Expression of the ace operon in Escherichia coli is triggered in response to growth rate-dependent flux-signal of ATP

Abstract

The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA-messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate ($\mu $) diminishes from µmax to ≤ 0.43h-1 ($ \pm 0.02;n4)\ $is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA, acetate kinase; pta, phosphotransacetylase; acs, acetyl CoA synthetase and aceA, ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism.

Keywords: ace operon; acetate-diauxic switch; acetogenic metabolism; flux signals; gluconeogenic metabolism; succinyl CoA synthetase.

Figure 1.

The role of signal metabolites, TF (CRP, and the pleiotropic ‘catabolite repressor activator’ protein (Cra) and allosteric controls (feedback inhibition and feed-forward activation) as well as catabolite inhibition and catabolite repression in the control of carbon flux among various enzymes of central metabolism during growth of E. coli on glucose minimal medium under aerobic conditions at 37°C. Note that catabolite repression of AcCoA-S; reaction 7, during growth of E. coli on glucose as a sole source of carbon is only triggered at a growth rate of 0.45h^-1 (Valgepea et al. 2010). Key: Large grey arrows indicate flux to intermediary metabolism for biosynthesis, while and indicate activation and inactivation, respectively. Enzymes are as follows: 1, Phosphoenolpyruvate (PEP)-glucose phosphotransferase system (PTS); 2. Phosphoglucoisomerase (PGI); 3. Phosphofructokinase (PFK); 4. Pyruvate kinase (PK); 5. Phosphotransacetylase (PTA); 6. Acetate kinase (AK); 7. Acetyl CoA synthetase (AcCoA-S); 8. Citrate synthase (CS); 9. Isocitrate dehydrogenase (ICDH); 10. Isocitrate lyase (ICL); 11. Malate synthase (MS) and 12. Malate dehydrogenase (MD).

Figure 2.

A composite graph highlighting: (A) the impact of growth rate (µ) on flux through SLP for ATP generation during growth of E. coli in continuous cultures on glucose (▴), pyruvate (○) or glycerol (▪) as a sole source of carbon under aerobic conditions at 37°C. (B) The effect of growth rate (µ) on the rate of substrate utilization and flux to acetate excretion during growth of E. coli in continuous cultures on glucose (▴, △), pyruvate (•, ○) or glycerol, (▪, □) minimal medium under aerobic conditions at 37°C.

Figure 3.

A composite graph highlighting: (A) effect of growth substrate and growth rate (µ) on the specific activity of ICL (µmol of glyoxylate -phenylhydrazone min^-1 mg^-1 protein at 27°C) during the growth of E. coli in continuous cultures on acetate, pyruvate or glycerol minimal medium under aerobic conditions at 37°C. Key: Shown in inset. (B) Effect of growth substrate and growth rate on the transcription of aceA-mRNA expressions (northern blots) during growth of E. coli in continuous cultures on acetate, pyruvate or glycerol as a sole source of carbon at 37°C under aerobic conditions. Intensity ratio of aceA-mRNA transcripts of the pyruvate and glycerol phenotype are shown relative to the aceA-mRNA signal shown by the acetate phenotype and are representative of several experiments (SD, 0.06; n4). Key: Shown on the right-hand side ordinate.

Figure 4.

Effect of growth substrate and growth rate on the transcription patterns (northern blots) of mRNA expressions of key gluconeogenic enzymes together with the enzymes involved in the excretion and uptake of acetate during growth of E. coli in continuous cultures on acetate, pyruvate, or glycerol as a sole source of carbon at 37°C under aerobic conditions. Key: As shown within the graph.

Figure 5.

The postulated role for the acetate-diauxic switch response regulators in coordinating the activation of AcCoA-S (reversible acetylation) and the partials inactivation (75%) of ICDH (reversible phosphorylation) to bring about successful adaptation and assimilation of the excreted acetate at the end of growth on acetogenic substrates. Key as follows: 1, NAD⁺-dependent protein deacetylase; 2, protein-lysine acetyltransferase; 3 and 4, reactions catalyzed by ACoA-S; 5 and 6, reversible phosphorylation reactions catalyzed by ICDH kinase/phosphatase; 7, ICDH; 8, ICL; 9, malate synthase; Glx, glyoxylate; OAA, oxaloacetate.