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Phosphate sink containing two-component signaling systems as tunable threshold devices.

Amin M, Kothamachu VB, Feliu E, Scharf BE, Porter SL, Soyer OS - PLoS Comput. Biol. (2014)

Bottom Line: Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling.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).

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom; Systems Biology Program, College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, United Kingdom.

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.

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Effect of CheS on the signal-response curve.The x- and y-axis show the ATP level and the corresponding steady state level of phosphorylated CheY2, respectively. The experimentally measured values are shown in circles (absence of CheS) and squares (presence of CheS). The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (absence of CheS) and with a solid line (presence of CheS; where the CheA-P to CheY1 phosphotransfer reaction rate constant (ks) and CheY1-P dephosphorylation rate constant (khs) were optimized for best fit to the experimental data; ks = 50 and khs = 0.067). See Figure S6 for alternative fits to these experimental data where we have individually modelled the effect of CheS altering only ks or khs. Error bars show the standard error of the mean obtained from three independent experiments.
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pcbi-1003890-g004: Effect of CheS on the signal-response curve.The x- and y-axis show the ATP level and the corresponding steady state level of phosphorylated CheY2, respectively. The experimentally measured values are shown in circles (absence of CheS) and squares (presence of CheS). The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (absence of CheS) and with a solid line (presence of CheS; where the CheA-P to CheY1 phosphotransfer reaction rate constant (ks) and CheY1-P dephosphorylation rate constant (khs) were optimized for best fit to the experimental data; ks = 50 and khs = 0.067). See Figure S6 for alternative fits to these experimental data where we have individually modelled the effect of CheS altering only ks or khs. Error bars show the standard error of the mean obtained from three independent experiments.

Mentions: Towards obtaining a better understanding of the role of CheS in the system and quantifying its potential effects on the signal-response curve, we first re-constituted CheS in the in vitro assay along with CheA, CheY1 and CheY2. Running phosphotransfer experiments in the presence or absence of CheS, we found that the presence of CheS in the system resulted in the sharpening of the signal-response curve (Figure 4), with the Hill coefficient increasing from 3.43, in the absence of CheS, to 7.61, in the presence of CheS. This increase in the Hill coefficient is in line with the observed capacity of two-component systems to display high levels of ultrasensitivity [36] and potentially bistability [37]–[39]. In an attempt to recapitulate these experimental findings in our mathematical model, we optimised two parameters: the rate of CheY1-P dephosphorylation (khs) and/or the rate of phosphotransfer between CheA and CheY1 (ks). We found that the experimentally observed sharpening of the signal-response curve by CheS can be best recapitulated by increasing both ks and khs (Figure 4), suggesting that CheS may increase both the rate at which CheA-P donates phosphoryl groups to the sink CheY and the rate at which the sink CheY dephosphorylates. These results suggest that the function of CheS is to sharpen the threshold of the sigmoidal signal-response curve given by the system comprising CheA, CheY1 and CheY2.


Phosphate sink containing two-component signaling systems as tunable threshold devices.

Amin M, Kothamachu VB, Feliu E, Scharf BE, Porter SL, Soyer OS - PLoS Comput. Biol. (2014)

Effect of CheS on the signal-response curve.The x- and y-axis show the ATP level and the corresponding steady state level of phosphorylated CheY2, respectively. The experimentally measured values are shown in circles (absence of CheS) and squares (presence of CheS). The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (absence of CheS) and with a solid line (presence of CheS; where the CheA-P to CheY1 phosphotransfer reaction rate constant (ks) and CheY1-P dephosphorylation rate constant (khs) were optimized for best fit to the experimental data; ks = 50 and khs = 0.067). See Figure S6 for alternative fits to these experimental data where we have individually modelled the effect of CheS altering only ks or khs. Error bars show the standard error of the mean obtained from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214558&req=5

pcbi-1003890-g004: Effect of CheS on the signal-response curve.The x- and y-axis show the ATP level and the corresponding steady state level of phosphorylated CheY2, respectively. The experimentally measured values are shown in circles (absence of CheS) and squares (presence of CheS). The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (absence of CheS) and with a solid line (presence of CheS; where the CheA-P to CheY1 phosphotransfer reaction rate constant (ks) and CheY1-P dephosphorylation rate constant (khs) were optimized for best fit to the experimental data; ks = 50 and khs = 0.067). See Figure S6 for alternative fits to these experimental data where we have individually modelled the effect of CheS altering only ks or khs. Error bars show the standard error of the mean obtained from three independent experiments.
Mentions: Towards obtaining a better understanding of the role of CheS in the system and quantifying its potential effects on the signal-response curve, we first re-constituted CheS in the in vitro assay along with CheA, CheY1 and CheY2. Running phosphotransfer experiments in the presence or absence of CheS, we found that the presence of CheS in the system resulted in the sharpening of the signal-response curve (Figure 4), with the Hill coefficient increasing from 3.43, in the absence of CheS, to 7.61, in the presence of CheS. This increase in the Hill coefficient is in line with the observed capacity of two-component systems to display high levels of ultrasensitivity [36] and potentially bistability [37]–[39]. In an attempt to recapitulate these experimental findings in our mathematical model, we optimised two parameters: the rate of CheY1-P dephosphorylation (khs) and/or the rate of phosphotransfer between CheA and CheY1 (ks). We found that the experimentally observed sharpening of the signal-response curve by CheS can be best recapitulated by increasing both ks and khs (Figure 4), suggesting that CheS may increase both the rate at which CheA-P donates phosphoryl groups to the sink CheY and the rate at which the sink CheY dephosphorylates. These results suggest that the function of CheS is to sharpen the threshold of the sigmoidal signal-response curve given by the system comprising CheA, CheY1 and CheY2.

Bottom Line: Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling.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).

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom; Systems Biology Program, College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus