The mechanism and function of programmed cell death in Caulobacter crescentus

细胞程序性死亡（PCD）是多细胞生物生长发育的重要过程。当被触发时，这个基因程序会导致从系统中清除多余的或潜在有害的细胞。最近，PCD也在细菌中进行了研究。不同的环境压力可能引起细菌亚群的选择性死亡，例如由于毒素-抗毒素系统或溶原性噬菌体的诱导。一种新的细菌PCD机制被描述为模式细菌新月形茎杆菌。严重的DNA损伤会触发所谓的SOS反应，这是一个阻止细胞周期并促进DNA修复的程序。当未解决的DNA损伤持续存在时，月牙草通过表达特定因子BapE做出反应。与SOS调控的其他成员相反，内切酶BapE不参与损伤修复，但通过基因组的断裂诱导细胞死亡。在我的工作中，我将利用bape诱导的C. crescentus细胞死亡作为模型来回答有关细菌PCD的基本问题。首先，我将通过分析控制BapE表达和活性的分子机制来确定PCD是如何被调节的。为此，我将确定bapE启动子的结构，以确定其转录控制元件。其次，我将通过表征BapE内切酶活性的分子细节来确定PCD执行的机制。由于BapE与任何特征的细菌蛋白没有明显的同源性，因此基因组DNA断裂的机制尚不清楚。我将筛选核酸内切酶的失活突变体，以确定DNA结合和DNA切割所需的BapE结构域。此外，我将通过染色质免疫沉淀法确定BapE染色体在体内的切割位点。第三，我将通过剖析BapE在Caulobacter生长和存活中的作用来解决PCD对细菌的功能。诱导细胞死亡是根杆菌对DNA损伤的应激反应的最终结果，类似于真核细胞凋亡途径。然而，单细胞生物程序性死亡的直接好处仍然难以捉摸。考虑到大多数细菌生活在群体中，并依赖于群体水平的特征来生存，细菌PCD可能对群落内Caulobacter的生长具有重要意义。为了进一步研究这个想法，我将使用微流体技术来研究茎状杆菌在多细胞生物膜群落中的生长和存活。用荧光BapE报告器成像茎状杆菌生物膜也将解决在结构群落中BapE表达的潜在空间调节。

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.