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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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The one HK – two RR motif as seen in the S. meliloti chemotaxis signaling pathway (A) A cartoon diagram of the CheA/CheY1/CheY2 system.The diagram is arranged to highlight the role of CheY1 as a phosphate sink for CheY2. Rate constants are shown on the relevant reactions. In the case of reversible reactions, two rate constants are given as kforward and kreverse. (B) Role of the sink, RR1 (CheY1) in signal termination (i.e. dephosphorylation of RR2 (CheY2)). The x- and y-axis show the time and the corresponding steady state levels of phosphorylated RR2, respectively. A value of ka was selected that resulted in ∼90% of the total RR2 being phosphorylated at steady state. At t = 0, ka was reduced to zero and the progress of the reaction to the new steady state simulated. The solid line represents the presence of the sink, while the dashed line shows the absence of the sink. (C) Signal-response relationship in the presence (solid line) and absence (dashed line) of sink, RR1 (CheY1). The x- and y-axis show the signal (ka) and the corresponding steady state level of phosphorylated RR2 (CheY2), respectively.
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pcbi-1003890-g001: The one HK – two RR motif as seen in the S. meliloti chemotaxis signaling pathway (A) A cartoon diagram of the CheA/CheY1/CheY2 system.The diagram is arranged to highlight the role of CheY1 as a phosphate sink for CheY2. Rate constants are shown on the relevant reactions. In the case of reversible reactions, two rate constants are given as kforward and kreverse. (B) Role of the sink, RR1 (CheY1) in signal termination (i.e. dephosphorylation of RR2 (CheY2)). The x- and y-axis show the time and the corresponding steady state levels of phosphorylated RR2, respectively. A value of ka was selected that resulted in ∼90% of the total RR2 being phosphorylated at steady state. At t = 0, ka was reduced to zero and the progress of the reaction to the new steady state simulated. The solid line represents the presence of the sink, while the dashed line shows the absence of the sink. (C) Signal-response relationship in the presence (solid line) and absence (dashed line) of sink, RR1 (CheY1). The x- and y-axis show the signal (ka) and the corresponding steady state level of phosphorylated RR2 (CheY2), respectively.

Mentions: While the implementation of the phosphate sink motif in diverse two-component systems could differ in the molecular details of the proteins involved and their exact kinetic rates, the sink mechanisms can be formulated as a general architectural motif (Figure 1A and S1A); a two-component system comprising a single HK and two RRs, namely the output-RR and the sink-RR (as referred to, in the rest of the text). We have developed a generic model of this motif and parameterized it using experimental measurements from the reaction kinetics of the S. meliloti chemotaxis and yeast osmoregulation systems (see Methods). To monitor temporal dynamics in the presence of a signal, we simulated two conditions, one with the sink-RR and one without the sink-RR. Using the “controlled comparison” approach [51], we simulated each scenario at a signal level that resulted in 90% phosphorylation of the output-RR at steady state. The signal was then removed and the half-time for the decay of phosphorylated output-RR measured. We found that under the experimentally measured parameters, the presence of the sink-RR decreases the half-time for the output-RR dephosphorylation by more than 2-fold in both S. meliloti and yeast (Figure 1B and S1B). These simulation results are consistent with previous experimental results [40], which led to the sink hypothesis, and show that in the experimentally observed parameter regime, a sink-RR can accelerate the dephosphorylation of the output-RR.


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)

The one HK – two RR motif as seen in the S. meliloti chemotaxis signaling pathway (A) A cartoon diagram of the CheA/CheY1/CheY2 system.The diagram is arranged to highlight the role of CheY1 as a phosphate sink for CheY2. Rate constants are shown on the relevant reactions. In the case of reversible reactions, two rate constants are given as kforward and kreverse. (B) Role of the sink, RR1 (CheY1) in signal termination (i.e. dephosphorylation of RR2 (CheY2)). The x- and y-axis show the time and the corresponding steady state levels of phosphorylated RR2, respectively. A value of ka was selected that resulted in ∼90% of the total RR2 being phosphorylated at steady state. At t = 0, ka was reduced to zero and the progress of the reaction to the new steady state simulated. The solid line represents the presence of the sink, while the dashed line shows the absence of the sink. (C) Signal-response relationship in the presence (solid line) and absence (dashed line) of sink, RR1 (CheY1). The x- and y-axis show the signal (ka) and the corresponding steady state level of phosphorylated RR2 (CheY2), respectively.
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Related In: Results  -  Collection

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pcbi-1003890-g001: The one HK – two RR motif as seen in the S. meliloti chemotaxis signaling pathway (A) A cartoon diagram of the CheA/CheY1/CheY2 system.The diagram is arranged to highlight the role of CheY1 as a phosphate sink for CheY2. Rate constants are shown on the relevant reactions. In the case of reversible reactions, two rate constants are given as kforward and kreverse. (B) Role of the sink, RR1 (CheY1) in signal termination (i.e. dephosphorylation of RR2 (CheY2)). The x- and y-axis show the time and the corresponding steady state levels of phosphorylated RR2, respectively. A value of ka was selected that resulted in ∼90% of the total RR2 being phosphorylated at steady state. At t = 0, ka was reduced to zero and the progress of the reaction to the new steady state simulated. The solid line represents the presence of the sink, while the dashed line shows the absence of the sink. (C) Signal-response relationship in the presence (solid line) and absence (dashed line) of sink, RR1 (CheY1). The x- and y-axis show the signal (ka) and the corresponding steady state level of phosphorylated RR2 (CheY2), respectively.
Mentions: While the implementation of the phosphate sink motif in diverse two-component systems could differ in the molecular details of the proteins involved and their exact kinetic rates, the sink mechanisms can be formulated as a general architectural motif (Figure 1A and S1A); a two-component system comprising a single HK and two RRs, namely the output-RR and the sink-RR (as referred to, in the rest of the text). We have developed a generic model of this motif and parameterized it using experimental measurements from the reaction kinetics of the S. meliloti chemotaxis and yeast osmoregulation systems (see Methods). To monitor temporal dynamics in the presence of a signal, we simulated two conditions, one with the sink-RR and one without the sink-RR. Using the “controlled comparison” approach [51], we simulated each scenario at a signal level that resulted in 90% phosphorylation of the output-RR at steady state. The signal was then removed and the half-time for the decay of phosphorylated output-RR measured. We found that under the experimentally measured parameters, the presence of the sink-RR decreases the half-time for the output-RR dephosphorylation by more than 2-fold in both S. meliloti and yeast (Figure 1B and S1B). These simulation results are consistent with previous experimental results [40], which led to the sink hypothesis, and show that in the experimentally observed parameter regime, a sink-RR can accelerate the dephosphorylation of the output-RR.

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