Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing

Maximum-depth sequencing (MDS), a new method of detecting extremely rare variants within a bacterial population, is used to show that mutation rates in Escherichia coli vary across the genome by at least an order of magnitude, and also to uncover mechanisms of antibiotic-induced mutagenesis. Knowledge of the spontaneous mutation rate of bacteria is important for the study of basic evolutionary processes and of potential value in various clinical settings. De novo mutations in bacteria are a difficult target for high-throughput sequencing, but now Justin Jee et al. describe a new method of detecting extremely rare variants within a bacterial population, termed maximum-depth sequencing (MDS), which can detect extremely rare variants within a bacterial population through error-corrected, high-throughput sequencing. The authors use this method to measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. MDS shows that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal mutation rate, but are repaired in vivo and are thus undetectable by conventional methods. Using MDS, the authors also uncover mechanisms of antibiotic-induced mutagenesis. In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing2,3, may be skewed by mutational ‘hot’ or ‘cold’ spots3,4. Both approaches are affected by numerous caveats5,6,7. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.