Quantitative studies of metabolic switches in enteric bacteria

描述（由申请人提供）：本研究涉及肠道细菌对碳源的分层使用，以及细菌从一种碳源切换到另一种碳源时发生的生长转变动力学。这种现象被称为双生长，是雅克·莫诺在65年前发现的。它通常被认为是由“分解代谢物抑制”引起的，这是一种在大肠杆菌中被分子表征的调节反应，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部门的代谢和细胞生长，而不是关于优先使用碳。本研究旨在阐明肠道细菌在环境中无限的碳源组合中选择的调节策略（通常首先选择快速代谢的碳源），以及当首选碳源耗尽时使它们能够快速切换碳源的动力学机制。该研究将采用多种方法进行：传统的生物化学和分子生物学方法来量化关键信号分子的池；定量蛋白质组学表征蛋白质合成和转换，导致蛋白质组在生长转变期间的重塑；合成遗传结构，使人们能够定量地探测变化的代谢通量和蛋白质负荷对生长转变的影响；基于微流体的方法在细胞水平上表征细胞生长、基因表达和信号分子；定量现象学方法发展粗粒度动力学模型，捕捉生理反应并将其与潜在的调节机制联系起来。这项工作本质上是我们实验室近年来开发的将基因表达与生长生理学联系起来的非常成功的方法的主要延伸，但从稳态到动力学领域。这项研究的成果是一个与生理行为的关键分子相互作用有关的双氧生长的定量预测模型，将为与各种其他问题相关的生长过渡动力学建模提供一个原型，这些问题包括从细菌对抗生素的反应到进入固定阶段后的分化和发育动力学。关于肠道细菌如何选择其碳偏好的具体知识可能会在代谢工程应用中被利用，以去除或改变碳消耗的顺序，而关于生长转变的调节策略的知识可能会导致旨在减缓生长恢复的新型抗菌策略。

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.