The mechanism and function of programmed cell death in Caulobacter crescentus

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Programmed cell-death (PCD) is an essential process for the growth and development of multi-cellular organisms. When triggered, this genetic program leads to the targeted removal of excess or potentially harmful cells from the system. More recently, PCD has also been studied in bacteria. Different environmental stresses may elicit the selective death of a subpopulation of bacteria, e.g. due to the induction of toxin-antitoxin systems or lysogenic prophages.A novel mechanism of bacterial PCD has been described for the model bacterium Caulobacter crescentus. Severe DNA damage triggers the so-called SOS response, a program that arrests the cell cycle and promotes DNA repair. As unresolved DNA damage persists, C. crescentus reacts by expressing the specific factor BapE. In contrast to other members of the SOS regulon, the endonuclease BapE does not contribute to damage repair but induces cell death by fragmentation of the genome.In my work, I will utilize the BapE-induced cell death in C. crescentus as a model to answer fundamental questions concerning bacterial PCD. First, I will determine how PCD is regulated by analyzing the molecular mechanisms governing BapE expression and activity. To this end, I will determine the architecture of the bapE promoter to identify its transcriptional control elements. Second, I will determine the mechanism by which PCD is executed by characterizing the molecular details of BapE endonuclease activity. As BapE does not share significant homology with any characterized bacterial protein, the mechanism underlying the fragmentation of genomic DNA remains unknown. I will screen for inactive mutants of the endonuclease in order to define domains of BapE required for DNA binding and DNA cleavage. In addition, I will determine the BapE chromosomal cleavage sites in vivo by chromatin immunoprecipitation.Third, I will address the function of PCD for the bacterium by dissecting the role of BapE for growth and survival of Caulobacter. The induction of cell death as a final consequence of the Caulobacter stress response to DNA damage resembles the eukaryotic apoptosis pathway. However, the immediate benefit of programmed death for a unicellular organism remains elusive. Taking into account that most bacteria live in populations and depend on population-level traits for their survival, bacterial PCD could have important implications for the growth of Caulobacter within a community. To follow up on this idea I will use microfluidics to study Caulobacter growth and survival in multicellular biofilm communities. Imaging Caulobacter biofilms with fluorescent BapE reporters will also address the potential spatial regulation of BapE expression within a structured community.