E. coli cells growing freely in glucose minimal media were switched to no carbon, acetate, succinate, or glycerol as indicated. After the indicated duration of glucose removal, the metabolome was quantitated by LC-MS. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log transformed. See for absolute concentration changes for key metabolites and for a heat map of all measured metabolites. Note that the decreased ATP/ADP and NADH/NAD+ ratios upon glucose removal thermodynamically drive carbon towards the bottom of glycolysis (i.e. towards PEP) (). G6P, glucose-6-phosphate; F6P. fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; DPG, 1,3-diphosphateglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; OAA, oxaloacetate; αKG, α-ketoglutarate; SucCoA, succinyl-coenzyme A. G6P and F6P, GAP and DHAP, and 3PG and 2PG were not differentiated by employed LC-MS method.

(a, b). Concentrations of unlabeled and U-¹³C-labeled PEP and FBP, showing rapid initial conversion from unlabeled to labeled forms upon the switch to U-¹³C-glucose, and then, upon glucose removal, accumulation of unlabeled PEP and depletion of labeled FBP. Cells were grown on glucose minimal media as in . Glycolytic intermediates were labeled by switching cells to U-¹³C-glucose for 8 min. Thereafter, glucose was removed and replaced with either no carbon (a, b) or acetate (c, d). Green = nonlabeled, red = U-¹³C labeled, blue = sum of nonlabeled and U-¹³C labeled. (c, d) Concentrations of unlabeled and U-¹³C-labeled lower glycolytic intermediates (sum of all measured compounds from FBP to PEP) starting 10 min after glucose removal. Points reflect data (mean ± range of N = 2 biological replicates); lines represent a fit of the data to equations described in Methods. The fitting enabled estimation of the decrease in PEP carboxylase flux relative to glucose-fed cells.

Each small plot shows activity of purified His-tagged PEP carboxylase as a function of FBP concentration. The six small plots differ in terms of the additional PEP carboxylase effectors present, building from FBP alone (left-most) to all known effectors (right-most) as indicated. The concentration of PEP (2 mM), as well as effectors acetyl-CoA (0.63 mM), aspartate (5.2 mM), malate (1.1 mM), and GTP (4.6 mM) were selected to match the physiological concentration in cells switched from glucose to acetate for 10 minutes (; see for other conditions). Reactions were carried out at 30°C and pH 8.5 in the presence of 10 mM bicarbonate and magnesium. In the plots, the x axis represents different FBP concentrations, and the logarithmic y axis represents specific PEP carboxylase activity in enzyme activity Units (μmol of product produced per minute) per mg of enzyme. Experimental data (mean ± range of N = 2) were fit to Hill-equations (lines). V_max/V_min represents observed range of PEP carboxylase activity as a function of FBP concentration, where V_max was measured at 15 mM FBP, matching the physiological concentration in cells growing on glucose, and V_min was measured at 0.45 mM FBP, matching the physiological FBP concentration in cells switched from glucose to acetate for 10 minutes. n_H and K_half represent the Hill-coefficient for FBP and the FBP concentration producing half-maximal activation.

(a). PEP carboxylase mutant R313Q has higher activity than wild type in the absence of FBP and is desensitized to FBP activation. The concentration of PEP and effectors and the y-axis units match the right-most plot in . (b). Expression of PEP carboxylase R313Q ablates the PEP spike upon carbon starvation (see for switch from glucose to acetate). E. coli strains in which endogenous PEP carboxylase has been replaced with inducible expression of wild type enzyme (Δppc/pCA24N-ppc, shown as PPC WT) or the FBP-insensitive R313Q mutant (Δppc/pCA24N-ppcR313Q, shown as PPC R313Q) were switched to no carbon. (c). Growth curves of Δppc/pCA24N-ppc and Δppc/pCA24N-ppcR313Q. Cells were grown in either steady glucose (closed symbols) or alternating between glucose minimal media and no carbon minimal media every 30 min (open symbols). The x axis represents time in hours, and the logarithmic y axis represents optical density (A600). (d, e). Cells were switched to U-¹³C-glucose for 8 minutes, followed by no carbon for 30 minutes, and finally re-addition of unlabeled glucose. Upon glucose re-addition, samples were collected and intracellular metabolites (labeled and unlabeled) quantitated by LC-MS. The x axis represents seconds after glucose re-addition (in logarithmic scale), and the y axis represents absolute intracellular concentration (mean ± range of N = 2 biological replicates); (d) Δppc/pCA24N-ppc, (e) Δppc/pCA24N-ppcR313Q. Throughout (b) to (e), experiments were performed in the presence of 100 μM IPTG.

PEP carboxylase does not productively contribute to growth on acetate or succinate, and thus optimal regulation would eliminate its activity. Persistent activity under these conditions would be expected to deplete PEP and upstream metabolites, to elevate TCA intermediates, and to reduce growth yields. (a) Intracellular metabolite concentrations in Δppc/pCA24N-ppc (PPC WT) and Δppc/pCA24N-ppcR313Q (PPC R313Q) as a function of added IPTG. Data are shown in heat map format, with each line a particular strain and carbon source. Metabolite levels were averaged, normalized to cells without recombinant enzyme expression (IPTG = 0), and the resulting fold changes log transformed. Data for Δppc/pCA24N-ppcR313Q at 200 μM IPTG is missing because the strain would not grow. (b) Growth parameters of Δppc/pCA24N-ppc and Δppc/pCA24N-ppcR313Q growing on succinate (left) and acetate (right). The x axis represents different IPTG concentration in μM, and the y axis represents parameters shown in the plots. Points reflect data (mean ± range of N = 2 biological replicates).