Metabolic Processes Account for the Majority of the Intracellular Water in Log-Phase <i>Escherichia coli</i> Cells As Revealed by Hydrogen Isotopes

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 (...

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

supporting

confidence: 94%

Section: Intracellular Water Isotopic Compositionmentioning

confidence: 65%

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

Wang

et al. 2016

“…Variations in the D/H ratio of intracellular water have been observed in rapidly-growing E. coli and were attributed to the accumulation of water derived from the oxidation of organic substrates (44). Given that substrate ␦D values did not approach ϩ200‰ or Ϫ300‰ in our study, and no systematic relationship between growth rate and fatty acid ␦D value was observed, the influence of varying intracellular water ␦D can be ruled out.…”

Section: Sources Of D/h Variability In Fatty Acidsmentioning

confidence: 50%

Large D/H variations in bacterial lipids reflect central metabolic pathways

Zhang¹,

Gillespie

Sessions

2009

202

268

Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500‰ in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately ؊200 to ؊400‰), photoautotrophic growth (؊150 to ؊250‰), heterotrophic growth on sugars (0 to ؊150‰), and heterotrophic growth on TCA-cycle precursors and intermediates (؊50 to ؉200‰) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH-and indirectly lipids-with characteristic isotopic compositions. If so, lipid ␦D values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by 13 C about pathways of carbon fixation.fatty acids ͉ fractionation ͉ metabolism ͉ hydrogen isotopes T he hydrogen-isotopic composition ( 2 H/ 1 H or D/H ratio, commonly expressed as a ␦D value) of lipids is being explored by scientists with diverse interests, including the origins of natural products (1, 2), biogeochemical cycles (3), petroleum systems (4), and paleoclimate (5-7). Because the D/H ratios of lipids are generally conserved over Ϸ10 6 -year time scales (8), they are a potentially useful tracer of biogeochemical pathways and processes in the environment. Most research to date has focused on higher plants, in which environmental water is the sole source of external hydrogen and consequently provides primary control over the D/H ratio of biosynthesized lipids (9). Although ␦D values for plant lipids and environmental water are generally well correlated, they are also substantially offset from each other. The biochemical basis for this lipid/water fractionation is not well understood. It is generally assumed to arise from a combination of isotope effects during photosynthesis and the biosynthesis of lipids (9-12), and is often treated as approximately constant to reconstruct isotopic compositions of environmental water as a paleoclimate proxy.There is, however, mounting evidence that the net D/H fractionation between lipids and water can vary by up to 150‰ in plants, even in the same organism (12-16). Modest fractionations associated with fatty acid elongation and desaturation ...

“…In addition, a minor portion (∼10-15%) of hydrogen atoms in proteins and AAs are known to be exchangeable (27), yet available data show that not all δ 2 H exchange rapidly in biological conditions (8,28). A deeper understanding of the cycling of water inside cell membranes (29), including mitochondria, and how this water is connected to external sources of water is needed. Compound-specific δ 2 H analysis of AA provides a tool to answer these questions, because these molecules are all synthesized in central metabolic processes.…”

Section: Discussionmentioning

confidence: 99%

Hydrogen isotopes in individual amino acids reflect differentiated pools of hydrogen from food and water in Escherichia coli

Fogel

Griffin

Newsome

2016

Hydrogen isotope (δ 2 H) analysis is widely used in animal ecology to study continental-scale movement because δ 2 H can trace precipitation and climate. To understand the biochemical underpinnings of how hydrogen is incorporated into biomolecules, we measured the δ 2 H of individual amino acids (AAs) in Escherichia coli cultured in glucose-based or complex tryptone-based media in waters with δ 2 H values ranging from −55‰ to +1,070‰. The δ 2 H values of AAs in tryptone spanned a range of ∼250‰. In E. coli grown on glucose, the range of δ 2 H among AAs was nearly 200‰. The relative distributions of δ 2 H of AAs were upheld in cultures grown in enriched waters. In E. coli grown on tryptone, the δ 2 H of nonessential AAs varied linearly with the δ 2 H of media water, whereas δ 2 H of essential AAs was nearly identical to δ 2 H in diet. Model calculations determined that as much as 46% of hydrogen in some nonessential AAs originated from water, whereas no more than 12% of hydrogen in essential AAs originated from water. These findings demonstrate that δ 2 H can route directly at the molecular level. We conclude that the patterns and distributions in δ 2 H of AAs are determined through biosynthetic reactions, suggesting that δ 2 H could become a new biosignature for studying novel microbial pathways. Our results also show that δ 2 H of AAs in an organism's tissues provides a dual tracer for food and environmental (e.g., drinking) water.hydrogen isotopes | aminio acids | diet | Escherichia coli