Conflicts between the DNA replication and repair machineries promote cell death in Gram-positive bacteria

Cellular proliferation relies on the successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilize regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In Escherichia coli, it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism Bacillus subtilis and the pathogen Staphylococcus aureus. In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in B. subtilis, temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarization and cell lysis. This phenotype could be suppressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In S. aureus, cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in S. aureus was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.