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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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Experimental validation for the role of the sink RR in shaping the signal-response curve.The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32P-ATP concentrations. (A) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ-32P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. (B) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.
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pcbi-1003890-g003: Experimental validation for the role of the sink RR in shaping the signal-response curve.The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32P-ATP concentrations. (A) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ-32P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. (B) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.

Mentions: To test the model findings experimentally, we re-constituted in vitro the CheA, CheY1 (sink-RR) and CheY2 (output-RR) proteins from S. meliloti. In vivo, CheA kinase activity is controlled by interaction with the signaling domain of chemoreceptor proteins [55]. Since it is experimentally difficult to re-constitute chemoreceptors in the in vitro system, we varied the kinase activity of CheA by varying the concentration of its substrate, ATP, as a proxy for the in vivo signal. This allowed us to monitor the steady state levels of phosphorylated CheY1 and CheY2 at different levels of kinase activity, i.e. to derive an experimental signal-response curve. We found excellent quantitative agreement between the signal-response curves resulting from the model and experiments. In the presence (absence) of CheY1, the steady state levels of phosphorylated CheY2 displayed a sigmoidal (hyperbolic) relation with increasing ATP levels (Figure 3). Thus, these experiments strongly suggest that the S. meliloti one HK – two RR motif displays a sigmoidal signal-response relationship in vivo and could potentially function as a threshold device.


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)

Experimental validation for the role of the sink RR in shaping the signal-response curve.The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32P-ATP concentrations. (A) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ-32P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. (B) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003890-g003: Experimental validation for the role of the sink RR in shaping the signal-response curve.The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32P-ATP concentrations. (A) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ-32P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. (B) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.
Mentions: To test the model findings experimentally, we re-constituted in vitro the CheA, CheY1 (sink-RR) and CheY2 (output-RR) proteins from S. meliloti. In vivo, CheA kinase activity is controlled by interaction with the signaling domain of chemoreceptor proteins [55]. Since it is experimentally difficult to re-constitute chemoreceptors in the in vitro system, we varied the kinase activity of CheA by varying the concentration of its substrate, ATP, as a proxy for the in vivo signal. This allowed us to monitor the steady state levels of phosphorylated CheY1 and CheY2 at different levels of kinase activity, i.e. to derive an experimental signal-response curve. We found excellent quantitative agreement between the signal-response curves resulting from the model and experiments. In the presence (absence) of CheY1, the steady state levels of phosphorylated CheY2 displayed a sigmoidal (hyperbolic) relation with increasing ATP levels (Figure 3). Thus, these experiments strongly suggest that the S. meliloti one HK – two RR motif displays a sigmoidal signal-response relationship in vivo and could potentially function as a threshold device.

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