remains translationally active after CRISPRi-mediated replication initiation arrest.

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. This process is tightly controlled by modulation of the availability or activity of DnaA and during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium . We successfully arrested replication in by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to . In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in . This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.