Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio

Abstract

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F₁-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS^- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F₁ subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS^-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F₁-ATPase (strain PB12AGD⁺). The analysis of the physiological and metabolic response of the PTS^- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

Keywords: Escherichia coli PTS−; F0-F1 ATPase synthase; [ATP]/[ADP] ratio; central carbon metabolism; transcriptional response.

Figure 1

Growth curves of the parental E. coli JM101 strain (●) and the PB12 (■) and PB12AGD⁺ (▲) derivatives conducted in bioreactors. These aerobic cultures were performed in M9 supplemented with glucose (2 g/L). All experiments were performed in duplicate, and the reported values represent the mean values of at least four independent experiments. The difference in measured metabolite concentrations among independent experiments was in the range of 1–5%.

Figure 2

Relative transcript levels of genes involved in glucose transport, carbon central metabolism, and fermentation pathways. RT–PCR values of those upregulated genes (1.7-fold or higher) are shown beside the gene’s name in parenthesis: The first value for PB12 and the second for PB12AGD⁺. The relative gene transcription value for JM101 is always equal to 1. Metabolite abbreviations: Glc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; P1,6dP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; 1,3-dGP, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phophoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PGLN, 6-phosphoglucono-δ-lactone; 6PGNT, 6-phophogluconate; KDPGNT, 2-keto-3-deoxy-6-phosphogluconate; RU5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; X5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; AcCoA, acetyl coenzyme A; CoA, coenzyme A; Ac-P, acetyl phosphate; ACE, acetate; LAC, lactate; FOR, formate; CIT, citrate; ICIT, isocitrate; GOx, glyoxylate; α-KG, α -ketoglutarate; SUC-CoA, succinyl-coenzyme A, SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; SA, shikimic acid; DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; cAMP, cyclicAMP. The corresponding names of the genes and the complete RT–qPCR values for all analyzed genes are shown in Table 3.

Figure 3

Relative transcript levels of genes involved in the respiratory chain and oxidative phosphorylation in PB12 and PB12AGD⁺ derivatives. RT-PCR values of those upregulated genes (1.7-fold or higher) are shown beside the gene’s name in parenthesis: The first value for PB12 and the second for PB12AGD⁺. The relative gene transcription value for JM101 is always equal to 1. NADH-I, NADH:quinone oxidoreductase I; FUM, fumarate, SUC, succinate. The corresponding names of the genes and the complete RT-qPCR values for all analyzed genes are shown in Table 3. The reaction mechanisms were adapted from the EcoCyc database (https://ecocyc.org, accessed on 8 January 2024) [4].