The mechanism and function of programmed cell death in Caulobacter crescentus

新月柄杆菌程序性细胞死亡的机制和功能

• 批准号: 258915568
• 负责人: Dr. Kathrin Fröhlich
• 金额: --
• 依托单位: Department of Molecular Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/258915568?language=en
• 关键词: mechanism; function; programmed; cell; death

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.

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，这种遗传程序会导致从系统中有针对性地清除多余或潜在有害的细胞。最近，PCD也在细菌中进行了研究。不同的环境胁迫可以诱导细菌亚群的选择性死亡，例如通过诱导毒素-抗毒素系统或溶原性噬菌体，以新月柄杆菌为模式菌，描述了一种新的细菌PCD机制。严重的DNA损伤会触发所谓的SOS反应，这是一种阻止细胞周期并促进DNA修复的程序。随着未解决的DNA损伤持续存在，C. crescentus通过表达特异性因子BapE来反应。与SOS调节子的其他成员相比，内切核酸酶BapE不参与损伤修复，而是通过基因组片段化诱导细胞死亡。crescentus作为一个模型来回答有关细菌PCD的基本问题。首先，我将通过分析BapE表达和活性的分子机制来确定PCD是如何调节的。为此，我将确定bapE启动子的结构，以确定其转录控制元件。第二，我将确定的机制，PCD是通过表征BapE核酸内切酶活性的分子细节执行。由于BapE与任何表征的细菌蛋白不具有显著的同源性，因此基因组DNA片段化的潜在机制仍然未知。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，还将通过染色质免疫沉淀法确定BapE在体内的染色体切割位点。第三，通过剖析BapE在柄杆菌生长和存活中的作用，阐明PCD在细菌中的功能。作为柄杆菌属对DNA损伤的应激反应的最终结果的细胞死亡的诱导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种群中，并依赖于种群水平的特征来生存，细菌PCD可能对群落中柄杆菌的生长具有重要意义。为了跟进这个想法，我将使用微流体来研究多细胞生物膜群落中的柄杆菌生长和存活。用荧光BapE报道分子成像柄杆菌生物膜也将解决结构化社区内BapE表达的潜在空间调节。