PTS network integrates other metabolic signals.

<p>(A) Measurement of the FRET response for cells expressing EIIA^Glc-CFP and MglA-YFP and stimulated with 100 μM of indicated non-PTS compounds. (B, C) Dose-response measurements for cells expressing EIIA^Glc-CFP and MglA-YFP (PTS) or CheZ-CFP and CheY-YFP (chemotaxis) pairs and stimulated by stepwise addition and subsequent removal of varying concentrations of glycerol (B) or pyruvate (C). FRET amplitude was normalized to the response at saturating stimulation. Data were fitted using a Hill equation (lines). Error bars indicate standard error of the mean of three independent experiments. The underlying data for Fig 4B and 4C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000074#pbio.2000074.s016" target="_blank">S1 Data</a>.</p

Effects of alanine substitutions on adaptation kinetics.

<p>Adaptation kinetics was measured upon stepwise addition (red arrow) and subsequent removal (blue arrow) of 10 μM MeAsp for Tar^EEEE (a), Tar^AEEE (b), Tar^EAEE (c), Tar^EEAE (d) and Tar^EEEA (e) expressed in VS181 (CheRB⁺) background.</p

Dynamic range measurement.

<p>(a) Example of the FRET measurement for Tar^AAEE expressed in VS181 (CheRB⁺) background. Cells were stimulated with sequentially increasing amounts of MeAsp in threefold steps, allowing the activity to adapt between the steps. The response to a saturating stimulus was tested before and after the experiment as a control. (b) Response amplitudes as a function of concentration for VS181 (CheRB⁺) strain carrying indicated receptors.</p

Interactions of EIIA^Glc with non-PTS transporters.

<p>(A) Summary of the observed interactions between EIIA^Glc and non-PTS transporters. (B, C) Measurement of the FRET response to 100 μM of indicated PTS and non-PTS sugars for cells expressing EIIA^Glc-CFP together with GalP-YFP (B) or MglA-YFP (C).</p

Chemotaxis of alanine-substituted Tar mutants on soft-agar gradient plates.

<p>(a) Gradient plate measurement. Gradient of MeAsp was established by applying 0.1 M solution in the middle of the minimal media plate (red dashed line), and allowing the chemical to diffuse. Receptorless UU1250 cells expressing indicated receptors were inoculated at either 1 or 3 cm from the line, and allowed to grow and chemotaxis. Tar^EEEE and Tar^AAAA were used as positive and negative controls, respectively. Greater colony extension towards the attractant reflects the chemotactic ability of the strain. The chemotactic bias is defined as the ratio of the up-gradient (+) to the down-gradient (-) extension. (b) The chemotactic bias for all receptors and inoculation positions.</p

Sugar Influx Sensing by the Phosphotransferase System of <i>Escherichia coli</i>

<div><p>The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in <i>Escherichia coli</i> and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in <i>E</i>. <i>coli</i>, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy.</p></div

Effects of alanine substitutions on the amplitude and sensitivity of Tar-mediated response.

<p>Responses were measured in CheRB+ (VS181) or CheRB^- (VH1) receptorless strains expressing Tar from inducible plasmid as a sole receptor. (a) Example of YFP/CFP ratio (proxy for the receptor activity) for a set of step stimulations with increasing concentrations of MeAsp in CheRB⁺ cells expressing Tar^EEAE. Addition (removal) of attractant is indicated with red (blue) arrows. The amplitude of the response increases until saturation. (b) Dose dependence of the relative kinase activity computed from (a) and fitted using a Hill equation (line) to obtain the value of EC₅₀, (MeAsp concentration at the half-maximal response). The change in YFP/CFP ratio (Δ<i>r</i>) was normalized to the maximum change in order to evaluate the relative kinase activity as 1 − Δ<i>r/</i>Δ<i>r</i>_max (see text for details). (c,d) Maximal amplitude of the response to an addition of attractant for all modified receptors in CheRB^- (c) or CheRB+ (d). (e,f) EC₅₀ computed as in (b) for all modified receptors in CheRB^- (e) or CheRB+ (f). Asterisks in (e) indicate Tar constructs for which the EC₅₀ could not be defined because of zero response (c). Vertical red lines in (c-f) separate groups of receptors with different number of methylation sites.</p

Behavior of cells with anisotropic tumbling model.

<p>(A) Distribution of cellular orientations prior to tumbles. The tumbling events are divided into 3 groups, by the number of CW-rotating motors involved in a tumble. The rose histograms are normalized by the number of counts. The inner black circle shows unbiased (isotropic) distribution as a reference. Cell orientation is given relative to the gradient. The gradient steepness is N1. (B) Average tumbling angle as a function of orientation along the gradient prior to tumbles. (C) Chemotactic drift velocity of cells in gradients of different steepness. Bars show the drift velocities of cells with 3 motors (left group) or 5 motors (right group) in the medium without a gradient (gray), in gradient N0 (blue), N1 (green) and N2 (red). Left bars show the isotropic model, right (hatched) bars – anisotropic model of tumbling. In the absence of gradient, the difference is within the error of estimation. Standard error of the mean is about 0.03. Cells in (A) and (B) have 3 motors, other parameters are as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000717#pcbi-1000717-t002" target="_blank">Tab. 2</a>. The number of simulated cells is in each case.</p

Parameters used in <i>E. coli</i> model.

<p>Parameters used in <i>E. coli</i> model.</p

Correlation between default uptake and metabolic efficiency of a carbon source.

<p>(A) The averaged relative response of EIIA^Glc–MglA, EIIAB^Man–EIIC^Man and EI–EIICBA^Nag FRET pairs to saturating concentration of indicated metabolites, normalized to the glucose response and plotted against the growth rate of exponential <i>E</i>. <i>coli</i> culture in minimal medium supplemented with the respective metabolite. Solid line indicates linear fit to the data, with R² = 0.83, <i>p</i> = 0.004. The underlying data for Fig 5' A can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000074#pbio.2000074.s016" target="_blank">S1 Data</a>. (B) Simulations of the growth rates at various basal uptake rates, computed for five different values of metabolic efficiency of a carbon source (marked by different colors). Note that the color code is not related to (A). Simulations were performed using the mathematical model described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000074#pbio.2000074.s015" target="_blank">S1 Text</a>. Inset shows the maximal growth rate as a function of the optimal basal uptake rate for different metabolic efficiency of the carbon source, with the same color code as in the main panel. (C) Correlation between the relative metabolic efficiency of the carbon source and its optimal basal uptake rate for simulated growth on two carbon sources.</p