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

描述（由申请人提供）：细菌在野外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更长期的营养不足。当缺乏营养或其他有利条件时，许多细菌试图通过激活次级分解代谢途径和转运体或释放细胞毒性化合物（包括抗菌剂）来获取替代碳和能源来源，以抵御竞争并清除资源。这些适应性反应中有许多碰巧对人类有害。CodY蛋白是主要的营养传感器和转录调节剂，负责控制低G+C革兰氏阳性细菌（包括许多顽固性人类病原体）中该适应性程序的表达。本应用旨在确定CodY如何处理多个输入信号，以控制转录在全球水平上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的其他因子。广泛的目标是阐明CodY激活和抑制众多位点转录的分子机制。本文描述的项目采用多学科方法，利用遗传学，生物化学，分子生物学和结构生物学来解决来自各个方向的生物学问题。报告融合、定量RT-PCR和微阵列将提供体内局部和全局的表达数据。包括凝胶迁移转移测定、dna酶I足迹、部分蛋白水解和体外转录在内的体外技术将证实和补充体内数据。与x射线晶体学（Anthony Wilkinson教授-约克大学）和细菌生理学（Uwe Sauer - ETH，苏黎世）专家的合作已经建立，以增加研究和发现的广度。剖析这个复杂的调控网络，研究破坏CodY活性背后的遗传和代谢回路的生理后果，将揭示细菌是如何决定激活发育和适应程序的。最近的工作已经记录了编码毒力决定因子的基因是cody依赖性抑制的直接目标。因此，可以增加CodY维持抑制毒力决定因素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源性疾病的新方法。

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.