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Regulation of the bacterial cell cycle in response to starvation
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2019. , p. 64
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-172600ISBN: 978-91-7797-823-7 (print)ISBN: 978-91-7797-824-4 (electronic)OAI: oai:DiVA.org:su-172600DiVA, id: diva2:1348495
Public defence
2019-10-18, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2019-09-25 Created: 2019-09-04 Last updated: 2019-09-17Bibliographically approved
List of papers
1. Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA
Open this publication in new window or tab >>Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA
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2015 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 11, no 7, article id e1005342Article in journal (Refereed) Published
Abstract [en]

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-166028 (URN)10.1371/journal.pgen.1005342 (DOI)
Available from: 2019-02-11 Created: 2019-02-11 Last updated: 2019-09-04Bibliographically approved
2. Regulation of DnaA and DNA replication at the onset of stationary phase in E. coli
Open this publication in new window or tab >>Regulation of DnaA and DNA replication at the onset of stationary phase in E. coli
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-172593 (URN)
Available from: 2019-09-04 Created: 2019-09-04 Last updated: 2019-09-06Bibliographically approved
3. Molecular Basis and Ecological Relevance of Caulobacter Cell Filamentation in Freshwater Habitats
Open this publication in new window or tab >>Molecular Basis and Ecological Relevance of Caulobacter Cell Filamentation in Freshwater Habitats
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2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 4, article id e01557-19Article in journal (Refereed) Published
Abstract [en]

All living cells are characterized by certain cell shapes and sizes. Many bacteria can change these properties depending on the growth conditions. The underlying mechanisms and the ecological relevance of changing cell shape and size remain unclear in most cases. One bacterium that undergoes extensive shape-shifting in response to changing growth conditions is the freshwater bacterium Caulobacter crescentus. When incubated for an extended time in stationary phase, a subpopulation of C. crescentus forms viable filamentous cells with a helical shape. Here, we demonstrated that this stationary-phase-induced filamentation results from downregulation of most critical cell cycle regulators and a consequent block of DNA replication and cell division while cell growth and metabolism continue. Our data indicate that this response is triggered by a combination of three stresses caused by prolonged growth in complex medium, namely, the depletion of phosphate, alkaline pH, and an excess of ammonium. We found that these conditions are experienced in the summer months during algal blooms near the surface in freshwater lakes, a natural habitat of C. crescentus, suggesting that filamentous growth is a common response of C. crescentus to its environment. Finally, we demonstrate that when grown in a biofilm, the filamentous cells can reach beyond the surface of the biofilm and potentially access nutrients or release progeny. Altogether, our work highlights the ability of bacteria to alter their morphology and suggests how this behavior might enable adaptation to changing environments.

National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-172586 (URN)10.1128/mBio.01557-19 (DOI)
Available from: 2019-09-04 Created: 2019-09-04 Last updated: 2019-09-04Bibliographically approved

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