In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty-first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

Antimicrobial resistance (AMR) rapidly develops against almost all available therapeutics. New antibiotics target essential processes in bacteria but fail to address the root of the problem: mutagenesis and subsequent evolution. We recently proposed that inhibiting evolution is the ultimate solution to preventing AMR development. Here, we describe the first compound that inhibits the evolution of AMR by directly targeting a highly conserved evolvability factor, Mfd. We previously found that this RNA polymerase-associated translocase is required for rapid AMR development across highly divergent pathogens. Through an in vivo screen of novel compounds, we identified a small molecule (referred to as ARM-1) that binds Mfd and prevents its RNA polymerase termination activity. Inhibition of Mfd activity by ARM-1 delays the development of mutations, both in pure culture and during infection. Importantly, our data show that ARM-1 prevents the evolution of AMR across highly divergent pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, which cause extremely difficult to treat infections due to AMR development. In summary, we describe a novel compound that could be developed into a clinically usable anti-evolution drug. This work shows that the mechanisms accelerating evolution are druggable, and that this strategy could prevent AMR development in the clinic.

Nucleotide excision repair (NER) is a highly conserved mechanism that removes lesions from DNA. This process has been studied for decades, however, almost all of the work on NER was performed in the presence of exogenous DNA damage. Under these conditions, NER is anti-mutagenic in bacteria. Here, we describe our findings on the role of NER in mutagenesis under endogenous conditions. Counter to dogma, we find that NER is actually pro-mutagenic. Our data suggest a hand-off mechanism between two different types of DNA polymerases that explains the mutagenic nature of NER. Additionally, NER is thought to occur in two different ways; 1) in a transcription-coupled manner where it plays a role in removing lesions that block RNA polymerase, and 2) in a process known as global genome NER, which is independent of transcription. Counter to the classical view, our genetic analyses of the relationship between NER and the RNA polymerase interacting DNA translocase, and evolvability factor, Mfd, indicate that most likely all NER is associated with transcription. Lastly, we show that NER is pro-mutagenic because of endogenous oxidative damage. Altogether, our data strongly suggest that oxidative damage induces a mutagenic NER mechanism, which then accelerates evolution across divergent bacterial species.