Large-scale 13C-flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Abstract

Despite our increasing topological knowledge on regulation networks in model bacteria, it is largely unknown which of the many co-occurring regulatory events actually control metabolic function and the distribution of intracellular fluxes. Here, we unravel condition-dependent transcriptional control of Escherichia coli metabolism by large-scale (13)C-flux analysis in 91 transcriptional regulator mutants on glucose and galactose. In contrast to the canonical respiro-fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the phosphoenol-pyruvate (PEP)-glyoxylate cycle. While 2/3 of the regulators directly or indirectly affected absolute flux rates, the partitioning between different pathways remained largely stable with transcriptional control focusing primarily on the acetyl-CoA branch point. Flux distribution control was achieved by nine transcription factors on glucose, including ArcA, Fur, PdhR, IHF A and IHF B, but was exclusively mediated by the cAMP-dependent Crp regulation of the PEP-glyoxylate cycle flux on galactose. Five further transcription factors affected this flux only indirectly through cAMP and Crp by increasing the galactose uptake rate. Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth.

Figure 1

Absolute metabolic fluxes in E. coli during aerobic growth on glucose (A) or galactose (B). Flux arrows are drawn in proportion to the substrate uptake rates for each condition. The numbers represent absolute flux values (mmol gCDW⁻¹ h⁻¹). One of two replicate experiments is shown (Supplementary Tables 2 and 3). The presented fluxes are from one of two independent experiments and were obtained by ¹³C-constrained flux analysis using the software FiatFlux (Zamboni et al, 2005). They were independently confirmed by flux estimation with a whole isotopologue model (Kleijn et al, 2005; van Winden et al, 2005). Generally, the deviation between the two independent experiments was 1–5%, and whole isotopologue sensitivity analysis through addition of Gaussian noise confirmed accurate estimation for all major fluxes (Supplementary Tables 2 and 3).

Figure 2

Average deviations of absolute fluxes and flux ratios in 91 mutants from the wild type. Deviations in absolute fluxes and flux ratios are %-differences compared with the wild-type values for each condition.

Figure 3

Absolute (A) and substrate uptake normalized (B) flux changes of key metabolic pathways in the 91 mutants compared with the wild type during growth on glucose (Δ) and galactose (□). The dashed line indicates the wild-type reference fluxes.

Figure 4

Influence of individual transcription factors on the flux distribution at key metabolic branch points for growth on glucose (Δ) and galactose (▪). The four branch points are the glucose-6-phosphate node on both hexoses (A), the acetyl-CoA node on glucose (B) and on galactose (C), and the isocitrate node on galactose (D). Values for unlabeled mutants are the average of two independent experiments obtained by ¹³C-constrained flux analysis (Supplementary Tables 2 and 3). For mutants with significantly altered flux distributions (labeled), a third independent experiment was conducted and the error bars represent standard deviations of these three independent experiments. Enzyme control mutants are given in italics. The wild-type values are highlighted by black circles.

Figure 5

Fraction of flux entering the PEP-glyoxylate cycle as a function of hexose uptake rate in batch (A) and chemostat (B) cultures. (A) Batch growth on galactose: the wild-type value is highlighted by a black circle. The PTS^Glc enzyme IIA^glc mutant (crr) is highlighted in red. Values are the average of two independent experiments obtained by ¹³C-constrained flux analysis (Supplementary Tables 2 and 3). For mutants with significantly altered flux distributions (labeled), a third independent experiment was conducted and the error bars represent standard deviations of these three independent experiments. (B) Chemostat growth on glucose and galactose: wild type on glucose (○), NagC mutant on galactose is highlighted in blue (⧫); values from batch experiments are additionally listed ( × ). Nomenclature: CS, chemostat culture; B, batch culture. Two biological replicates were conducted for each condition and error bars represent the deviation between the two experiments.

Figure 6

Intracellular cAMP concentrations relative to the wild type on galactose. Intracellular cAMP levels for mutants grown on batch galactose and the wild type grown on batch glucose relative to intracellular cAMP levels for the wild type grown on galactose. Intracellular cAMP levels were determined from two biological replicates for each strain from two separate shake flask experiments. Error bars represent the deviation between the two experiments.

Figure 7

Growth optimality on glucose (Δ) and galactose (▪). Growth optimality of 81 transcription factors and 10 sigma- and anti-sigma factors compared with the wild type (highlighted by a black circle) on (A) glucose and (B) galactose. Lines indicate equal biomass productivity (g (g hexose h)⁻¹). Error bars represent standard deviations from at least three independent experiments.