Quantitative studies of metabolic switches in enteric bacteria

描述(申请人提供)：这项研究解决了肠道细菌对碳源的分层使用，以及当细菌从一个碳源转换到另一个碳源时发生的生长转变的动力学。这种现象被称为对位生长，由雅克·莫诺在65年前发现。它通常被认为是“分解代谢抑制”的结果，这是一种调控反应，在大肠杆菌中具有很好的分子特征，并在微生物中广泛传播。然而，最近的研究表明，分解代谢抑制是关于协调碳代谢与其他新陈代谢和细胞生长，而不是优先使用碳水化合物。这项研究旨在阐明肠道细菌在环境中无限组合的碳源(通常首先选择快速代谢的碳源)中进行选择的调节策略，以及使它们能够在首选碳源耗尽时快速切换碳源的动力学机制。这项研究将结合使用多种方法进行：传统的生化和分子生物学方法来量化关键信号分子的池；定量蛋白质组学来表征蛋白质的合成和周转，从而导致生长过渡期间蛋白质组的重塑；合成的遗传结构，使人们能够定量地探索变化的代谢通量和蛋白质负荷对生长过渡的影响；基于微流体的方法来在逐个细胞的水平上表征细胞生长、基因表达和信号分子；以及定量现象学方法来开发捕捉生理反应并将其与潜在的调控机制相联系的粗粒度动力学模型。这项拟议的工作实质上是我们实验室近年来开发的一种非常成功的方法的重大扩展，该方法将基因表达与生长生理联系起来，但从稳态领域到动态领域。这项研究的成果是一个将关键分子相互作用与生理行为联系起来的二元生长的定量预测模型，将提供一个原型，用于模拟与从细菌对抗生素的反应到分化动力学和进入静止阶段后的发展有关的各种其他问题的生长过渡动力学。有关肠道细菌如何选择其碳偏好的专门知识可被用于代谢工程应用，以消除或改变碳消耗的顺序，而关于生长过渡调节策略的知识可能导致旨在减缓生长恢复的新类别的抗菌策略。

DESCRIPTION (provided by applicant): This research addresses the hierarchical usage of carbon sources by enteric bacteria, and the kinetic of growth transition that occurs when bacteria switch from one to another carbon source. This phenomenon, known as diauxic growth, was discovered by Jacques Monod 65 years ago. It was commonly thought to result from "catabolite repression", a regulatory response well characterized molecularly in E. coli and wide spread among microbes. However, recent studies establish that catabolite repression is about coordinating carbon metabolism with other sectors of metabolism and cell growth, and not about prioritizing the use of carbons. This research aims to elucidate the regulatory strategies enteric bacteria employ to choose among an infinite combination of carbon sources in the environment (often taking the fast-metabolizing carbons first), and the kinetic mechanism that enable them to switch carbon source rapidly when the preferred one runs out. The research will be carried out using a combination of approaches: traditional biochemical and molecular biology approaches to quantify the pools of key signaling molecules; quantitative proteomics to characterize protein synthesis and turnover that result in proteome-wide remodeling during growth transitions; synthetic genetic constructs that allow one to quantitatively probe the effect of changing metabolic fluxes and protein loads on growth transitions; microfluidic-based approaches to characterize cell growth, gene expression, and signaling molecules at a cell-by-cell level; and quantitative phenomenological approaches to develop coarse- grained kinetic models that capture the physiological responses and relate them to the underlying regulatory mechanisms. The proposed work is in essence a major extension of the highly successful approach our lab has developed in recent years to relate gene expression to growth physiology, but from the steady state to the kinetic domains. The output of this research, a quantitative predictive model of diauxic growth relating key molecular interactions to physiological behaviors, will provide a prototype for modeling the kinetics of growth transitions relevant to a wide variety of other problems ranging from the response of bacteria to antibiotic, to the kinetics of differentiation an development after entering the stationary phase. The specific knowledge on how enteric bacteria select their carbon preferences may be exploited in metabolic engineering applications to remove or alter the order of carbon consumption, while knowledge on the regulatory strategies of growth transitions may lead to new classes of antimicrobial strategies aimed at slowing down growth recovery.