A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens.

All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels-a condition that is not satisfied under many "real-world" circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated gene. We found that in this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a mutant. In , the reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene , as well as the specific mutational signature associated with Finally, the reporter was used to measure the mutation rates of clinical isolates carrying different alleles of Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis. Measurements of mutation rates-i.e., how often proliferating cells acquire mutations in their DNA-are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.