Network Governing Sporulation during Myxococcus Development

在自然界中，细菌作为群体的成员存在，在这个群体中，细胞对来自彼此和环境的信号做出反应。微生物群落影响全球过程，如土壤、水和空气之间元素的循环，以及海洋的初级生产力；它们影响着生态系统和所有生活在其中的生物。对微生物如何控制复杂行为以应对彼此及其环境的理解有限，阻碍了我们利用它们来控制污染和气候，以及增加生物能源和粮食生产的能力。了解细菌如何整合信号并做出适当的反应是一项具有重要现实意义的根本性挑战。操纵微生物群落以改善生活和解决全球问题将取决于细菌如何相互作用及其环境的知识。为了进一步了解数千种黄粘球菌如何相互作用以及它们的环境，实验和数学建模方法将用于更好地了解控制孢子形成的生化开关。本科生和研究生以及一名博士后将接受跨学科的研究培训，并通过向五年级女生演讲、参加密歇根州立大学科学节、以及由当地高中参加的非正式科学cafei系列研讨会等方式向非科学家进行推广。技术描述。黄粘球菌提供了一个有吸引力的实验体系。饥饿时，细胞改变基因表达和新陈代谢，相互传递信号，改变运动，形成多细胞丘（新生子实体），一些杆状细胞分化成球形休眠孢子。其他细胞留在子实体的外部，成为外周杆，大多数细胞发生裂解。此前由美国国家科学基金会资助的研究重点是了解在子实体发育过程中，基因是如何响应c信号而受到调节的。c信号传导涉及CsgA，一种与细胞表面相关并介导细胞间短距离信号传导的蛋白质。c信号不仅调节基因表达，还调节细胞运动、裂解和产孢。几个c信号依赖的启动子被发现在MrpC和FruA两个转录因子的组合控制下。最近，一个这样的启动子被发现驱动devT的转录，其产物正调控mrpC。由于MrpC激活了fruA的转录，这三种蛋白质形成了一个网络。理解这个网络是如何整合多种信号和控制多种行为的，这是一个艰巨的挑战，需要系统分析，包括建模和合成生物学方法，如重新连接自然发生的网络。待验证的中心假设是MrpC-FruA-DevT （MFD）网络整合了饥饿和c信号，作为控制孢子形成承诺的生化开关（或开关的一部分）。为了解决这一假设，提出了以下目标：1)测量野生型和突变体承诺前和承诺期间MFD网络成分和输出的动态变化，并利用这些数据建立MFD网络的数学模型；2)利用该模型探索该网络是否可以作为一个开关，变得不可逆，并通过测量发育细胞添加营养物质后MFD网络的动态来验证这一点。3)利用该模型探索MFD网络是否能够实现超灵敏的响应，并通过测量MFD网络响应作为添加c信号或营养物质的函数来验证这一点；4)构建综合重连线MFD网络，测量其动态响应，并使用重连线网络的数学模型分析结果。

In nature, bacteria exist as members of communities in which cells respond to signals from each other and the environment. Microbial communities impact global processes like cycling of elements between soil, water, and air, and primary productivity of the oceans; they impact ecosystems and all the organisms that inhabit them. Limited understanding of how microbes control complex behaviors in response to each other and their environment impedes our ability to harness them for pollution and climate control, and for increased bioenergy and food production. Understanding how bacteria integrate signals and respond appropriately is a fundamental challenge of great practical significance. Manipulation of microbial communities to improve life and solve global problems will depend on knowledge of how bacteria interact with each other and their environment. To advance knowledge of how thousands of Myxococcus xanthus bacteria interact with each other and their environment, experimental and mathematical modeling methods will be used to better understand the biochemical switch which controls spore formation. Undergraduate and graduate students, and a postdoctoral scholar will receive interdisciplinary research training as a result of this project, and there will be outreach to non-scientists through presentations to 5th grade females, participation in the MSU Science Festival, and an informal Science Café-style seminar series involving local high schools.Technical Description. Myxococcus xanthus provides an attractive experimental system. When starved, cells change their gene expression and metabolism, send signals to each other, alter their movements to construct multicellular mounds (nascent fruiting bodies), and some of the rod-shaped cells differentiate into spherical, dormant spores. Other cells remain outside of fruiting bodies as peripheral rods and the majority of cells undergo lysis. Previous work funded by the National Science Foundation focused on understanding how genes are regulated in response to C-signaling during fruiting body development. C-signaling involves CsgA, a protein that becomes associated with the cell surface and mediates short-range signaling between cells. C-signaling regulates not only gene expression, but cell movements, lysis, and sporulation. Several C-signal-dependent promoters were discovered to be under combinatorial control of two transcription factors, MrpC and FruA. Recently, one such promoter was found to drive transcription of devT, whose product positively regulates mrpC. Since MrpC activates transcription of fruA, the three proteins form a network. Understanding how this network integrates multiple signals and governs multiple behaviors during M. xanthus development is a formidable challenge requiring systems analysis, including modeling and synthetic biology approaches like rewiring the naturally occurring network. The central hypothesis to be tested is that the MrpC-FruA-DevT (MFD) network integrates the starvation and C-signals, functioning as a biochemical switch (or part of the switch) that governs commitment to spore formation. To address this hypothesis, the following aims are proposed: 1) measure dynamical changes in the MFD network components and output before and during commitment of wild type and mutants, and use the data to build a mathematical model of the MFD network, 2) use the model to explore whether the network can operate as a switch that becomes irreversible and test this by measuring MFD network dynamics after addition of nutrients to developing cells, 3) use the model to explore if the MFD network can achieve an ultrasensitive response and test this by measuring MFD network response as a function of added C-signal or nutrients, and 4) construct synthetically rewired MFD networks, measure their dynamical responses, and analyze the results using mathematical models of rewired networks.