Abstract Initiation of bacterial DNA replication takes place at the origin of replication, a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis . For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response. Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis , which might be a valuable tool for biotechnological applications. Importance Even though bacteria are constantly replicating under laboratory conditions, natural environments expose them to various stresses like lack of nutrients, high salinity, and pH changes, which can keep them in non-replicating states. Non-replicating states can allow bacteria to become less sensitive or tolerant to antibiotics (persisters), remain inactive in specific niches for an extended period (dormancy), and adapt to some hostile ecosystems. Non-replicating states have been studied due to the possibility of repurposing energy to produce additional metabolites or proteins. Using CRISPRi targeting bacterial replication initiation sequences, we successfully arrested the replication of B. subtilis . We observed that non-replicating cells continued growing but not dividing, and the initial arrest did not induce global stress conditions such as SOS or stringent response. Notably, these cells continued their metabolic activity and translation. This study provides comprehensive insights into the physiological response of replication initiation blockage in B. subtilis .

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest