Conservation of the metabolomic response to starvation across two divergent microbes

Abstract

We followed 68 cellular metabolites after carbon or nitrogen starvation of Escherichia coli and Saccharomyces cerevisiae, using a filter-culture methodology that allows exponential growth, nondisruptive nutrient removal, and fast quenching of metabolism. Dynamic concentration changes were measured by liquid chromatography-tandem mass spectrometry and viewed in clustered heat-map format. The major metabolic responses anticipated from metabolite-specific experiments in the literature were observed as well as a number of novel responses. When the data were analyzed by singular value decomposition, two dominant characteristic vectors were found, one corresponding to a generic starvation response and another to a nutrient-specific starvation response that is similar in both organisms. Together these captured a remarkable 72% of the metabolite concentration changes in the full data set. The responses described by the generic starvation response vector (42%) included, for example, depletion of most biosynthetic intermediates. The nutrient-specific vector (30%) included key responses such as increased phosphoenolpyruvate signaling glucose deprivation and increased ␣-ketoglutarate signaling ammonia deprivation. Metabolic similarity across organisms extends from the covalent reaction network of metabolism to include many elements of metabolome response to nutrient deprivation as well.Escherichia coli ͉ metabolomics ͉ nitrogen/carbon metabolism ͉ Saccharomyces cerevisiae ͉ starvation response T he pathways of cellular metabolism are close to identical across widely divergent organisms (1). The prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae share essentially the same metabolic network (2) despite radically different compartmentation. The concentrations and fluxes of metabolites depend on the interactions among this conserved network structure, the cellular environment, and species-specific factors, such as the location, activities, and regulation of metabolic enzymes. Except for a few classic examples, e.g., central carbon metabolism in E. coli and yeast (3-5) and nitrogen assimilation in bacteria (6-8) the effect of environmental nutrient perturbations on the cellular metabolome have not been directly measured.We explored exponentially growing E. coli and S. cerevisiae cultures suddenly deprived of their carbon or nitrogen sources. Metabolic composition can change in as little as a few seconds (9, 10). To get accurate snapshots of the metabolome, we devised a method of growing cells directly on filters to avoid time-consuming procedures (e.g., centrifugation or filtration) before quenching of biochemical activity (11). Transfer of a filter from a plate containing growth medium into cold organic solvent quickly quenches metabolism; transfer to a plate with a different medium composition allows a quick change of the nutrient environment (Fig. 1).For measurement of a number of metabolites simultaneously, we used a liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-MS/...

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