Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4

Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the ¹⁸O/¹⁶O ratio of PO₄ in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30–40% of O in PO₄ comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.Metabolic water, more precisely defined as an isotopically distinct flux of O (and H) produced during metabolism (1), has been studied extensively as an alternative water source contributing to body water in animals, such as desert mammals, insects, and migrating birds (2–6), but does not easily lend itself to direct measurement. In recent years, interest in metabolic water has been extended to its oxygen isotopic composition (¹⁸O/¹⁶O ratio or δ¹⁸O value) and contribution to body water because this information is crucial to the interpretation of biomineral e.g., carbonate—CaCO₃—and phosphate—Ca₃(PO₄)₂] oxygen isotopic compositions used heavily in paleoclimate/paleohydrological research (7–14). This includes biomineral shells of aquatic marine organisms that are preserved in the geologic record and used to infer Earth’s climate history (15–19).A core assumption of applications of biomineral oxygen isotopic compositions to infer environmental conditions is that the ¹⁸O:¹⁶O ratio is controlled by exchange of oxygen isotopes between oxyanions comprising the biomineral and ambient water in bodily fluids (i.e., body water) (9, 20–24). In multicellular eukaryotic organisms, body water includes all water found in various body compartments (e.g., intravascular/intercellular) and bodily fluids (e.g., blood plasma, urine, breath vapor), including water produced by metabolism, and is averaged over the entire organism (25, 26). Blood is the largest reservoir of body water in mammals. Accordingly, most previous studies have been based on measurements of total body water in blood, in urine, or in breath CO₂ that has exchanged and presumably equilibrated with body water, and, thus, not on direct measurement of intracellular water.A strong linear relationship has been observed between δ¹⁸O values of body water determined from mammal blood and δ¹⁸O values of extracellular (ingested) water, which is equivalent to local meteoric water (1, 27–30). A contribution of metabolic water to body water can be detected using this linear relation based on deviation of the slope from a value of 1 (7, 8, 28, 30–32). Based on slopes and modeling calculations, the percentage of metabolic water has been determined to vary between 7% and 56% among different mammal species (7, 8, 30–34).In contrast to macroorganisms and mammals, microorganisms are primarily unicellular (e.g., bacteria and archaea), and, thus, body water is equivalent to intracellular water, which has traditionally been assumed to be identical to water in the surrounding extracellular medium (35), without consideration of a metabolic water component.Here we present two independent lines of evidence for a significant contribution of metabolic water to the intracellular water pool of bacterial cells, based on a new approach for detecting the contribution of metabolic water by direct sampling of the intracellular environment through measurement of the ¹⁸O:¹⁶O ratio of PO₄ (i.e., δ¹⁸O_PO4) in intracellular molecules (e.g., DNA) and also PO₄ in total biomass. Our findings are consistent with recent results of Kreuzer-Martin et al. (27, 36) indicating a metabolic water contribution to the intracellular water of Escherichia coli cells of as much as 70% during the log phase of growth and up to 27% for cells in the stationary phase. Knowledge of the amount and also the isotopic composition of a metabolic water component of body/intracellular water is important to studies of cell physiology and metabolism in which the ¹⁸O:¹⁶O ratio of both water and oxyanions such as CO₂/CO₃ and PO₄ are used to track metabolic processes and reaction pathways.