Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (␦ 18 O) and hydrogen (␦D) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their ␦ 18 O and ␦D values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. O) that form the organic matter of those organisms. For example, isotope ratios have been used to trace the origins of migratory butterflies (1), birds (2, 3), and elephants (4, 5). Point-of-origin information can span several spatial scales. As an example, the physiological differences between C 3 and C 4 photosynthetic pathways, which result in large 13 C͞ 12 C differences, allow one to trace the flow of this organic carbon as differential dietary inputs to animals (6), the transport of carbon across ecosystems (7), and, ultimately, the movement of region-specific carbon back into the atmosphere (8-10). This sourcing aspect of stable isotopes has also been applied to forensic issues, such as determining the pointof-origin of illicit drugs (11, 12) and adulteration of foods and alcoholic beverages (13-15).Continentality, storm-track trajectories, and moisture origins result in substantial geographic gradients in the ␦ 18O and ␦D values of precipitation and, therefore, of local waters (16). Several maps have been produced that show the extent of isotopic variations in waters at the continental and global scales (17, 18). Again, organisms often record the isotopic composition of these source waters in their organic compounds. In trees, for example, the isotopic composition of cellulose is correlated with that of source water (19,20). Similar examples are found in animals where oxygen isotope ratios of bone and blood are closely related to that of the local water (21).We hypothesized that microorganisms, too, should show a record of their growth environment in their cellular components. If relationships between growth environment and cellular isotope composition could be elucidated, it might become possible to draw conclusions about the waters in which the microbes had been grown, particularly for genetically identical organisms cultured in different geographic regions. We used the growth of Bacillus subtilis in laboratory culture as a model system. B. subtilis is a nonpathogenic, endospore-forming, Gram-positive soil bacterium. Because B. subtilis can grow on a variety of nutrients, we focused first on the relationship between the ␦ 18O and ␦D values in the microbial products (cells or spores) and those ratios in the water used to make the culture media. We then tested the values predicted by laboratory model with cultures grown in different geographical regions across the United States. MethodsOur experimental organism was B. subtilis strain 6051 (American ...

The transport of water into and out of unicellular organisms is a seemingly simple process in which water diffuses either through pores or directly across the membrane. Because of the rates at which water can theoretically diffuse, it is generally accepted that intracellular water is indistinguishable from extracellular water. Using stable isotope ratio mass spectrometry, we directly tested this assumption. Here we demonstrate that under active growth, up to 70% of the intracellular water in log-phase Escherichia coli cells was actually generated during metabolism and was isotopically distinct from extracellular water. The contribution of metabolism to intracellular water was substantially less in stationaryphase or quiescent cells.stable isotope ͉ mass spectrometry ͉ water transport S table isotopes are commonly used as a tool for probing complex biological and environmental processes. For instance, at the cellular level, hydrogen, carbon, nitrogen, and oxygen isotopes are often used to dissect reaction mechanisms and as tracers to follow metabolic pathways. On a more global level, these same isotopes are used in ecological studies to probe the relationship among environmental nutrients, water, and organic molecules and biological tissues. Information from these studies can be used to deduce information about the growth environment of an organism or source of a sample from its isotope ratios (1-3). A common approach to environmental reconstruction by geochemists is the analysis of isotope ratios of biomarkers, resistant biogenic molecules, to infer the conditions under which the metabolites were formed (4). Because the hydrogen and oxygen isotope ratios of precipitation are strongly influenced by environmental variables, these elements are of particular utility in both paleoclimate and forensic studies.Studies of isotopic relationships between growth water and organic components of organisms are often carried out under laboratory conditions to determine fractionation factors between organism and environment. The results from these studies are then used to infer information about the growth environment that led to the production of the same compounds isolated from environmental samples. Two implicit assumptions are nearly always made during environmental reconstruction. The first is that intracellular water is isotopically identical to extracellular water (5, 6). This idea is based on the notion that water inside of a cell should be in rapid equilibrium with water outside of the cell. The second assumption is that the isotopic fractionations determined from cellular studies performed in the laboratory will be equivalent to those ratios measured in the natural environment.We recently questioned the validity of these two common assumptions when studying the biosynthesis of heme O. When Escherichia coli cells were grown in the laboratory in 2ϫ LB prepared with 95% H 2 18 O, Ϸ40% of the heme O molecules contained a labeled oxygen atom in the 17-hydroxyethylfarnesyl moiety (7), despite the fact that this oxygen atom ...

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.