Life without Replication in Bacillus subtilis

Abstract

Bacterial replication, as the starting point of the cell cycle, enables the duplication of genomic information and ensures that each daughter cell receives one copy of the parental genetic code. Replication is initiated at the origin of replication (oriC), which contains several sequences called DnaA boxes that serve as binding sites for the master initiator protein DnaA.

Absence or failure of DNA replication can lead to bacterial growth arrest or cell death. Our knowledge of the cellular effects of replication arrest comes primarily from studies using thermosensitive mutants, the addition of antibiotics, or the induction via nutrient depletion. Although these studies are useful, they do not provide a global picture of the mere absence of replication initiation in the cell, but are biased by the additional stress conditions. In this study, we aimed to uncover the global effect of life without replication in the model bacterium Bacillus subtilis.

For this purpose, we developed two strategies. First, we generated a bacterial strain in which the oriC can be removed by an inducible and customized CRISPR-Cas9 system. After successful deletion of oriC, the cells were no longer able to initiate new rounds of DNA replication and they expressed a fluorescent reporter protein that enabled us to select and characterize the cells by flow cytometry and time-lapse microscopy. Thereby, we were able to produce the deletion of essential sequences for replication. Unfortunately, we found that the recovery and enrichment of non-replicating cells was insufficient and challenging to proceed with a proteomic characterization of these cells. Nevertheless, we characterized a potential tool for the characterization of essential genes and genetic elements in bacteria.

In the second part of this work, we blocked replication using a CRISPRi approach by targeting DnaA boxes 6 and 7 with a specific sgRNA and dCas9 expression, which are essential for replication. We characterized the phenotype of these cells and analyzed global changes in the proteome by quantitative Mass Spectrometry. Thereby, we could observe the replication arrested cells were elongated and did not activate neither SOS nor stringent response. Additionally, we confirmed that translation activity of these cells is upregulated. These results expand our understanding of potential direct or indirect connections between replication and translation. At the same time, these experiments provide and a good starting point for better research of non-replicating but translationally active B. subtilis strains. These could also lead to interesting biotechnological applications, such as construction of improved producer strains.