Metabolic profiling of Escherichia coli under deuterium oxide-induced stress

Introduction Some bacteria tolerate deuterium oxide (D 2 O) and are able to grow efficiently, while others have slower growth phenotypes. Thus, for some bacteria D 2 O perturbs hydrogen-dependent enzymatic processes through kinetic isotope effects, yet the resulting metabolic adaptations in bacteria are not fully understood. In particular, the biochemical factors underlying strain-specific tolerance to D 2 O remain unclear. Objectives This study aimed to characterise the global metabolic responses of D 2 O-susceptible and D 2 O-tolerant Escherichia coli isolates, and to identify pathways and metabolites associated with isotopic stress tolerance. Methods Five uropathogenic E. coli isolates and the reference strain E. coli K-12 MG1655, were grown in minimal medium supplemented with glucose and casamino acids containing 0%, 40%, and 80% D 2 O. Growth profiles were monitored, and intracellular metabolites were extracted at strain- and condition-specific time points corresponding approximately to the midpoint of the maximum observed OD₆₀₀ . Untargeted LC-MS analysis was performed in both positive (ESI+) and negative (ESI-) ionisation modes, and data were processed using established metabolomics workflows. Statistical analyses included PCA, PC-DFA, two-way ANOVA (FDR < 0.05), fold-change assessment, and pathway enrichment analysis. Results Growth profiling showed strain-dependent and progressively greater inhibition with increasing D 2 O concentration, with stronger effects in the susceptible isolates. Metabolomic analysis identified 44 significantly altered metabolites across nine metabolic pathways, including central carbon metabolism, amino acid degradation, nucleotide metabolism, and redox processes. Susceptible strains showed pronounced changes in metabolites associated with glutathione and polyamine metabolism, nicotinate and nicotinamide metabolism, nucleotide metabolism and central-carbon-associated processes. In contrast, tolerant strains generally showed more moderate abundance changes in several of these metabolite groups. Additional responses in susceptible isolates included changes in amino acid-, dipeptide- and polyamine-associated metabolites, whereas tolerant isolates generally showed more moderate responses. Conclusions D 2 O exposure was associated with significant abundance changes in metabolites involved in central carbon-associated, amino acid, nucleotide, nicotinate and nicotinamide, glutathione and polyamine metabolism in E. coli . The metabolic trajectories of susceptible versus tolerant isolates diverge significantly, revealing strain-specific adaptive strategies. These findings provide a biochemical basis for isotopic stress tolerance and enhance our understanding of how bacteria remodel metabolism under heavy-water exposure.