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

Abstract

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.

Keywords: Escherichia coli; aminio acids; diet; hydrogen isotopes.

Fig. S1.

Relationship between the hydrogen isotopic composition of bulk E. coli cells and the isotopic composition of the water in the growth medium. Cells were grown on either defined glucose (○) or tryptone-based (●) media.

Fig. 1.

(A) δ²H of individual AAs from glucose-grown E. coli cells. (B) δ²H of individual AAs from tryptone-grown cells. The AAs classified as nonessential for eukaryotes are plotted on the left side of the graph, and AAs considered to be essential are plotted on the right side. Parallel lines indicate similarities in biosynthetic δ²H fractionation.

Fig. 2.

(A) Modeled contribution of hydrogen from water versus organic hydrogen source based on δ²H of individual AAs from E. coli grown on glucose. (B) Expanded view. Isotope fractionations for water (α_W) and food (α_F) are estimates following the method of Session and Hayes (15), using the linear regression slope associated with water- and glucose-labeling experiments.

Fig. 3.

Calculated %H (f_w) originating from water in microbes grown on either glucose or tryptone. Dashed lines represent the values of f_w that correspond to that point on the curve at α_F or α_W. Estimates of α_W and α_F follow the method of Session and Hayes (15), using the linear regression slope associated with water- and tryptone-labeling experiments.

Fig. 4.

Calculated proportion of hydrogen originating from water in microbes grown on either glucose or tryptone based on models presented in Figs. 2 and 3.

Fig. 5.

Conceptual dendrogram of AA relationships. AAs in italics are considered to be essential AAs in eukaryotes. The length of the horizontal lines indicates the relative complexity of steps in the AA biosynthetic pathway.