Fig. 2: Induction of the glycerol pathway activates the transcription factor Cra.

a Growth of the base strain (E. coli ΔglpK with the pBAD glycerol plasmid) at different induction levels (0, 0.1, and 0.5% ara). The base strain was cultured in n = 3 shake flasks and samples for metabolomics and proteomics were collected at the time points indicated by red arrows. Dots are means and lines are standard deviations of the OD₆₀₀ in n = 3 shake flask cultures. b Intracellular concentration of dihydroxyacetone phosphate (DHAP) and fructose-1,6-bisphosphate (FBP). Data are normalized to the 0% culture. Dots are data of n = 3 independent shake flask cultures and bars are the mean. c Fructose-1-phosphate (F1P) and FBP inhibit the activity of the transcription factor Cra. Cra activates the expression of genes encoding gluconeogenic enzymes (e.g., ppsA) and represses those of glycolytic enzymes. d Proteome data showing the relative abundance of proteins in the base strain with 0.1% ara and 0.5% ara. Data are normalized to the base strain with 0% ara. Δcra is the proteome of a cra deletion strain, normalized to the wild-type strain. Dots are means of samples from n = 3 independent shake flask cultures (a). Shown are only protein levels with a relative standard deviation smaller than 20%. Blue dots are enzymes that belong to glycolysis or gluconeogenesis in the iML1515 model. e The glycerol pathway was expressed in the Δcra strain. The Δcra strain was cultured in 96-well plates. Growth was measured in a plate reader at different induction levels of GPD1 (0, 0.1, 0.3, 0.5, 1, and 2% ara). Glycerol in the medium was measured after 24 h. Growth rates were determined by regression analysis between 5 and 10 h. Growth curves and dots show the means of n = 2 plate reader cultures. Source data are provided in the Source Data file.