(A–H) Extracellular metabolite levels and uptake/excretion rates. The experimental measurements (dots) were fitted (orange curves) as described. Different colors in the scatter plots represent individual biological replicates. In the case of carbon dioxide production rate, the individual measurements are not shown because there are over 10 000 data points. The regressions curves were derived and divided by the cellular dry weight at each time point to calculate the specific rate of biomass production and the specific rates of uptake/excretion of each metabolite (shown in blue). Shaded areas represent the 95% confidence intervals for each curve and the vertical dashed lines show the time points chosen for the determination of intracellular flux ranges and Gibbs-free energies of reaction. Glucose depletion at t=0 h is indicated with a vertical solid line.

(A–C) Intracellular concentrations of metabolites participating in glycolysis (A), the TCA/glyoxylate cycle (B) and PP pathway (C). The concentrations of each metabolite were scaled appropriately to show the general trend in each pathway. Phosphoenolpyruvate, which presents a different behavior than the general trend, is shown in the inset of (A). The time points chosen for the determination of intracellular flux ranges and Gibbs energies of reactions are depicted with dashed lines. Glucose depletion (t=0 h) is indicated with solid vertical lines. Further information (abbreviations used and the actual values of concentrations for each metabolite) is available in .

(A–E) Graphical representation of the metabolic flux distribution in the central carbon metabolism at the indicated time points. Reaction names are taken from the model used for the flux variability analysis (). The arrow thickness is related to the average of the maximal and minimal value of the flux through each reaction relative to the growth rate at each time point, as follows: dashed lines for fluxes lower than 0.2 mmol/gDW, for values between 0.2 and 20 mmol/gDW the thickness is proportional to the flux and for fluxes higher than 20 mmol/gDW the thickness increments every 5 mmol/gDW (exact values are provided in ). Reactions that are far from equilibrium (i.e., with a Gibbs-free energy of at least 5 kJ/mol in either direction) are indicated with filled boxes, while empty boxes denote reactions that are close to equilibrium (i.e., with a Gibbs-free energy of at most 5 kJ/mol in both directions). The three main events of the adaptation process are indicated with roman numerals: I, decreased glycolytic flux and production of storage compounds; II, onset of the glyoxylate cycle; III, halting of the PP pathway. (F) Flux ranges through the reactions shown in (A–E). The reactions are grouped according to the pathway to which they belong and each group is separated by solid lines. The pathways, from top to bottom, are alternative carbon metabolism, glycolysis/gluconeogenesis, PP pathway, pyruvate metabolism, TCA/glyoxylate cycle and anaplerosis. The relative fluxes were scaled within each group to fit the interval between −1 and 1, taking into account the maximal and minimal values within each pathway. The maximal and minimal values for the reactions labeled as ‘(m+c)' make reference to the net flux through that reaction in the cytoplasm plus the same reaction in the mitochondria, and the fluxes labeled as ‘Storage' represent the amount of glucose residues being consumed (or produced) by trehalose and glycogen synthesis (or hydrolysis).

(A) Heat map showing the changes in protein abundance at different time points. Colors show the degree of upregulation (green) or downregulation (red) in terms of the logarithm to base two of the fold change with respect to the reference state (t=−3.8 h). Brackets indicate clusters that are enriched in enzymes from particular pathways (). (B) Comparison between the expression levels of TCA/glyoxylate cycle enzymes that are localized in the cytoplasm (solid lines) and those that are not (dashed lines). Colors indicate the reactions catalyzed by those enzymes. The shaded areas represent the flux ranges through ICL (a glyoxylate cycle-specific reaction) in yellow and through AKGDam (a TCA cycle-specific reaction) in cyan at different time points. (C) Schematic representation of the reactions in the glyoxylate cycle and its regulation. Reactions in red were found to be far from equilibrium at all time points. The thickness of the green arrows indicates fold change of protein abundances upon glucose exhaustion. (D) −log(P-value) of the most significant transcription factors as revealed by the hypergeometric mean test (over-representation). Relevant transcriptions factors were grouped according to their behavior during the diauxic shift (see ) and the average −log (P-value) of each group was plotted against time. The horizontal dotted line denotes the threshold corresponding to a P-value of 0.01. Glucose exhaustion and the biomass curve are also shown. Roman numerals in (A, B and D) indicate the occurrence of the three main events during the adaptation (see text).

Time-course reconstruction of the different events that lead to the adaptation based on the results presented in this report (regular text and solid arrows) and also supported by studies that were previously published (italics and dashed arrows) (, ; ; ; ; ; ; ; ).