Amplification and suppression of noise in B. subtilis developmental gene circuits

描述（由申请人提供）：“噪声”-细胞成分的随机波动-是单细胞水平生命的基本方面。噪声在生物系统中扮演着双重角色：一方面，它允许细胞做出受外部条件影响的随机决策。例如，通过对压力的反应形成孢子，单个细胞对它们未来的环境进行概率性的“赌注”。另一方面，噪音会干扰依赖于精确遗传调控的发育过程。细胞内噪声最近已被检测和量化在简单的合成电路。在这里，我们建议直接分析自然遗传电路中的噪声，这些遗传电路做出概率性的细胞命运决定并经历精确的发育过程。枯草芽孢杆菌提供了这样做的独特机会：它使用充分表征的遗传回路来概率性地启动感受态和孢子形成的分化程序。在孢子形成过程中，它也经历了一个紧密协调、抑制噪音的发育过程。本研究的目的是了解B。枯草芽孢杆菌基因电路放大噪声以概率性地调节感受态事件和孢子形成起始，以及它们如何抑制噪声以在孢子形成期间产生有序的事件序列。我们将应用定量延时荧光显微镜技术来观察单细胞中的基因电路动态，并“重新布线”电路来测试特定的预测。整个方法将由基础基因电路的数学模型驱动。我们将具体解决三个问题：（1）在能力调节的情况下，我们将测试的假设，噪声驱动的兴奋性产生概率和短暂的能力发作。(2)在孢子形成启动的情况下，我们将测试的假设，嵌套的正反馈回路，连同噪音，产生时间变异的决定，开始孢子形成。(3)在孢子形成的发育过程中，我们将确定如何抑制野生型细胞中的噪音，但决定部分渗透（PP）突变体（突变体中，一些细胞成功完成孢子形成，而其他细胞死亡）的命运。在这个模型系统中确定的电路水平的策略可能会在更复杂的生物体中进行分化和发展。与公共卫生的相关性：传染病的传播取决于替代遗传程序的概率激活，例如细菌中的感受态、孢子形成和抗生素持久性，以及病毒的潜伏期。该提案将解决单个细胞随机进入这些交替状态的机制。此外，该提案还将调查导致部分突变（仅发生在某些受影响的个体中）的机制。在人类疾病中也发现了部分性痴呆。

DESCRIPTION (provided by applicant): "Noise" - random fluctuation of cellular components - is a fundamental aspect of life at the single cell level. Noise plays a dual role in biological systems: On the one hand, it allows cells to make random decisions biased by external conditions. For instance, by sporulating in response to stress individual cells make probabilistic 'bets' about their future environment. On the other hand, noise can interfere with developmental processes that depend on precise genetic regulation. Intracellular noise has recently been detected and quantified in simple synthetic circuits. Here we propose to analyze noise directly within natural genetic circuits that make probabilistic cell-fate decisions and undergo precise developmental processes. Bacillus subtilis presents a unique opportunity to do so: It uses well-characterized genetic circuits to probabilistically initiate the differentiation programs of competence and sporulation. It also undergoes a tightly-coordinated, noise-suppressing developmental process during sporulation. The goal of this research is to understand how B. subtilis gene circuits amplify noise to probabilistically regulate competence events and sporulation initiation, and how they suppress noise to generate an ordered sequence of events during sporulation. We will apply quantitative time-lapse fluorescent microscopy techniques to observe gene circuit dynamics in single cells, and 're-wire' circuits to test specific predictions. The overall approach will be driven by mathematical models of underlying gene circuits. We will specifically address three problems: (1) In the case of competence regulation, we will test the hypothesis that noise- driven excitability generates probabilistic and transient competence episodes. (2) In the case of sporulation initiation, we will test the hypothesis that nested positive feedback loops, together with noise, generate temporal variability in the decision to initiate sporulation. (3) In the developmental process of sporulation, we will determine how noise is suppressed in wild-type cells but determines the fate of partially penetrant (PP) mutants (mutants in which some cells successfully complete sporulation, while others die). The circuit-level strategies identified in this model system are likely to operate in more complex organisms that undergo differentiation and development. Relevance to Public Health: The spread of infectious diseases depends on probabilistic activation of alternative genetic programs, such as competence, sporulation, and antibiotic persistence in bacteria, and latency in viruses. This proposal will address the mechanism by which individual cells randomly enter these alternate states. In addition, the proposal will investigate the mechanisms leading to the partial penetrance (occurring only in some affected individuals) of mutations. Partial penetrance is also found in human diseases.