The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insuffi- cient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide. IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

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.

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.

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the alpha-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor sigma(32) , demonstrating that the DnaK-dependent inactivation of sigma(32) is a growth requirement. While most mutations occurred in the rpoH gene encoding sigma(32) , we also identified mutations affecting sigma(32) activity or stability in trans, providing important new insight into the regulatory mechanisms controlling sigma(32) activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of sigma(32) , and a mutation leading to increased levels of the house keeping sigma(70) that outcompete sigma(32) for binding to the RNA polymerase. We demonstrate that sigma(32) inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has coopted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information.