Figure 1.
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.
Figure 2.
The effect of parameter changes on the “sigmoidality” of the signal-response curve.
The level of sigmoidality, Hill coefficient, is shown as a heat map on each panel. (A) Effect of varying the forward and reverse phosphotransfer rates for the sink RR (CheY1; x-axis; kS and y-axis; krS). (B) Effect of varying the total concentration of the output RR (CheY2; y-axis) and sink RR (CheY1; x-axis). (C) Effect of changing the phosphotransfer rate (ks) from CheA to the sink protein (CheY1) on the signal response curve. Each curve is coloured to match the parameter values indicated by the coloured spots on the heatmap shown in panel (A). (D) Effect of changing the concentration of the sink protein (CheY1) on the signal response curve. Each curve is coloured to match the parameter values indicated the coloured spots on the heatmap shown in panel (B).
Table 1.
The parameters used for the model of the S. meliloti phosphate sink.
Figure 3.
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.
Figure 4.
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.
Table 2.
The strains and plasmids used in this study.