Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli

Abstract

Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

FIG. 1.

Biochemical reaction network for central carbon metabolism in E. coli. The arrowheads indicate the assumed reaction reversibility. The inset provides an overview of central metabolic genes that are regulated by the global regulators which we investigated. Negative transcriptional regulation and positive transcriptional regulation are indicated by minus signs and plus signs that follow the gene abbreviations, respectively. Only regulated genes are indicated in the network for clarity.

FIG. 2.

Growth rate dependence of the flux ratio for serine originating from glycine. Data for the isogenic mutants, the original mutants, and all reference strains are included. The errors bar for the ratio were estimated from redundant mass distributions (16). The errors bars for the growth rate indicate standard deviations for at least two independent cultivations.

FIG. 3.

Metabolic flux distribution in aerobic (A), anaerobic (B), and anaerobic nitrate-respiring (C) batch cultures of the reference strain (top values) and the ArcA mutant (bottom values) on glucose. For the anaerobic conditions (B and C), the original ArcA mutant and its reference strain were used. For clarity only the lower part of metabolism is shown. Net molar fluxes (expressed in millimoles per gram per hour) were determined by ¹³C-constrained flux analysis from two separate experiments with 100% [1-¹³C]glucose and with a mixture of 20% [U-¹³C]glucose and 80% unlabeled glucose. The standard deviations were less than 0.2 mmol g⁻¹ h⁻¹ (A) and less than 0.3 and 0.5 mmol g⁻¹ h⁻¹ for the reference strain and the ArcA mutant, respectively (C). In panel B, the standard deviations for TCA cycle andacetate formation were 0.1 mmol g⁻¹ h⁻¹. For other fluxes in both the reference strain and the mutant, the standard deviation was 0.7 mmol g⁻¹ h⁻¹. The asterisks indicate ill-determined fluxes with confidence intervals as large as the fluxes themselves. Fluxes to biomass building blocks and extracellular products are enclosed in boxes.

FIG. 4.

Metabolic flux distribution in the Crp mutant (bottom values) and the parent strain (top values) (A) and in the Cya mutant (bottom values) and the parent strain (top values) (B) during exponential aerobic growth on glucose. For clarity only the lower part of metabolism is shown. Net molar fluxes (expressed in millimoles per gram per hour) were determined by ¹³C-constrained flux analysis from two separate experiments with 100% [1-¹³C]glucose and with a mixture of 20% [U-¹³C]glucose and 80% unlabeled glucose. In all cases the standard deviation was less than 0.2 mmol g⁻¹ h⁻¹. Fluxes to biomass building blocks and extracellular products are enclosed in boxes.