Deuterium enhances mutation fixation in Escherichia coli through a transition-biased mechanism.

Deuterium (D) alters biochemical reaction kinetics through the hydrogen/deuterium kinetic isotope effect (KIE), but its role in spontaneous mutagenesis remains controversial. Here, we investigated the effects of deuterium on mutagenesis in Escherichia coli strains AB1157 and BMH71-18 under either deuterium-labeled medium (D-broth) or unlabeled medium (H-broth) conditions. The growth rates of both strains in D-broth were reduced by 2.77- and 3.90-fold, respectively, compared with those in H-broth. Stationary-phase viability was not significantly affected in either strain, indicating that deuterium inhibits growth kinetically rather than through cytotoxicity. Spontaneous mutation frequency was evaluated using rifampicin resistance (Rifᴿ). A significant 5.57-fold increase in RifR mutant frequency was observed in BMH71-18 under deuterated conditions, whereas no significant increase was detected in AB1157. Whole-genome sequencing was therefore focused on BMH71-18, the strain showing the clearest mutational response to deuteration. Whole-genome sequencing of randomly selected colonies revealed a 5.35-fold increase in mutation events under deuterated conditions. Single-base substitutions predominated in both conditions, with transition mutations accounting for more than 90% of all substitutions. In contrast, no significant differences were detected in transversions, small insertions, deletions, or structural variants. These findings are consistent with the possibility that deuterium promotes mutation fixation by prolonging the persistence of rare tautomeric states during DNA replication. Collectively, our results suggest that deuterium enhances mutation fixation in E. coli rather than acting as a primary mutagen and is associated with a strongly transition-biased mutation spectrum.