TOWARD A COMPREHENSIVE UNDERSTANDING OF MICROBIAL METABOLISM

Abstract

A comprehensive understanding of metabolism remains challenging. Even in the best understood model microbes, some pathways remain ill-defined. For those best defined pathways, their regulation remains incompletely understood. New tools that allow direct measurement of metabolites and their fluxes by liquid chromatography-mass spectrometry hold the potential to address these limitations. Using a combination of metabolomics, genetics, proteomics, biochemistry and modeling, metabolism and its regulation of model bacterium Escherichia coli and yeast Saccharomyces cerevisiae were investigated.

For example, nucleotide degradation is a universal metabolic capability of any organism. However, the involved pathway was poorly characterized. Herein, a yeast protein not previously associated with nucleotide degradation, Phm8, was found to convert nucleotide monophosphates into nucleosides. A carefully mapping of the downstream steps showed that this pathway eventually salvages carbons into the pentose phosphate pathway. Deletion of Phm8 or downstream steps of this pathway resulted in metabolite depletion and impaired survival of starving yeast.

Glycolysis is the best-studied metabolic pathway and its regulation has been extensively characterized. The fate of the last intermediate of glycolysis, phosphoenolpyruvate (PEP), controls much of cellular metabolism, e.g. the balance of glycolysis and gluconeogenesis. In both E. coli and yeast, removal of glucose results in a paradoxical increase in PEP, which goes up the most of any canonical metabolite. The switch-like inhibition of the PEP consuming enzymes cannot be explained by previously emphasized regulations. In contrast, the heretofore under-appreciated allosteric regulation predominates in both organisms, with PEP consumption activated in an ultrasensitive manner by the upstream glycolytic intermediate, fructose-1,6-bisphosphate. Mutations that eliminate this regulation do not impair growth on steady glucose, but they render microbes defective in gluconeogenesis and in growth in an oscillating glucose environment. Thus, microbial central metabolism is intrinsically programmed with ultrasensitive feed-forward regulation that enables rapid adaptation to changing environmental conditions.