Cyclic AMP-dependent catabolite repression is the dominant control mechanism of metabolic fluxes under glucose limitation in Escherichia coli

Abstract

Although a whole arsenal of mechanisms are potentially involved in metabolic regulation, it is largely uncertain when, under which conditions, and to which extent a particular mechanism actually controls network fluxes and thus cellular physiology. Based on (13)C flux analysis of Escherichia coli mutants, we elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, CreB, CreC, Crp, Cya, Fnr, Hns, Mlc, OmpR, and UspA on aerobic glucose catabolism in glucose-limited chemostat cultures at a growth rate of 0.1 h(-1). The by far most relevant control mechanism was cyclic AMP (cAMP)-dependent catabolite repression as the inducer of the phosphoenolpyruvate (PEP)-glyoxylate cycle and thus low tricarboxylic acid cycle fluxes. While all other mutants and the reference E. coli strain exhibited high glyoxylate shunt and PEP carboxykinase fluxes, and thus high PEP-glyoxylate cycle flux, this cycle was essentially abolished in both the Crp and Cya mutants, which lack the cAMP-cAMP receptor protein complex. Most other mutations were phenotypically silent, and only the Cra and Hns mutants exhibited slightly altered flux distributions through PEP carboxykinase and the tricarboxylic acid cycle, respectively. The Cra effect on PEP carboxykinase was probably the consequence of a specific control mechanism, while the Hns effect appears to be unspecific. For central metabolism, the available data thus suggest that a single transcriptional regulation process exerts the dominant control under a given condition and this control is highly specific for a single pathway or cycle within the network.

FIG. 1.

Central carbon metabolism in E. coli. Arrowheads indicate the assumed reaction reversibility. The insets provide an overview of central metabolic genes that are regulated by the investigated global regulators. Plus and minus signs indicate positive and negative transcriptional regulation, respectively. Only regulated genes in the central metabolism are shown for clarity. PP, pentose phosphate; CoA, coenzyme A; OAA, oxaloacetate; P, phosphate.

FIG. 2.

Biomass yields of selected global regulator mutants in glucose-limited chemostat culture at a dilution rate of 0.1 h⁻¹. Values are averages from two to six independent cultivations, and the error bars represent the standard deviations. The light gray box indicates the standard deviation from six reference strain experiments. Variations in the dilution rate between 0.09 and 0.13 h⁻¹ were corrected by using linear regression on the glucose uptake rate for yield calculations (equation 3 and Table 1 in reference 35).

FIG. 3.

Origin of key metabolic intermediates in E. coli global regulator mutants during continuous, glucose-limited growth. The fraction of serine derived through the EMP pathway and the fraction of pyruvate derived through the ED pathway were obtained from experiments with 50% [1-¹³C]glucose and 50% natural glucose. All other ratios were from experiments with 20% [U-¹³C]glucose and 80% natural glucose. For [U-¹³C]glucose experiments, the bars represent the average of two to four independent cultivations and the error is the standard deviation. For all [1-¹³C]glucose experiments, the bars represent a single experiment and error bars are the experimental error calculated from redundant mass distribution (13, 35). The light gray boxes indicate the range of values obtained from three experiments with the parent strain.

FIG. 4.

Metabolic net fluxes in (from top to bottom) the E. coli reference strain and the Cra, Crp, and Cya mutants (A) and for the reference strain and the Hns and Mlc mutants (B) in glucose-limited continuous cultures at a dilution rate of 0.1 h⁻¹. Flux values are normalized to the specific glucose uptake rate (given in the first box). Values are the mean of two to four independent experiments, and the errors indicate the standard deviation for these independent experiments. Only selected fluxes are shown for clarity. P, phosphate; CoA, coenzyme A.