Oxygen isotopes indicate most intracellular water in log-phase Escherichia coli is derived from metabolism

Abstract

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 ...

Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO 4

“…Our findings are consistent with recent results of Kreuzer-Martin et al (27,36) indicating a metabolic…”

“…In contrast to previous studies reporting direct measurement of intracellular water from only one microbial strain, E. coli (27,36), we tested several different strains of bacteria to look for species-/strain-specific effects and also used the technique of multilabeled water isotope probing (MLWIP), used in previous studies of metabolic water in rats (30) and further developed in our group to probe organophosphorus and phosphoenzyme reaction mechanisms (23,(42)(43)(44). Using this technique, multiple (three or more) labeled-water probes having different oxygen isotopic composition are used to interrogate reaction mechanisms and trace oxygen transfers between compounds in reactions.…”

“…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 CO 2 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 δ 18 O values of body water determined from mammal blood and δ 18 O values of extracellular (ingested) water, which is equivalent to local meteoric water (1,(27)(28)(29)(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)(31)(32).…”

“…Here we used the MLWIP method, combined with graphical analysis, to both identify a metabolic water component in the intracellular water pool and to confirm the transfer of the oxygen isotopic signature of intracellular water to phosphate (PO 4 ) in DNA and cellular biomass. In previous studies (27), direct oxygen isotope analysis of intracellular water was achieved by extracting water from whole cells using a vacuum cryodistillation method. In the present study, we measured the oxygen isotopic composition of PO 4 in DNA and total cellular biomass, which should reflect complete exchange of all 4 O atoms in PO 4 with O in water due to the action of intracellular PPase (Fig.…”

Hydrogen and oxygen isotope ratios in human hair are related to geography

“…Hydrogen and oxygen in follicle and gut water are each derived from multiple sources. The ␦ 2 H values of cells and their surrounding water environment differ because of the noninstantaneous exchange of intracellular and extracellular water (24). Therefore, we consider two water pools in a human, when describing the ways in which the H isotope ratio of tap water (␦ 2 H we ) in the geographic environment influences the H isotope ratio of water in the follicle (␦ 2 H wf ).…”