Functioning of a metabolic flux sensor in Escherichia coli

Abstract

Regulation of metabolic operation in response to extracellular cues is crucial for cells' survival. Next to the canonical nutrient sensors, which measure the concentration of nutrients, recently intracellular "metabolic flux" was proposed as a novel impetus for metabolic regulation. According to this concept, cells would have molecular systems ("flux sensors") in place that regulate metabolism as a function of the actually occurring metabolic fluxes. Although this resembles an appealing concept, we have not had any experimental evidence for the existence of flux sensors and also we have not known how these flux sensors would work in detail. Here, we show experimental evidence that supports the hypothesis that Escherichia coli is indeed able to measure its glycolytic flux and uses this signal for metabolic regulation. Combining experiment and theory, we show how this flux-sensing function could emerge from an aggregate of several molecular mechanisms: First, the system of reactions of lower glycolysis and the feedforward activation of fructose-1,6-bisphosphate on pyruvate kinase translate flux information into the concentration of the metabolite fructose-1,6-bisphosphate. The interaction of this "flux-signaling metabolite" with the transcription factor Cra then leads to flux-dependent regulation. By responding to glycolytic flux, rather than to the concentration of individual carbon sources, the cell may minimize sensing and regulatory expenses. R egulation of metabolic operation is crucial for cells' survival. The canonical view is that this regulation occurs in response to extracellular cues, where, for instance, nutrient-specific transmembrane or intracellular receptors sense the presence of a nutrient and transfer respective commands to the regulatory machinery (1-5).Recently, however, a novel impetus for metabolic regulation was proposed: cells could regulate their metabolism as a function of the actually occurring intracellular metabolic fluxes (6, 7). According to this concept, changes in extracellular nutrient abundances would first-in a rather passive manner-result in changes in intracellular metabolic fluxes. In a second instance, the metabolic fluxes would be sensed by molecular systems ("flux sensors"), which in turn would transmit the sensed "flux signal" to the regulatory machinery that consequently would adjust metabolic operation (6). On the basis of a detailed mathematical model of Escherichia coli's central metabolism and its regulation, Kotte et al. suggested that this organism would have such flux sensors in place that establish a correlation between a metabolic flux and the concentration of certain so-called flux-signaling metabolites, which in turn affect the activity of transcription factors and thus would allow for transcriptional regulation in a flux-dependent manner.Using intracellular flux to regulate metabolism is an appealing concept as it would omit the need for nutrient-specific sensors for many different nutrients, and would allow the integration of multiple nutrient inputs directl...

Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

“…Lastly, the mechanism of fitness enhancement of the crr IS element mutation identified here requires further study. One possibility is that the reduced glycolytic flux in Δpgi could be sensed (46) by the PTS system-for example, via perturbation of the PEP/Pyr concentration ratio-and result in feedback inhibition of glucose uptake. Another is that the accumulated G6P (23) could induce a maladaptive regulatory response via CRP activation by P ∼ EIIB Glc .…”

Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

“…The amino acid mutations are located in the binding cavity of the effector. This finding suggests that the mutation site might affect the target gene expression by affecting the affinity of Cra for the effector FBP34. As shown in Fig.…”

Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB) In Vivo Justified by Intracellular Amino Sugar Metabolite Concentrations

“…The main exception was the concentration of fructose-1,6-bis-phosphate (FBP), which was slightly lower on GlcNAc than on glucose and 2-to 3-fold lower on GlcN. FBP has been described as a metabolic flux sensor (4) and so its lower concentration reflects the lower growth rate on GlcN, compared to glucose or GlcNAc (Fig. 6A).…”

“…Allosteric activation by the substrate of the previous reaction constitutes part of a feed-forward loop. In the case of glycolysis, the function of a feed-forward loop is to sense flux through the pathway (4), and feed-forward regulation in general speeds up the response time to extrinsic signals (5). Several of the enzymes of glycolysis and gluconeogenesis have been shown to respond allosterically to changes in concentrations of the metabolites of glycolysis and thus allow a rapid response to changes in metabolic flux (6,7).…”