Performance evaluation of COMMGEN.

<p>(<b>a</b>) Evaluation of GSM ability to predict growth phenotypes. Predictive ability of initial GSMs (blue), basic consensus models (red), and automatically created refined consensus model (green) according to the metrics defined in the text. The test data comprised gene knockout data (<i>B</i>. <i>subtilis</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref003" target="_blank">3</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref036" target="_blank">36</a>], <i>P</i>. <i>putida</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref008" target="_blank">8</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref050" target="_blank">50</a>], <i>M</i>. <i>tuberculosis</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref051" target="_blank">51</a>], <i>S</i>. <i>cerevisiae</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref049" target="_blank">49</a>]), biolog data (<i>B</i>. <i>subtilis</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref003" target="_blank">3</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref036" target="_blank">36</a>], <i>P</i>. <i>putida</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref008" target="_blank">8</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref033" target="_blank">33</a>]) and auxotrophies (<i>P</i>. <i>putida</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref050" target="_blank">50</a>]). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.s004" target="_blank">S3 Protocol</a> for details. (<b>b,c</b>) Comparison of manual (yeast consensus model [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref020" target="_blank">20</a>] based on the IGSMs iMM904 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref037" target="_blank">37</a>] and iLL672 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref038" target="_blank">38</a>]) and automatic consensus model generation with namespace matching only, or with COMMGEN. (<b>b</b>) Numbers of common reactions and metabolites for manual curation, name space conversion, and automatically created refined consensus model. (<b>c</b>) Incidences of inconsistent reaction classes identified by COMMGEN.</p

Models used in this study and classification of inconsistencies.

<p>(<b>a</b>) Overview of the used initial GSMs. (<b>b</b>) Instances of identical metabolites with different MnXRef identifiers. (<b>c</b>) Non-identical metabolites that perform identical functions in the network context. (<b>d</b>) Alternative modeling of polymers. (<b>e</b>) Nested and encompassing reactions. (<b>f</b>) Alternative usage of redox pairs. (<b>g</b>) Alternative reactions with consequences for redox metabolism. (<b>h</b>) Partially overlapping reactions differing in phosphate products. (<b>i</b>) Lumped vs. non-lumped representation of a pathway. (<b>j</b>) Invalid transport reaction (IR08663). (<b>k</b>) Alternative transport reactions for putrescine. (<b>l</b>) Alternative transport reactions for glycine. (<b>m</b>) Invalid boundary reaction (R841). Circles represent chemical species, arrows chemical reactions, and grey boxes different compartments. Red nodes indicate instances of identical species within the network context whose alternative names are separated by horizontal lines. Rectangular boxes contain the original reaction names, rounded rectangles their corresponding GPRs, where '&' represents a logical AND, and '|' a logical OR. Edges with filled circles represent reversible reactions. Stoichiometric coefficients unequal to one are indicated at their respective arrows. The shown reactions originate from GSMs of four different organisms: <i>B</i>. <i>subtilis</i> (<b>d</b>), as represented in iYO844 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref003" target="_blank">3</a>] (blue) and iBSu1103 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref036" target="_blank">36</a>] (orange); <i>M</i>. <i>tuberculosis</i> (<b>m</b>), as represented in iNJ661 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref014" target="_blank">14</a>] (blue) and GSMN_TB [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref007" target="_blank">7</a>] (orange);<i>P</i>. <i>putida</i> (<b>b,c,e,f,h,I,j,k</b>), as represented in iJN746 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref033" target="_blank">33</a>] (blue) and iJP962 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref010" target="_blank">10</a>] (orange); and <i>S</i>. <i>cerevisiae</i> (<b>g,l</b>), as represented in iIN800 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref048" target="_blank">48</a>] (blue) and iMM904 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref018" target="_blank">18</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref037" target="_blank">37</a>] (orange) and iND750 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref049" target="_blank">49</a>] (pink).</p

Subnetwork analysis for <i>P</i>. <i>putida</i>.

<p>(<b>a</b>) Example error of ‘naïve’ iGSM merging where the initial <i>P</i>. <i>putida</i> BCM contains a biologically inaccurate carbon dioxide fixation cycle due to incorrect directionalities in the IGSMs. This error is automatically resolved as COMMGEN assigns reaction directionalities opposite to those shown with dashed reaction arrows. (<b>b</b>) Example for a new metabolic function in the consensus model. <i>P</i>. <i>putida</i> can grow on L-quinate as its sole carbon source. Neither of the initial models captures this behavior, whereas the consensus model provides the necessary, complementary reactions.</p

Application of COMMGEN to <i>P</i>. <i>putida</i> GSMs.

<p>(<b>a</b>) Automatic inconsistency identification and reconciliation substantially increases consensus and reduces inconsistencies. Reactions are classified into consensus reactions (green) and unique reactions involving no (blue), a single (orange), or multiple (red) inconsistencies. (<b>b, c</b>) Characteristics of the refined consensus model as in (<b>a</b>) without network-based metabolite matching (<b>b</b>), or after manually addressing the remaining inconsistencies (<b>c</b>). (<b>d</b>) Numbers of reversible (‘+’) and irreversible (‘-‘) reactions in the RCM, grouped by the four possible combinations of reversibilities in the IGSMs. (<b>e</b>) Numbers of active and inactive reactions in the RCM, grouped by being active (‘+’) or inactive (‘-‘) in the IGSMs.</p

COMMGEN framework.

<p>(<b>a,b</b>) Overview of COMMGEN workflow and available methods. The COMMGEN methods are either fully automatic (+), conditionally or optionally automatic (+/-), or they always require manual intervention (-). (<b>c</b>) Performance of the metabolite matching methods if run without manual intervention, leading to ROC-curves of the classification of metabolites as identical or non-identical based on their network context. Lines correspond to different fractions of the network information being randomly discarded: black, 0%; red, 30%; green, 60%; blue, 90%. The shades indicate the standard deviations in the classification. The data presented here was obtained using the <i>Pseudomonas putida</i> GSMs iJP962 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref010" target="_blank">10</a>] and iJN746 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.ref033" target="_blank">33</a>]; analysis results for the other sets of GSMs and additional information can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005085#pcbi.1005085.s006" target="_blank">S5 Protocol</a>.</p

Fluorescent nematodes observed in the flow device.

<p>A) Nematodes floating over the wells while the chamber is filled with liquid. The fluorescent oesophagus in the front side of the nematode is clearly visible. B) Nematode trapped in a well filled with fluorescent <i>E. coli</i> cells after removing the liquid from the chamber. C) Next day: A nematode after consuming all fluorescent bacteria from the well, resulting in observable fluorescence in the nematode intestine.</p

Schematic representation of the flow device.

<p>A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036982#pone.0036982.s001" target="_blank">File S1</a> for more views of the device.</p

Co-cultivation of cells separated by a microsieve.

<p>Increase of GFP expression of inducible cells on the sieve after inoculation of inducer cells below. Graph plotted with the image analysis and processing tool ImageJ. The x-axis corresponds to time and the y-axis shows the detected GFP signal (in arbitrary units). Below: a number of representative images of the microsieve. The time points at which the images were taken are indicated with an asterisk.</p

Overview of the BioBrick parts used.

*<p>co-cultivated with RFP containing BioBrick part as control for leakage.</p

Oscillating GFP expression observed in microdish wells.

<p>Fluorescent <i>E. coli</i> cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.</p