Thias Oberg Boesen:
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During bacterial growth, chromosomes must be replicated to be passed on to daughter cells. Bacteria have the ability to adapt to a range of different growth rates, depending on the nutrient availability. At faster growth rates, when the generation time is shorter than the time it takes to replicate a chromosome, bacteria can grow with overlapping replication cycles and still initiate replication one and only once per cell cycle. Decades of research have provided insight into how chromosome replication is regulated and the role of the main initiator protein, DnaA, in determining timely initiation of replication. Key aspects of the robustness of initiation control mechanisms remains to be elucidated. The aim of this PhD thesis was to explore the importance of extrinsic regulators effecting the nucleotide bound status of DnaA and how initiation control is governed when these are absent.
Paper I serves as a general introduction to chromosome replication and reviews the current knowledge on the mechanisms of replication initiation control and the role of the main initiator protein, DnaA.
In paper II we investigated the robustness of replication initiation control in E. coli, by creating mutants lacking the ability to actively convert DnaAATP to DnaAADP and vice versa. Using a combination of population wide flow cytometry and single-cell measurements of cell-cycle parameters, allowed us to gain a deeper understanding of the initiation dynamics and cell-to-cell variability of these mutants. Surprisingly, we found that eliminating all extrinsic elements mediating conversion between active and inactive DnaA results in cells that are not only viable, but show initiation characteristics similar to that of the wild-type. The mechanisms of controlling timely initiation in gram-positive bacteria appear to be fundamentally different from those found in E. coli, since no homologues of the extrinsic regulators of DnaA activity have been identified.
To further our understanding on how initiation of replication is regulated when DnaA activity depends solely on intrinsic ATPase activity, In paper III we characterized a previously described temperature sensitive DnaA mutant in Staphylococcus aureus. The mutant is unable to initiate chromosome replication at the non-permissive temperature and by selecting for 6 suppressors of this phenotype, we identified and characterized specific mutations involved in helicase loading, suggesting that the binding of the adaptor proteins prior to loading acts as a checkpoint for replication initiation. In addition, we also find that the YabA protein, previously described as a negative regulator of replication initiation in Bacillus subtilis, has little to no effect in Staphylococcus aureus. Inhibitors of transcription or translation, such as antibiotics, have long been a useful resource in cellular physiology research. Experiments involving the suppression of transcription or translation have contributed to our understanding of the regulation of chromosome replication in conjunction with growth and division in the field of cell cycle control.
In paper IV, we sought to understand the impact of rifampicin treatment, preventing new initiations, on cell cycle dynamics in single-cells and how cells ensure completion of ongoing replication cycles. Here we find that cells on average display a volume increase of approximately 20%, decoupled from protein synthesis. In addition, we find that the pool of synthesized dNTP’s in the cell at time of rifampicin addition was insufficient for completion of chromosome replication, but mRNA salvage alone can provide dNTP’s sufficient for completion.
In summary, the findings reported in these papers provide novel insight into replication initiation control dynamics in E. coli and S. aureus and elaborated on the current knowledge of cellular responses to transcription- or translation inhibiting antibiotics used for studying the bacterial cell-cycle.