18 O-Informed Metabolomics Reveals Microbial Phosphoryl-Transfer Dynamics and Stress-Induced Changes

Microbial metabolism provides critical ecosystem functions, yet characterizing microbial metabolic activity in environmental systems remain challenging. Since phosphoryl-transfer reactions are central to energy metabolism, biosynthesis, and metabolic regulation, they have been proposed as a proxy for overall metabolic activity. Some phosphoryl-transfer reactions are known to contribute to the oxygen isotope fractionation between phosphate and water, leaving an isotopic signature of metabolic activity. Although these reactions are well characterized at the level of individual enzymes, their dynamics within the phosphoryl-transfer network remain difficult to resolve, limiting their use for assessing metabolic activity in the environment. Here, we present an 18 O stable isotope labeling-based metabolomics workflow to trace phosphoryl-transfer reactions in Escherichia coli . Cells are grown in labeled medium containing ∼10 atom% H 2 18 O under optimal and heat-stressed conditions, and 18 O incorporation into ATP and other phosphate-containing metabolites is quantified from isotopologue distributions. ATP is labeled within one minute of label introduction and approaches the enrichment of intracellular water in stationary phase, indicating ATP turnover as the main entry point of 18 O into the phosphoryl-transfer network. The label rapidly propagates to other nucleotides, sugar phosphates, redox cofactors, and all other measured phosphate-containing metabolites within three hours of label introduction. Normalizing metabolite enrichment to ATP enrichment reveals phosphoryl group turnover that changes with growth phase, indicating net phosphoryl flux through gluconeogenesis into the pentose phosphate pathway (PPP) during exponential growth. Heat stress induces moderate effects on growth and central carbon metabolism, but transiently accelerates label accumulation in the non-oxidative PPP and the turnover of NAD + and NADP + . These results show that 18 O-informed metabolomics provides a systematic view of phosphoryl-transfer dynamics and reveals microbial metabolic adaptation under stress, with implications for tracking microbial stress responses and phosphorus cycling in changing environments.