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Coping with Stress Coping with Stress Regulation of the Caulobacter crescentus cell cycle in response to environmental cues Kristina Heinrich Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 21 September 2018 at 10.00 in Vivi Täckholmsalen, NPQ-huset, Svante Arrhenius väg 20 A. Abstract All organisms have to respond to environmental changes to maintain cellular and genome integrity. In particular, unicellular organisms like bacteria must be able to analyze their surroundings and rapidly adjust their growth mode and cell cycle program in response to environmental changes, such as changes in nutrient availability, temperature, osmolarity, or pH. Additionally, they have to compete with other species for nutrients and evade possible predators or the immune system. Bacteria exhibit a myriad of sophisticated regulatory pathways that allow them to cope with various kinds of threats and ensure their survival. However, the precise molecular mechanisms underlying these responses remain in many cases incompletely described. This thesis focuses on the mechanisms that adjust growth and cell cycle progression of Caulobacter crescentus under adverse conditions. In paper I we describe a mechanism by which environmental information is transduced via the membrane-bound cell cycle kinase CckA into the cell division program of C. crescentus. This mechanism ensures rapid dephosphorylation and clearance of the cell cycle master regulator CtrA under salt and ethanol stress. The downregulation of CtrA leads to a cell division block and cell filamentation, which provides a growth advantage under these conditions. Cell filamentation of C. crescentus can also be observed in the late stationary phase, in which a small subpopulation of cells transforms into helical shaped filaments. In these cells not only CtrA but all major cell cycle regulators are cleared (paper II), leading to a situation in which cells block their cell cycle but continue to grow. We found that a combination of different stresses, namely phosphate starvation, high pH, and excess nitrogen, triggers this response. These stresses can also be observed in C. crescentus’ natural freshwater habitat during algae blooms. Furthermore, our results indicate that filamentous cells are able to reach beyond biofilm surfaces, possibly enabling cells to reach nutrients and to release progeny. While our studies highlight that cell filamentation is a common bacterial response to stress, some stress conditions, such as acute proteotoxic stress, lead to a growth arrest. In paper III we show that the regulatory interaction between the major chaperone DnaK and the heat shock sigma factor σ32 adjusts growth rate in response to changes of the global protein folding state. We show that high σ32 activity inhibits growth by re-allocating cellular resources from proliferative to maintenance functions. Under stress conditions when σ32 is active, this re-allocation likely helps cells to survive. However, under non- stress conditions unrepressed σ32 activity is detrimental. We demonstrate that in the absence of stress, the DnaK chaperone is absolutely necessary to limit σ32 activity and in this way to allow rapid proliferation. In summary, the described studies highlight critical pathways that allow C. crescentus to integrate environmental information with cell cycle and growth regulation and shed new light onto the mechanisms by which bacteria adapt to their environment and in this way ensure their survival. Keywords: Caulobacter crescentus, bacteria, cell cycle, stress, filamentation, cell morphology, DNA replication, cell division. Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-158108 ISBN 978-91-7797-381-2 ISBN 978-91-7797-380-5 Department of Molecular Biosciences, The Wenner-Gren Institute Stockholm University, 106 91 Stockholm COPING WITH STRESS Kristina Heinrich Coping with Stress Regulation of the Caulobacter crescentus cell cycle in response to environmental cues Kristina Heinrich ©Kristina Heinrich, Stockholm University 2018 ISBN print 978-91-7797-381-2 ISBN PDF 978-91-7797-380-5 Cover illustration by Kristina Heinrich Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Meinen Eltern SUMMARY All organisms have to respond to environmental changes to maintain cellular and genome integrity. In particular, unicellular organisms like bacteria must be able to analyze their surroundings and rapidly adjust their growth mode and cell cycle program in response to environmental changes, such as changes in nutrient availability, temperature, osmolarity, or pH. Additionally, they have to compete with other species for nutrients and evade possible predators or the immune system. Bacteria exhibit a myriad of sophisticated regulatory pathways that allow them to cope with various kinds of threats and ensure their survival. However, the precise molecular mechanisms underlying these responses remain in many cases incompletely described. This thesis focuses on the mechanisms that adjust growth and cell cycle progression of Caulobacter crescentus under adverse conditions. In paper I we describe a mechanism by which environmental information is transduced via the membrane-bound cell cycle kinase CckA into the cell division program of C. crescentus. This mechanism ensures rapid dephosphorylation and clearance of the cell cycle master regulator CtrA under salt and ethanol stress. The downregulation of CtrA leads to a cell division block and cell filamentation, which provides a growth advantage under these conditions. Cell filamentation of C. crescentus can also be observed in the late stationary phase, in which a small subpopulation of cells transforms into helical-shaped filaments. In these cells not only CtrA but all major cell cycle regulators are cleared (paper II), leading to a situation in which cells block their cell cycle but continue to grow. We found that a combination of different stresses, namely phosphate starvation, high pH, and excess nitrogen, triggers this response. These stresses can also be observed in C. crescentus’ natural freshwater habitat during algae blooms. Furthermore, our results indicate that filamentous cells are able to reach beyond biofilm surfaces, possibly enabling cells to reach nutrients and to release progeny. While our studies highlight that cell filamentation is a common bacterial response to stress, some stress conditions, such as acute proteotoxic stress, lead to a growth i arrest. In paper III we show that the regulatory interaction between the major chaperone DnaK and the heat shock sigma factor 32 adjusts growth rate in response 32 to changes of the global protein folding state. We σshow that high activity inhibits growth by re-allocating cellular resources from proliferativeσ to maintenance functions. Under stress conditions when 32 is active, this re-allocation likely helps 32 cells to survive. However, under non-stressσ conditions unrepressed activity is detrimental. We demonstrate that in the absence of stress, the DnaKσ chaperone is absolutely necessary to limit 32 activity and in this way to allow rapid proliferation. In summary, the describedσ studies highlight critical pathways that allow C. crescentus to integrate environmental information with cell cycle and growth regulation and shed new light onto the mechanisms by which bacteria adapt to their environment and in this way ensure their survival. ii POPULÄRVETENSKAPLIG SAMMANFATTNING En organism måste producera en avkomma för att säkerställa sin arts överlevnad. Encelliga organismer, såsom bakterier, åstadkommer detta mestadels genom att dela sig på mitten för att bilda två i stort sett likadana dotterceller. Innan en cell kan dela sig måste dess arvsmassa kopieras i en process som kallas DNA-replikation. DNA:t måste sedan separeras och transporteras in i de båda cellhalvorna. Cellen växer fortfarande under DNA-replikationen för att säkerställa att dottercellerna kommer att vara lika stora som modercellen efter celldelningen. Celldelningen avslutas med bildandet av en ny cellvägg som delar modercellen och separerar den till två dotterceller. Dessa steg bildar tillsammans cellcykeln. Bakterier är överlevnadsspecialister. De klarar sig i extremt varma eller kalla omgivningar såsom vulkaniska områden eller arktiska vatten, i Döda havets hårda omgivningar med höga saltkoncentrationer eller i värdorganismer, till exempel människor, där de framgångsrikt undkommer eller övervinner immunförsvaret. Bakterier uppnår dock inte allt detta enbart genom att snabbt reproducera sig utan också genom att känna av och anpassa sig till sin omgivning. Vid stressande förändringar i deras närmiljö svarar de inte bara med att dra igång processer för att förhindra och reparera cellskador utan även med förändringar i cellcykeln. DNA- replikation, celldelning och celltillväxt regleras så att bakterien kan anpassa sig till de nya förhållandena i omgivningen. Detta gör det möjligt för bakterier att kontrollera intracellulära processer som kan påverkas direkt eller indirekt av olika stressfaktorer och därigenom överleva tills förutsättningarna ändras till det bättre. Celler måste kunna känna av sin omgivning för att kunna svara på den. Hos bakterier möjliggörs detta av proteiner i bakteriernas cellvägg. Vid stress interagerar dessa proteiner med andra cellulära proteiner som för signalen vidare in i cellen vilket innebär att cellen kan genomföra förändringar
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