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

Significance Hydrogen isotope (δ2H) values of bulk tissues have become valuable tracers for studying migration and movement patterns in animals, but the biochemical mechanism for how hydrogen is incorporated into heterotrophic organisms is not well understood. We grew Escherichia coli as a model organism on two different substrates, and then measured δ2H values of individual amino acids (AAs) in cellular material. The δ2H values of AAs were highly variable within a simple microbial culture. Using isotopic fractionation models, we show that AA δ2H provide tracers of an organism’s environmental (e.g., drinking) water, as well as its food, information of prime interest to ecologists. The work is also of significance to microbial physiologists studying metabolic pathways in microbes from extreme environments. Hydrogen isotope (δ2H) analysis is widely used in animal ecology to study continental-scale movement because δ2H can trace precipitation and climate. To understand the biochemical underpinnings of how hydrogen is incorporated into biomolecules, we measured the δ2H of individual amino acids (AAs) in Escherichia coli cultured in glucose-based or complex tryptone-based media in waters with δ2H values ranging from −55‰ to +1,070‰. The δ2H values of AAs in tryptone spanned a range of ∼250‰. In E. coli grown on glucose, the range of δ2H among AAs was nearly 200‰. The relative distributions of δ2H of AAs were upheld in cultures grown in enriched waters. In E. coli grown on tryptone, the δ2H of nonessential AAs varied linearly with the δ2H of media water, whereas δ2H of essential AAs was nearly identical to δ2H 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 δ2H can route directly at the molecular level. We conclude that the patterns and distributions in δ2H of AAs are determined through biosynthetic reactions, suggesting that δ2H could become a new biosignature for studying novel microbial pathways. Our results also show that δ2H of AAs in an organism’s tissues provides a dual tracer for food and environmental (e.g., drinking) water.