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

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.