Acetate Metabolism in a pta Mutant of Escherichia coli W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival

Abstract

In order to study the physiological role of acetate metabolism inEscherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,d-lactate, and l-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by thepta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The ptamutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

Characterization of the Acetate-Producing Pathways in Escherichia coli

“…With the ackA-pta pathway deleted, acetyl-CoA accumulates (Vadali et al, 2004), which may inhibit pyruvate dehydrogenase (Kakuda et al, 1994b). Because the poxB pathway is largely inactive in exponential growth, an overflow results at the pyruvate node, thereby inhibiting glucose uptake through the phosphotransferase system (Avison et al, 2001;Chang et al, 1999). During the exponential phase, cultures of CD61 consume glucose slower than cultures of MG1655 ( Figure 3).…”

Enhancement of lactate and succinate formation in adhE or pta-ackA mutants of NADH dehydrogenase-deficient Escherichia coli

“…The effect of ndh is stronger in the log phase comparison, and this effect may be due to the activation of ndh in log phase by Fis (Jackson et al 2004). Strains with mutations in the genes of the pta-ack pathway have been studied for their application in cultures where acetate reduction is desired (Kakuda et al 1994;Chang et al 1999;Yang et al 1999a). Introduction of the pta-ackA mutation into ndh, nuoA, or the ndh nuoA strains produced strains that yielded higher production of lactate and succinate in log phase cultures.…”

A third mode of surface‐associated growth: immobilization of Salmonella enterica serovar Typhimurium modulates the RpoS‐directed transcriptional programme

“…S2H). Salmonella did not catabolize tryptophan, glutamate, glycine or alanine under the growth conditions used in this study (static, 25°C) (Tables S1 and S2), whereas E. coli cells do utilize these amino acids during growth at 37°C (Prüß et al, 1994;Prüß and Matsumura, 1996;Chang et al, 1999;Sezonov et al, 2007).…”