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

CodY 的生理后果：革兰氏阳性菌的主要调节因子。

• 批准号: 7901561
• 负责人: Shaun R Brinsmade
• 金额: $ 5.05万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901561
• 关键词: Address; Alleles; Amino Acid Substitution; Amino Acids; Anabolism; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Physiology; Binding; Binding Sites; Biochemistry; Biological; Biological Assay; Branched-Chain Amino Acids; Carbon; Cells; Collaborations; Companions; Complement; Complex; DNA Binding; DNA Binding Domain; DNase-I Footprinting; Data; Development; Disease; Energy-Generating Resources; Famines; Gel; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Glucose; Glutamine; Goals; Gram-Positive Bacteria; Growth; Guanosine Triphosphate; Homeostasis; Housing; Human; In Vitro; Isoleucine; Laboratories; LacZ Genes; Lead; Leucine; Logic; Metabolic; Metabolism; Methods; Molecular; Molecular Biology; Molecular Profiling; Monitor; Mutation; Nitrogen; Nutrient; Nutritional status; Operon; Pathway interactions; Phase; Physiological; Process; Production; Proteins; Proteolysis; Regulation; Regulon; Reporter; Repression; Research; Resources; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Source; Structure; System; Techniques; Testing; Universities; Valine; Variant; Virulence; Virulence Factors; Work; X-Ray Crystallography; analog; antimicrobial; antitermination; cytotoxic; gel mobility shift assay; genetic regulatory protein; in vivo; interdisciplinary approach; member; microbial; mimicry; pathogen; post-doctoral training; prevent; professor; programs; protein complex; research study; response; sensor; structural biology; three dimensional structure; transcription factor; transcription termination; ward

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.

描述（由申请人提供）：野生细菌很少发现自己处于促进有效和稳健生长的条件下。短时期的养分过剩会导致更长时期的养分限制。当被剥夺营养或其他有利条件时，许多细菌试图通过部署策略来适应，以通过激活次级分解代谢途径和转运蛋白或通过释放细胞毒性化合物（包括抗菌剂）来获得替代碳和能源，以抵御竞争和争夺资源。这些适应性反应中的许多对人类都是有害的。CodY蛋白是负责控制低G+C革兰氏阳性细菌（包括许多寄生的人类病原体）中该适应性程序的表达的主要营养传感器和转录调节因子。该应用旨在确定CodY如何处理多个输入信号，以在全局水平上控制转录，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定义在不同生长条件下改变CodY活性的生理重要性，并将鉴定CodY的其他协同效应物。广泛的目标是阐明CodY功能激活和抑制转录在许多位点的分子机制。本文所述的项目采用多学科方法，使用遗传学，生物化学，分子生物学和结构生物学，从各个方向解决生物学问题。报告融合，定量RT-PCR和微阵列将提供本地和全球的表达数据在体内。体外技术，包括凝胶迁移率变动分析，DNA酶I足迹，部分蛋白水解，并在体外转录将确认和补充体内数据。与X射线晶体学（约克大学的安东尼威尔金森教授）和细菌生理学（苏黎世的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，将揭示细菌如何决定激活发育和适应程序。最近的工作已经记录了编码毒力决定因子的基因作为CodY依赖性抑制的直接靶点。因此，可以增加CodY维持毒力决定因子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。