Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway (original) (raw)

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13 C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli. More than a century ago, Harden reported that the enterobacterium Escherichia coli excretes acetate when growing on excess fermentable sugars 1. This phenomenon has been extensively investigated due to its physiological and applicative importance 2-7. In E. coli, the main, constitutive, pathway of acetate production involves a combination of the phosphate acetyl-transferase (Pta) and acetate kinase (AckA). This way, acetyl-coA is converted into acetyl-phosphate then into acetate which is excreted 7. Another route to form acetate is through oxidative decarboxylation of pyruvate by pyruvate oxidase PoxB 8,9. E. coli is also able to consume acetate as a carbon and energy source to support growth. Acetate can be metabolized by two alternative pathways: the reversible Pta-AckA pathway (a low affinity route with a K M for acetate of 7-10 mM) 2,10 , or the high affinity, irreversible acetyl-coA synthetase, Acs (with a K M for acetate of 200 μ M) 2,11,12. Both pathways lead to the formation of acetyl-CoA (Fig. 1). E. coli cells growing on excess glucose produce acetate but consume it only after the glucose is totally consumed 7. This diauxic behavior is due to the catabolite repression exerted by glucose on acetate utilization. When glucose is in excess, the EIIA component of the phosphoenolpyruvate-carbohydrate phosphotransferase system PTS (the main glucose transport system in E. coli), mostly exists in its unphosphorylated form. This leads to the inhibition of adenylyl cyclase. Therefore cAMP levels are low and the transcriptional activator cAMP receptor protein (CRP), which is needed to transcribe acs, is inactive. The repression of acs expression prevents acetate consumption during the period of growth on glucose. In the absence of glucose, cAMP is produced and binds to CRP, which leads to acs expression and allows cells to consume acetate. Consistent with the control of acs expression, simultaneous consumption of acetate and glucose is observed when catabolite repression is partially impaired 13 or weakened 14-17. In these conditions, acs is expressed and acetyl-CoA synthetase (Acs) is active, enabling acetate consumption to occur. The activity of Acs, in concert with the constitutive activity of Pta and AckA, results in setting up a metabolic cycle (Pta-AckA-Acs cycle) in which the acetate produced from glucose by Pta-AckA can be utilized by Acs 15-17. This cycle leads to the simultaneous production and consumption of acetate. Due to catabolite repression, the simultaneous consumption of glucose and acetate is normally expected not to occur. However, it was recently observed that acetate can be taken up and metabolized during exponential growth of wild-type E. coli K-12 strains on a mixture of glucose and acetate 18. This observation was made in conditions