Regulation of the bacterial cell cycle in response to starvation

Abstract: Bacteria have adapted to diverse environments, which are often unpredictable and constantly changing. In order to survive, bacteria need to make the most of nutrients while they are available, while being prepared to rapidly change their behaviour when conditions take a turn for the worse. One of the most central processes that must be regulated to ensure survival when conditions change is the cell cycle, the succession of DNA replication, chromosome segregation and cell division connecting growth and proliferation.In this thesis, we investigate how environmental information, specifically nutrient availability, is used to modulate cell cycle progression. In Paper I, we uncover a mechanism used by Caulobacter crescentus to arrest DNA replication in response to nutrient depletion. We find that the essential replication initiator protein DnaA is eliminated under these conditions, and determine that this occurs by a mechanism based on constant degradation of DnaA by the protease Lon. This constant degradation is coupled with regulated translation of the dnaA mRNA to decrease DnaA synthesis as nutrient levels decrease. We found that this regulated translation of dnaA depends on its long 5′ untranslated region.The replication initiator DnaA is conserved in almost all bacteria, and although some aspects of its regulation are maintained, others work differently in distantly related bacteria. In Paper II, we investigate how the enteric bacterium Escherichia coli regulates DNA replication at the onset of the stationary phase. We found that although DnaA is eliminated as growth slows, this downregulation is not required to arrest replication. We also found that the signalling molecule ppGpp, which is produced in response to starvation, is required for the elimination of DnaA at entry to stationary phase. High ppGpp levels lead to a block of replication initiation, however we found that chromosome content is still dramatically reduced at the onset of stationary phase in the absence of ppGpp, indicating that a ppGpp-independent mechanism is involved.While bacteria are usually studied over short timeframes and under optimal conditions in the laboratory, in nature, bacteria are often found in environments where only very slow growth is possible. In Paper III, we investigate a change in morphology observed to occur in a small subpopulation of cells in cultures of C. crescentus after extended incubation in the stationary phase. These cells form long, helical filaments. We determined that the filamentation arises as a result of a block of DNA replication and cell division while growth continues, and that this can be induced by a combination of conditions in the medium: low phosphate, high pH and excess ammonium. We find that these conditions occur in freshwater lakes during persistent algal blooms in the summer months, indicating that this response might occur in the wild.In summary, this thesis provides new insight into the mechanisms bacteria use to adapt their cell cycle, and specifically, DNA replication to changes in their environment, how bacteria are able to change their morphology by disrupting the coupling between growth and the cell cycle, and investigates how this morphological plasticity may be advantageous in natural environments.

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