By:
Frederic Dominique Schramm, MBW, Stockholm University
Supervisor:
Kristina Jonas, MBW, Stockholm University
Opponent:
Franz Narberhaus, Department of Microbial Biology, Ruhr University Bochum, Germany
Committee:
Diarmaid Hughes, Department of Medical Biochemistry and Microbiology, Uppsala University
Claes von Wachenfeldt, Department of Biology, Lund University
Sabrina Büttner, MBW, Stockholm University
Chairman:
Ylva Engström, MBW, Stockholm University

Stress response regulation and protein aggregate inheritance in Caulobacter crescentus

Abstract
Many stress conditions a cell encounters threaten the continuation of basic biological processes ultimately endangering its survival. Heat shock and antibiotic exposure can lead to a sudden surge of protein un- and misfolding, while nutrient starvation directly causes a lack of energy and molecular building blocks. Our understanding of how cells integrate environmental stress signals, execute protective functions and handle persistent damage is still far from comprehensive. In this thesis the model bacterium Caulobacter crescentus was used to answer basic questions about the regulation and execution of bacterial stress responses and damage clearance.

Persistent larger protein aggregates can be maintained as remnants of a past stress exposure and in all of the few bacteria studied to date these particles collect at the poles. In the symmetrically dividing bacterium E. coli this aggregate localization pattern was shown to lead to an old pole lineage-specific retention. In paper I, we studied aggregate formation and inheritance in an asymmetrically dividing bacterium. While aggregates are dissolved by molecular chaperones following moderate heat stress, intense stress induces the emergence of long-lived aggregates. Surprisingly, we find that the majority of persistent aggregates do not collect at the old poles but instead describe a mechanism by which they are constantly displaced towards the new pole. This causes inheritance of aggregates by old and new pole cells at a stable rate without lineage-specific retention, a previously unknown pattern of aggregate inheritance in bacteria.

While we found that deletion of most chaperones in C. crescentus does not affect viability in the absence of stress, the mechanistic basis for why DnaK, like in other bacteria, is also required in the absence of stress remains unclear. In paper II, we show that DnaK's function as a negative regulator of the heat shock sigma factor σ32 is essential for viability at physiological temperatures and uncover potential new layers of  σ32 regulation. We find that the σ32-dependent response comprises a reallocation of resources from proliferative to maintenance functions and in addition to its known function in blocking DNA replication also affects other processes like protein translation, a process vulnerable to proteotoxic stress. Prolonged unrestricted activity of this stress response induced by the absence of DnaK is lethal. We conclude that while DnaK is essential for protein folding at elevated temperatures, its evolutionarily newer function in balancing the cell's proliferative and maintenance programs is a requirement for survival.

Growth and cell cycle progression is also regulated in response to nutrient limitation. Like under heat shock conditions, we show in paper III that carbon starvation during entry into stationary phase leads to a block of DNA replication for which, in contrast to heat stress, the molecular basis was not yet understood. We find that downregulation of DnaA levels is achieved by an as yet unknown nutrient availability sensing process involving the 5' untranslated region, inhibiting translation of the dnaA mRNA, which combined with constant degradation of DnaA by the protease Lon results in its elimination. This study provided new mechanistic insight into nutrient-dependent control of DNA replication and shows that the same regulatory outcomes can be achieved through different means depending on the stress response.

In conclusion this thesis describes the discovery of an unanticipated alternative way of protein aggregate inheritance with implications for our view on damage segregation in bacterial populations as well as new mechanistic insight into how cells balance proliferative with protective functions in response to heat shock and nutrient limitation.

Keywords: stress, protein aggregation, cellular aging, molecular chaperones, heat shock response, DnaK, sigma factor, suppressor genes, DNA replication, starvation, Caulobacter crescentus.