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一个意想不到的细胞调控的动态图景，这是以前被传统的细胞群体平均技术所掩盖的。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应激反应和分化等过程。在这样的系统中，转录因子在连续的离散和明显随机的脉冲序列中被激活。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，以及它们如何在同一细胞中相互作用的一般问题。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，通常同时使用脉冲来调节关键的应激反应，包括孢子形成。实验方法结合定量延时荧光显微镜，电路重新布线和移植，以及激酶的光遗传操作

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.