No abstract

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 18 O/ 16 O ratio of PO 4 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 PO 4 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 | isotope probing | phosphate oxygen isotopes M etabolic 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 ( 18 O/ 16 O ratio or δ 18 O value) and contribution to body water because this information is crucial to the interpretation of biomineral [e.g., carbonate-CaCO 3 -and phosphate-Ca 3 (PO 4 ) 2 ] oxygen isotopic compositions used heavily in paleoclimate/paleohydrological research (7)(8)(9)(10)(11)(12)(13)(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)(16)(17)(18)(19).A core assumption of applications of biomineral oxygen isotopic compositions to infer environmental conditions is that the 18 O: 16 O 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)(21)(22)(23)(24). In multicellular eukaryotic organisms, body water includes all water found in various body compartments (...