Figure 1.
Schematic Describing the Experimental and Computational Approach for the Analysis of Redundant Pathways in Central Metabolism Involving Genetic, Physiological, Biochemical Methods and In Silico Modeling
Figure 2.
Metabolism of G. sulfurreducens with Respect to Possible Electron and Carbon Donors, and Electron Acceptors
MQ, menaquinone pool.
Figure 3.
Optimal Equivalent Reactions Sets Studied
The sets were identified in the metabolism of G. sulfurreducens using the FVA analysis during acetate oxidation with either fumarate or Fe(III) citrate as the acceptor, (A) pyruvate to acetyl-CoA and (B) succinyl-CoA to succinate; and non-optimal central metabolism alternate pathways studied, (C) the redundant pathways for conversion of malate to oxaloacetate and (D) the pathways for synthesis of phosphoenolpyruvate (PEP) from pyruvate. The energetically favorable pathways selected in the model simulations are enclosed in the red box.
Ack, Acetate kinase; Adk1, Adenylate kinase; Ato, Acetyl CoA transferase; Fdh, Formate dehydrogenase; Me, Malic enzyme; Mdh, Malate dehydrogenase; Pc, Pyruvate carboxylase; Pdh, Pyruvate dehydrogenase; Pfl, Pyruvate formate lyase; Por, Pyruvate oxidoreductase; Ppa, diphosphatase; Ppck, Phosphoenolpyruvate carboxykinase; Ppdk, pyruvate phosphate dikinase; Ppsa, PEP synthase; Pta, Phosphotransacetylase; Sucoas, Succinyl-CoA synthetase.
Table 1.
Bacterial Strains and Plasmids Used in This Study
Figure 4.
Growth Curves for Wild Type Strain
(A) WT, (B) POR1, pyruvate ferredoxin oxidoreductase; (C) PPCK1, phosphoenolpyruvate carboxylase; (D) MDH1, malate dehydrogenase; (E) PTA1, phosphotransacetylase; (F) ATO1, acetyl-CoA transferase 1; (G) ATO2, acetyl-CoA transferase 2; (H) ATO3, acetyl-CoA transferase 1 and 2 mutant strains grown in medium containing fumarate as the electron acceptor, and acetate (•), acetate and hydrogen (), pyruvate (□), pyruvate and hydrogen (▴), acetate and pyruvate (○), or acetate, pyruvate, and hydrogen (▵) as electron donors. Growth was measured at A600 over time. Data are means of triplicates.
Figure 5.
Fe(III) Citrate Reduction of Wild Type Strain
(A) WT; (B) POR1, pyruvate ferredoxin oxidoreductase; (C) PPCK1, phosphoenolpyruvate carboxylase; (D) MDH1, malate dehydrogensae; (E) PTA1, phosphotransacetylase; (F) ATO2, acetyl-CoA transferase 1; (G) ATO2, acetyl-CoA transferase 2; (H) ATO3, acetyl-CoA transferase 1 and 2 mutant strains in Fe(III) citrate medium at 120 h using different electron donors. Log-phase cultures grown using fumarate as electron acceptor, and acetate (WT, PPCK1, ATO1, and ATO2 strains), acetate and hydrogen (MDH1 and ATO3 strains), and acetate and pyruvate (POR1 strain) as electron donors/carbon sources, were used as inoculum (3%). The data are the means for triplicate cultures.
Table 2.
Specific Activities of Different Enzymes Measured in Cultures of the WT and Mutant Strains Grown Using Fumarate as the Electron Acceptor, and the Electron Donors Allowing the Best Growth of the Corresponding Mutant
Table 3.
Comparison of the Experimental Growth Phenotypes with In silico Predictions from Two Cases: Those Utilizing the Model as Published in Mahadevan et al. (2006), and Those Obtained with the Additional Constraints Incorporated, Derived from the Analysis of the Experimental Data (In Vivo/In Silico/In Silico with Revised Constraints)
Figure 6.
Central Metabolism in G. sulfurreducens Showing the Alternative Metabolic Pathways Studied (Dotted/Dashed Lines)
The metabolic pathway in G. sulfurreducens after the refinement of the network based on the comparison of the in silico predictions with the physiological data from the mutant strains is shown. The underlined reactions represent cases for which the maximum allowed flux was constrained to the wild type levels. The boxes around the enzyme names highlight those proteins that were eliminated. The reactions with the “X” are constrained to have zero flux. Abbreviations are explained in Table S4.