Stochastic Pulsing in Microbial Regulation and Differentiation

描述（由申请人提供）：单细胞研究已经开始揭示细胞调节的意外动态图像，该图像先前被细胞群平均的传统技术所掩盖。特别是，不同的系统似乎使用动态的，脉动的激活机制来控制过程，如应激反应和分化。在这样的系统中，转录因子在离散的和明显随机的脉冲的持续序列中被激活。这些系统的输入调制脉冲的频率、持续时间或幅度以控制下游过程。脉动调节的潜在普遍性引发了脉冲调制系统如何工作，它们为细胞提供什么功能以及它们在同一细胞中如何相互作用的一般性问题。我们将在枯草芽孢杆菌中解决这些问题，因为它包含多个电路，这些电路通常同时使用脉冲来调节关键的应激反应，包括孢子形成的启动。实验方法结合联合收割机定量延时荧光显微镜，电路重新布线和移植，以及激酶的光遗传学操作，所有这些都在 与基础遗传电路的数学建模密切相关。使用这些技术，我们将首先分析如何脉冲磷酸化的主转录因子Spo0A使细胞延迟启动孢子形成的多个细胞周期，以响应突然的营养限制，并使用工程光激活激酶来分析交替脉冲动力学的影响。然后我们将研究B的一般应激反应系统。由交替的西格玛因子B介导的枯草芽孢杆菌对单独产生不同脉冲调制响应的不同输入进行整合。我们将在E. coli来识别足以对信号进行频率编码的最小电路。最后，我们将更广泛地研究替代西格玛因子动力学，以了解细胞如何以及为什么在同一细胞中同时以脉动方式调节许多西格玛因子。特别是，我们将测试的假设，并行脉冲调节，使这些监管机构有效地“时间共享”有限的RNA聚合酶的资源。这些结果将提供一种全面的方法来理解随机脉冲在细菌细胞中的作用，并确定适用于不同物种和细胞类型的更一般的基于脉冲的设计原则。

DESCRIPTION (provided by applicant): Single-cell studies have begun to reveal an unexpectedly dynamic picture of cellular regulation that was previously obscured by traditional techniques that average over cell populations. In particular, diverse systems appear to use dynamic, pulsatile activation mechanisms to control processes such as stress response and differentiation. In such systems, transcription factors are activated in a sustained sequence of discrete and apparently stochastic pulses. Inputs to these systems modulate the frequency, duration, or amplitude of pulses to control downstream processes. The potential generality of pulsatile regulation provokes the general questions of how pulse modulation systems work, what functions they provide cells, and how they interact with one another in the same cell. We will address these questions in Bacillus subtilis, because it contains multiple circuits that use pulsing, often simultaneously, to regulate key stress responses, including the initiation of sporulation. Experimental approaches combine quantitative time-lapse fluorescence microscopy, circuit re- wiring and transplantation, and optogenetic manipulation of kinases, all in close connection with mathematical modeling of the underlying genetic circuits. Using these techniques we will first analyze how pulsed phosphorylation of the master transcription factor Spo0A enables cells to defer the initiation of sporulation for multiple cell cycles in response to sudden nutrient limitation, and use an engineered light activated kinase to analyze the effects of alternative pulse dynamics. We will then study the way in which the general stress response system of B. subtilis, mediated by the alternative sigma factor ¿B, integrates distinct inputs which individually generate different pulse modulation responses. We will reconstitute this system in E. coli to identify a minimal circuit sufficient for frequency encoding of signals. Finaly, we will study alternative sigma factor dynamics more broadly, to understand how and why cells regulate many sigma factors in a pulsatile fashion simultaneously in the same cell. In particular, we will test the hypothesis that parallel pulse-regulation enables these regulators to effectively 'time-share' the limiting resource of RNA polymerase. Together these results will provide a comprehensive approach to understand the role of stochastic pulsing in bacterial cells and identify more general pulse-based design principles applicable across species and cell types.