<p><b>A</b>) Validation of mutant phenotype predictions. The colors show the predicted gene relevance for spleen colonization (red, essential; orange, contributing; blue, non-detectable; see text for definitions). Comparison of model predictions with 738 experimental <i>Salmonella</i> mutant phenotypes revealed 92% prediction accuracy (inner dark colors) but also 61 discrepancies (pale outer colors). Numbers (correct/total number of experimentally validated predictions) are also given. <b>B</b>) Potential reasons for inaccurate phenotype predictions (redu, unrealistic redundancy; biom, incomplete biomass/maintenance issues; part, partially contributing functions; toxic, accumulation of toxic upstream metabolites; gap, missing enzyme; or exp, possibly inaccurate experimental data). For detailed descriptions see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s016" target="_blank">Table S10</a>. <b>C</b>) Detection of enzymes with predicted differential relevance for optimal <i>Salmonella</i> in vivo growth. Enzyme relevance was classified by parsimonious enzyme usage flux-balance analysis (pFBA) (ess, essential enzymes; optima, enzymes predicted to be used for optimal in vivo growth; ELE, enzymatically less efficient enzymes that will increase flux if used; MLE, metabolically less efficient enzymes that will impair growth rate if used; zeroFlux, enzymes that cannot be not used in vivo). Filled bars represent enzymes that were detected by <i>Salmonella</i> ex vivo proteomics, open bars represent enzymes that were not detected. Statistical significance of the relationship between enzyme classes and the proportion of detected proteins was determined using the Chi square trend test. <b>D</b>) Feasibility of predicted reaction rates. For each reaction, the range of flux rates compatible with full <i>Salmonella</i> growth was determined using Flux-Variability Analysis. The circles represent the most economical state with minimal total flux (see text). Predicted reaction rates are compared to corresponding catalytic capacities calculated form experimental enzyme abundance and turnover numbers (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s008" target="_blank">Table S2</a>). The reddish area represents infeasible fluxes. Reactions with substantial infeasible fluxes in the most economic simulated state are labeled (1, formyltetrahydrofolate dehydrogenase; 2, phosphoserine aminotransferase; 3, glycerol dehydrogenase). <b>E</b>) Predicted flux ranges and corresponding catalytic capacities after constraining all reactions to feasible fluxes (except for the three aminoacyl tRNA ligations mentioned in the text). <b>F</b>) Relative flux ranges of the initial unrestrained (straight line) and the enzyme capacity-restrained (dotted line) models. For each reaction, the flux range was divided by the respective flux value in the most economical state. Reactions that carried no flux in the most economical state were not considered. Statistical significance of the difference between both distributions was tested using the Mann-Whitney U test.</p
