Physiological consequences of CodY: a master regulator in gram-positive bacteria.

描述(申请人提供)：野外的细菌很少发现自己处于促进高效和强劲生长的条件下。短期的营养过剩会导致营养限制期更长。当缺乏营养或其他有利条件时，许多细菌试图通过部署策略来获得替代碳和能源，要么激活次级分解代谢途径和转运蛋白，要么释放包括抗菌剂在内的细胞毒性化合物，以抵御竞争和寻找资源。这些适应性反应中的许多恰好对人类有害。Cody蛋白是主要的营养感受器和转录调节因子，负责控制这种适应性程序在低G-C革兰氏阳性细菌中的表达，包括一些顽固的人类病原体。这项应用旨在确定Cody如何处理多个输入信号，以在全球水平上控制转录，从而改变中枢代谢的重要方面。此外，本申请中提出的研究将确定在不同生长条件下改变Cody活性的生理重要性，并将确定Cody的其他协效应子。我们的主要目标是阐明Cody激活和抑制多个位点转录的分子机制。这里描述的项目采取了多学科的方法，使用遗传学、生物化学、分子生物学和结构生物学从不同的方向解决生物学问题。报告融合、定量RT-PCR和微阵列将提供体内局部和全局表达数据。包括凝胶迁移率变化分析、DNase I足迹、部分蛋白分解和体外转录在内的体外技术将证实和补充体内数据。已经与X射线结晶学(安东尼·威尔金森教授-约克大学)和细菌生理学(苏黎世Uwe Sauer-ETH)专家建立了合作关系，以增加研究和发现的广度。解剖这个复杂的调控网络，研究扰乱Cody活性背后的遗传和代谢电路的生理后果，将揭示细菌是如何决定激活发育和适应程序的。最近的工作证明，编码毒力决定因素的基因是Cody依赖抑制的直接目标。因此，能够增加Cody保持对毒力决定因素的抑制的能力的方法和生物活性化合物可以导致控制和预防微生物来源的疾病的新方法。

DESCRIPTION (provided by applicant): Bacteria in the wild seldom find themselves in conditions that promote efficient and robust growth. Short periods of nutrient surplus give rise to even longer periods of nutrient limitation. When deprived of nutrients or otherwise favorable conditions, many bacteria attempt to adapt by deploying strategies to acquire alternative carbon and energy sources either by activating secondary catabolic pathways and transporters or by unleashing cytotoxic compounds, including antimicrobials, to ward off competition and scavenge for resources. Many of these adaptive responses are coincidently harmful to humans. The CodY protein is the master nutrient sensor and transcriptional regulator responsible for controlling the expression of this adaptive program in low G+C gram-positive bacteria, including a number of recalcitrant human pathogens. This application aims to determine how CodY processes multiple input signals for controlling transcription on a global level to alter important aspects of central metabolism. In addition, research proposed in this application will define the physiological importance of altering CodY activity under different growth conditions, and will identify additional coeffectors of CodY. The broad goal is to elucidate the molecular mechanism by which CodY functions to activate and repress transcription at numerous loci. The projects described herein take a multidisciplinary approach using genetics, biochemistry, molecular biology and structural biology to address biological questions from a variety of directions. Reporter fusions, quantitative RT-PCR and microarrays will provide local and global expression data in vivo. In vitro techniques including gel mobility shift assays, DNase I footprinting, partial proteolysis, and in vitro transcription will confirm and complement in vivo data. Collaborations with experts in X-ray crystallography (Professor Anthony Wilkinson - University of York) and bacterial physiology (Uwe Sauer - ETH, Zurich) have been established to increase the breadth of research and discovery. Dissecting this complex regulatory network and studying the physiological consequences of disrupting genetic and metabolic circuitry underlying CodY activity will reveal how bacteria decide to activate developmental and adaptive programs. Recent work has documented genes encoding virulence determinants as direct targets of CodY-dependent repression. As such, methods and bioactive compounds that can increase the capacity of CodY to maintain repression of virulence determinants can lead to new ways to control and prevent illnesses of microbial origin.