Plasmids and strains used and the associated literature source.

<p>Plasmids and strains used and the associated literature source.</p

A cartoon diagram of the CheA3-CheA4-CheY6 split kinase system.

<p>The diagram is arranged so to highlight the role of free CheA3 acting as a branching point for the two arms that form competing cycles leading to phosphorylation and dephosphorylation of CheY6. Rate constants are shown on the relevant reactions. In the case of reversible reactions, two rate constants are given (<i>k</i>_forward/<i>k</i>_reverse).</p

Effects of varying key parameters of the model and addition of different phosphatases.

<p>The x- and y-axis show the signal (<i>k₅</i>) level and the corresponding steady state CheY6-P level respectively. Each panel shows a signal-response analysis for varying model parameters (A–C) or the inclusion of additional phosphatases (D). The results of the basic model are shown in red. Where present, the dark region indicates the region of unstable steady states and hence the presence of bistability. Arrows on panels A, B and C indicate increasing value of the changed parameter. (<b>A</b>) The on rate (<i>k₁</i>) for CheA3:CheA4 complex formation was varied from basic model value [100(µMs)⁻¹] to 10, 1, and 0.208. (<b>B</b>) Concentration of CheA4 was varied from 30 µM, 40 µM (basic model) and 80 µM. (<b>C</b>) The rate of CheA3 mediated dephosphorylation of CheY6-P (<i>k₁₁</i>) was varied from 1 s⁻¹, 2.5 s⁻¹ (basic model) and 5s⁻¹. (<b>D</b>) The basic model has free CheA3 as the sole phosphatase; the effect of having either CheA3-P or CheA3:CheA4 and CheA3:CheA4:ATP as additional phosphatases is shown. See also Figures S1, S2, S3, S4 for additional sensitivity analyses.</p

Time-course analyses.

<p>The model is simulated with increasing and decreasing signal levels (<i>k₅</i>) in course of time. <i>k₅</i> is increased from 2 to 6 and decreased in similar fashion at indicated time points (top most, left panel), and changes in each species were measured (as indicated on each panel). The dotted line represents the highest signal level, with equal signal steps on each side of it. The noted asymmetry around this line shows the presence of hysteresis in the system. The x- and y-axis represent time and species concentration respectively, where the latter is normalized by the appropriate total protein levels.</p

Literature source and parameter values used in the analysis of the basic model.

<p>Literature source and parameter values used in the analysis of the basic model.</p

Experimental validation for the role of the sink RR in shaping the signal-response curve.

<p>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. (<b>A</b>) 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>B</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.</p

The effect of parameter changes on the “sigmoidality” of the signal-response curve.

<p>The level of sigmoidality, Hill coefficient, is shown as a heat map on each panel. (<b>A</b>) Effect of varying the forward and reverse phosphotransfer rates for the sink RR (CheY1; x-axis; <i>k_S</i> and y-axis; <i>k_rS</i>). (<b>B</b>) Effect of varying the total concentration of the output RR (CheY2; y-axis) and sink RR (CheY1; x-axis). (<b>C</b>) 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). (<b>D</b>) 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).</p

The one HK – two RR motif as seen in the <i>S. meliloti</i> chemotaxis signaling pathway (A) A cartoon diagram of the CheA/CheY1/CheY2 system.

<p>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 <i>k_forward</i> and <i>k_reverse</i>. (<b>B</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 <i>k_a</i> was selected that resulted in ∼90% of the total RR2 being phosphorylated at steady state. At t = 0, <i>k_a</i> 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. (<b>C</b>) Signal-response relationship in the presence (solid line) and absence (dashed line) of sink, RR1 (CheY1). The x- and y-axis show the signal (<i>k_a</i>) and the corresponding steady state level of phosphorylated RR2 (CheY2), respectively.</p

The parameters used for the model of the <i>S. meliloti</i> phosphate sink.

<p>The parameters used for the model of the <i>S. meliloti</i> phosphate sink.</p

Effect of CheS on the signal-response curve.

<p>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 (<i>k_s</i>) and CheY1-P dephosphorylation rate constant (<i>k_hs</i>) were optimized for best fit to the experimental data; <i>k_s</i> = 50 and <i>k_hs</i> = 0.067). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003890#pcbi.1003890.s006" target="_blank">Figure S6</a> for alternative fits to these experimental data where we have individually modelled the effect of CheS altering only <i>k_s</i> or <i>k_hs</i>. Error bars show the standard error of the mean obtained from three independent experiments.</p