signal-response relationship they embed. Around the core HK-RR interaction, different two-component systems have diverse architectures, which could underpin specific signal processing capabilities. For example, the commonly observed phosphorelays, where the flow of phosphoryl groups from the HK to the RR is relayed through several proteins, are believed to allow signal integration and specific response properties such as control of noise and ultrasensitivity[32]–[36]. Other architectural features such as presence of a bifunctional or a split HK, formation of specific “dead-end” complexes and also transcriptional feedbacks have been shown to allow ultrasensitivity and even bistability[37]–[39]. Of such different architectural features, one that has not attracted much attention is the “sink” system, where two RRs can compete for the phosphoryl group from a single HK. This architectural motif has been identified in several microbial and plant systems[40]–[44]. In the Sinorhizobium meliloti chemotaxis pathway, the two response regulators CheY1 and CheY2 are phosphorylated by their cognate kinase CheA. Of these, only CheY2 in its phosphorylated form can bind to the flagellar motor and control its rotation[40]. Both CheYs can also perform reverse phosphotransfer, where they return the phosphoryl group to CheA. Given its high phosphorylation rate( from HK), low reverse phosphorylation rate( to HK), and the observation that the S. meliloti chemotaxis system lacks a dedicated phosphatase, it is proposed that CheY1 acts as a sink that accelerates dephosphorylation of CheY2[40]. A similar situation is described in the Rhodobacter sphaeroides and Helicobacter pylori chemotaxis pathways[41],[42] and the yeast osmoregulation pathway[43],[45]. In the latter case, the HK, SLN1 autophosphorylates in response to changes in the membrane structure and phosphorylates two downstream RRs, SSK1 and SKN7. In vitro phosphotransfer studies found similar dynamics as in the S. meliloti chemotaxis pathway with SKN7 displaying significant reverse phosphotransfer to SLN1, while SSK1 showing no such activity[45]. Interestingly, both SSK1 and SKN7 are functionally active in this system, with SSK1 activating the downstream HOG1 MAP kinase cascade[46],[47] and SKN7 acting as a transcription factor for genes involved in various stress related responses[48],[49]. Here, we use mathematical and experimental approaches to identify the full signal processing capabilities of this two-component system. We first develop a generic model of the one HK – two RR motif and perform both analytical and simulation-based analyses. These reveal that this system is capable of both enhancing signal termination time and implementing a threshold signal-response relationship, i. e. the system displays a sigmoidal signal-response relationship in which the steady state levels of the phosphorylated output RR remains low until a threshold level of signal is crossed. We then verify these dynamics experimentally by in vitro re-constitution of the two-component proteins from the chemotaxis pathway of S. meliloti. Using this in vitro setup, we further show that specific properties of the threshold dynamics can be controlled through the concentrations of the core components, as well as through presence of an auxiliary protein that is known to bind the HK in S. meliloti[50]. These findings allow better understanding of the physiological responses mediated by phosphate sink-containing two-component systems in microbes and plants, and will facilitate design of synthetic threshold devices using two-component signalling proteins.
Discussion:
We have analyzed the system dynamics of an architectural motif found in bacterial two-component signalling pathways where a single HK can reversibly phosphorylate two RRs. We have shown that this one HK-two RR motif can accelerate signal termination, i. e. act as a sink, as hypothesized before[40], but more interestingly, allows the system to exhibit a sigmoidal signal-response relationship. This high level of sigmoidality and corresponding high Hill coefficients can be explained by the effect of the sink-RR on the response dynamics. Due to differing phosphotransfer kinetics, as signal levels increase from zero and kinase activity increases, phosphoryl groups are preferentially given to sink-RR rather than the output-RR( Figure S3). This continues until the sink-RR becomes saturated with phosphoryl groups i. e. is completely phosphorylated. At this threshold point, phosphorylation levels of the output-RR rise dramatically with increasing signal, giving the observed sigmoidal response. We have shown that such threshold behavior is observed under experimentally measured parameters from the S. meliloti chemotaxis and yeast osmoregulation pathways. Further, theoretical analyses showed that the presence of a sigmoidal signal-response relationship necessitates two conditions on the system;( i) the sink-RR to be present and( ii) kS>krS, where kS and krS are the forward and reverse phosphotransfer rate constants of the sink-RR respectively. Factors that promote operation of the sink e. g. increasing the kinetic preference of the kinase for the sink-RR over the output-RR, and/or increasing the rate at which the sink can autodephosphorylate all increase this sigmoidality by sharpening the transition at the threshold point( Figure 4). We verified these findings experimentally, showing that the auxiliary protein, CheS in the S. meliloti chemotaxis pathway, can modulate levels of sigmoidality( resulting in Hill coefficients of 7. 6) by sharpening the response threshold. These findings have important implications for understanding bacterial physiology and designing synthetic signaling circuits. In broad terms, the findings of this study will have implications for any two-component signaling circuit where multiple response regulators compete for phosphorylation by a single phosphodonor. This includes the cases where the HK acts as the phosphodonor, as well as the cases where this function is performed by an Hpt domain or protein. These include the majority of bacterial chemotaxis systems( which employ CheY and CheB as response regulators)[40]–[42], fungal osmoregulatory circuits[43] and certain plant signaling systems[44]. Additional examples include the E. coli kinases NarX and NarQ that can both phosphorylate the response regulators, NarL and NarP[56]. Similarly, in Caulobacter crescentus, the kinases DivJ and PleC can each phosphorylate the two response regulators, DivK and PleD[57]. The present study indicates that these systems might be acting as a threshold device, whereby cells commit to a specific outcome only above certain signal thresholds. Alternatively, the threshold behavior could be used for regulating the noise characteristics of the system[35],[58]. It is important to note however, that the one HK – two RR architectural motif is able to display sigmoidal signal-response relationships, but does not preclude hyperbolic relationships. In other words, this motif cannot be taken as proof for threshold behavior but should be taken as indicative and be considered in experimental design when analyzing the response dynamics in associated signaling systems. Synthetic biology has so far concentrated on designing small circuits based on transcriptional regulation. While two-component systems have been recognized as potential candidates for synthetic design, the main efforts have concentrated on engineering chimeric proteins and interaction specificity[15],[17],[27]–[31]. Our findings show that a system dynamics perspective can allow understanding of the signal processing capabilities of natural bacterial signaling pathways and new avenues for reengineering these. Exploiting the single HK- two RR system in the construction of synthetic signaling circuits will require coupling of an appropriate output( e. g. an RR that can act as a transcription factor) to a useful signal that can control HK activity. This could be accomplished through mutational alterations on the signal and output of an existing natural system( such as the one used here), using chimeric proteins, or by artificially engineering phosphate sinks into existing two-component systems. Two-component proteins are highly modular, and evolution seems to have exploited this feature in creating diverse architectures in signaling. Studies such as those provided here should allow us to understand these functionalities and ultimately lead to their application in synthetic biology.
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 .