Dynamical interrogation of the Bacillus subtilis sporulation network using an engineered light-switchable promoter system

 描述(由申请人提供)：理解和操纵控制细胞如何做出决定的生化事件的能力是开发微生物感染、自身免疫性疾病、癌症和发育缺陷新型治疗方法的核心。枯草芽孢杆菌在饥饿时分化为耐胁迫的、代谢惰性的孢子，并在受到各种胁迫时激活一个单独的基因一般应激反应途径。枯草杆菌产孢量和胁迫反应是研究和控制分化的创新新技术的理想模式途径。尽管已知核心基因回路中的基本调控相互作用，但我们缺乏对主要调控蛋白动态变化如何导致细胞信息处理的系统水平的了解，也缺乏最终指导这些大规模细胞决策的方法。我们的中心假设是，动态扰动和实时观察基因回路中的蛋白质活动的能力将产生关于细胞决策和分化的关键见解。为此，我们建议开发一种技术来询问枯草杆菌孢子形成和胁迫反应基因电路的信号特性，该技术使用时变的光信号来编程在活细胞中异常明确的基因表达动力学。作为第一个目标，我们将重新设计一个我们先前在大肠杆菌中建立的绿/红光可切换的双组分系统，以控制转录并在枯草杆菌中产生动态的基因表达功能。通过将这个系统从大肠杆菌转移到进化遥远的枯草杆菌，我们还将获得关于 需要考虑将这些强大的光遗传工具转移到其他模式生物和临床上重要的物种。作为第二个目标，我们将使用我们的光学方法来分析主要孢子形成调节剂的不同激活速度如何影响电路功能和所产生的表型。我们期望明确地证明：i)孢子形成程序的成功执行不仅需要适当的稳态水平，还需要适当的关键调节因子的动态变化，以及ii)最近观察到的主要胁迫反应调节因子的脉冲被用来诱导许多目标基因的比例激活水平。在第三个目标中，我们将使用或光学方法来研究最近描述的主孢子形成调节因子在种群和单细胞水平上的脉动动力学的生物学意义。特别是，我们将评估我们最近的假设，即孢子形成脉冲必须发生在DNA复制之后，以确保孢子遗传染色体，以及一个流行的假设，即必须达到超过阈值的主孢子形成调节器的浓度，细胞才能致力于孢子形成。由于枯草杆菌产孢量和应激反应回路在蜡状芽胞杆菌、炭疽杆菌和艰难梭菌等医学上重要的芽胞形成细菌中广泛保守，我们的结果不仅将使人们对细胞决策的理解有所突破，而且还将为设计新的抗菌剂提供基础。

 DESCRIPTION (provided by applicant): The ability to understand and manipulate the biochemical events controlling how cells make decisions is central to the development of novel treatments for microbial infections, autoimmune diseases, cancer, and developmental defects. The bacterium Bacillus subtilis differentiates into stress-resistant, metabolically inert spores upon starvation, and activates a separate gene general stress response pathway when challenged with various stresses. B. subtilis sporulation and stress response are ideal model pathways for which to develop innovative new technologies to study and control differentiation. Though the basic regulatory interactions in the core gene circuits are known, we lack a systems-level understanding of how dynamical changes in the major regulatory proteins result in cellular information processing and the ultimate guide these large-scale cellular decisions. Our central hypothesis is that the ability to dynamically perturb and observe protein activities in gene circuis in real time will yield crucial insights about cellular decision-making and differentiation. To thi end, we propose to develop a technology for interrogating the signaling properties of the B. subtilis sporulation and stress response gene circuits that uses time-varying light signals to program exceptionally well-defined gene expression dynamics in live cells. As a first aim, we will re-engineer a green/red light-switchable two-component system we previously built in E. coli to control transcription and generate dynamical gene expression functions in B. subtilis. By moving this system from E. coli to the evolutionary distant B. subtilis, we will also gain insights on the considerations needed to move these powerful optogenetic tools to other model organisms and clinically important species. As a second aim, we will use our optical method to analyze how different rates of activation of the major sporulation regulators impact the circuit functions and resulting phenotype. We expect to unequivocally demonstrate that i) successful execution of the sporulation program requires not only proper steady-state levels, but also proper dynamics of key regulators and that ii) recently observed pulsing in the major stress response regulator are used to elicit proportional activation levels of many target genes. In the third aim, we will use or optical method to investigate the biological significance of the recently described pulsatile dynamics of the master sporulation regulator at the population and single cell levels. In particular, we will evaluate our recent hypothesis that the sporulation pulses must occur after DNA replication to ensure that spores inherit chromosomes and a prevailing hypothesis that a supra-threshold concentration of the master sporulation regulator must be reached for cells to commit to sporulation. Since the B. subtilis sporulation and stress response circuits are widely conserved among medically important spore-forming bacteria including B. cereus, B. anthracis, and C. difficile, our result will not only enable breakthroughs in the understanding of cellular decision-making, but also provide a basis for design of new antibacterial agents.