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?-Ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition.

ABSTRACT: Microbes survive in a variety of nutrient environments by modulating their intracellular metabolism. Balanced growth requires coordinated uptake of carbon and nitrogen, the primary substrates for biomass production. Yet the mechanisms that balance carbon and nitrogen uptake are poorly understood. We find in Escherichia coli that a sudden increase in nitrogen availability results in an almost immediate increase in glucose uptake. The concentrations of glycolytic intermediates and known regulators, however, remain homeostatic. Instead, we find that ?-ketoglutarate, which accumulates in nitrogen limitation, directly blocks glucose uptake by inhibiting enzyme I, the first step of the sugar-phosphoenolpyruvate phosphotransferase system (PTS). This inhibition enables rapid modulation of glycolytic flux without marked changes in the concentrations of glycolytic intermediates by simultaneously altering import of glucose and consumption of the terminal glycolytic intermediate phosphoenolpyruvate. Quantitative modeling shows that this previously unidentified regulatory connection is, in principle, sufficient to coordinate carbon and nitrogen utilization.

SUBMITTER: Doucette CD

PROVIDER: S-EPMC3218208 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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α-Ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition.

Doucette Christopher D CD Schwab David J DJ Wingreen Ned S NS Rabinowitz Joshua D JD

Nature chemical biology 20111016 12

Microbes survive in a variety of nutrient environments by modulating their intracellular metabolism. Balanced growth requires coordinated uptake of carbon and nitrogen, the primary substrates for biomass production. Yet the mechanisms that balance carbon and nitrogen uptake are poorly understood. We find in Escherichia coli that a sudden increase in nitrogen availability results in an almost immediate increase in glucose uptake. The concentrations of glycolytic intermediates and known regulators ...[more]

PMID: 22002719

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Project description:Fungi employ extracellular enzymes to initiate the degradation of organic macromolecules into smaller units and to acquire the nutrients for their growth. As such, these enzymes represent important functional components in terrestrial ecosystems. While it is well-known that the regulation and efficiency of extracellular enzymes to degrade organic macromolecules and nutrient-acquisition patterns strongly differ between major fungal groups, less is known about variation in enzymatic activity and carbon/nitrogen preference in mycorrhizal fungi. In this research, we investigated variation in extracellular enzyme activities and carbon/nitrogen preferences in orchid mycorrhizal fungi (OMF). Previous research has shown that the mycorrhizal fungi associating with terrestrial orchids often differ from those associating with epiphytic orchids, but whether extracellular enzyme activities and carbon/nitrogen preference differ between growth forms remains largely unknown. To fill this gap, we compared the activities of five extracellular enzymes [cellulase, xylanase, lignin peroxidase, laccase, and superoxide dismutase (SOD)] between fungi isolated from epiphytic and terrestrial orchids. In total, 24 fungal strains belonging to Tulasnellaceae were investigated. Cellulase and xylanase activities were significantly higher in fungi isolated from terrestrial orchids (0.050 ± 0.006 U/ml and 0.531 ± 0.071 U/ml, respectively) than those from epiphytic orchids (0.043 ± 0.003 U/ml and 0.295 ± 0.067 U/ml, respectively), while SOD activity was significantly higher in OMF from epiphytic orchids (5.663 ± 0.164 U/ml) than those from terrestrial orchids (3.780 ± 0.180 U/ml). Carboxymethyl cellulose was more efficiently used by fungi from terrestrial orchids, while starch and arginine were more suitable for fungi from epiphytic orchids. Overall, the results of this study show that extracellular enzyme activities and to a lesser extent carbon/nitrogen preferences differ between fungi isolated from terrestrial and epiphytic orchids and may indicate functional differentiation and ecological adaptation of OMF to local growth conditions.

Project description:?-Ketoglutarate (?KG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae ?KG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (??5) showed that ?KG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of ?KG was further investigated under ?KG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated ?KG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of ?KG. Consumption of ?KG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative ?KG transporter is repressed by glucose. Fully 13C-labeled ?KG was introduced as a tracer during a glucose/?KG co-feeding chemostat to trace ?KG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C ?KG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular ?KG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on ?KG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of ?KG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic ?KG as precursor, both observed sinks should be minimized to increase the putative yields.

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?-Ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition.

Publications

α-Ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition.

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