A multidisciplinary approach to elucidating gene function in a model Gram-positiv

描述（由申请人提供）：由于它们栖息的生态位的多样性，它们的多功能性以及它们的成员对人类健康构成的威胁，革兰氏阳性细菌在微生物学的多个方面都是至关重要的。尽管如此，在任何革兰氏阳性细菌的全基因组范围内，还没有系统的努力来阐明基因功能和关系。枯草芽孢杆菌是研究革兰氏阳性菌的首要模式生物，因为它提供了强大的经典和分子遗传学，高分辨率的细胞生物学，以及一个庞大而充满活力的研究人员群体。来自其他模式生物的证据表明，全基因组方法加速了功能发现。GO资助的机会催化了一个基础广泛的研究人员社区，汇集他们的专业知识，创建一个集中的、协调的、多学科的努力，以开发实施全面基因功能工作所需的基因组资源。这一举措将为今后在其他模型和致病菌方面的类似努力提供蓝图。我们将：1；构建用于全球表型分析的基因组尺度工具。这些将包括两个条形码的零突变文库，一个有序过表达文库，300个与yfp融合的启动子文库和一个所有必需基因的c端表位标记文库。2. 开发并提供关键表型方法的原理验证实验。这些将包括高通量遗传相互作用分析和化学遗传谱；整体细胞学表型；转录分析。3. 整合和分析跨物种的数据。每个全球表型数据集将与生物信息学家合作进行分析。这种分析将产生概率功能相互作用图和转录网络，将提供有关基因功能和关系的关键信息。这些将用于革兰氏阳性有机体之间以及革兰氏阳性和革兰氏阴性之间的比较分析。4. 建立枯草芽孢杆菌数据库（subtilisHUB）。我们将构建一个基于web的数据资源来支持正在进行的功能注释。该网站将记录所有基因组工具的构建，存储协议和数据集，并提供基础设施，以实现基因本体分配，文献挖掘和社区讨论。5. 参与微生物学家社区的基因组资源和方法。一个广泛的研究专家社区将利用这些工具、方法和资源启动基因组规模的项目。

DESCRIPTION (provided by applicant): Because of the diversity of niches they inhabit, their versatility, and the threat their members pose to human health, Gram-positive bacteria are of paramount importance to multiple facets of microbiology. Nonetheless, there has been no systematic effort to elucidate gene functions and relationships on a genome-wide scale in any Gram-positive bacterium. Bacillus subtilis is the premier model organism for studies of Gram-positive bacteria because it offers powerful classical and molecular genetics, high-resolution cell biology, as well as a large and vibrant community of researchers. Evidence from other model organisms demonstrates that genome-wide approaches accelerate functional discovery. The opportunity for GO funding has catalyzed a broad-based community of researchers to pool their expertise to create a focused, coordinated, multidisciplinary effort to develop the genomic resources necessary to implement a comprehensive gene function effort. This initiative will serve as the blueprint for similar efforts in other model and pathogenic bacteria in the future. We will: 1. Construct genome-scale tools for global phenotypic analysis. These will include two bar-coded null mutant libraries, an ordered over-expression library, 300 promoter fusions to yfp and a C-terminal epitope- tagged library for all essential genes. 2. Develop and provide proof-of-principle experiments for key phenotyping methods. These will include high-throughput genetic interaction analysis and chemical genetic profiling; global cytological phenotyping; and transcriptional profiling. 3. Integrate and analyze the data across species. Each global phenotyping dataset will be analyzed in collaboration with bioinformaticists. This analysis will generate probabilistic functional interaction maps and transcriptional networks that will provide key information about gene functions and relationships. These will be used for comparative analysis among Gram-positive organisms and between Gram-positives and Gram- negatives. 4. Develop a B. subtilis database (subtilisHUB). We will build a web-based data resource to support ongoing functional annotation. This site will document the construction of all the genomic tools, house protocols and datasets, and provide infrastructure to enable Gene Ontology assignments, literature mining, and community discussion. 5. Engage the community of microbiologists in the genomic resources and approaches. A broad community of research specialists will launch genome-scale projects taking advantage of the tools, methodologies, and resources. PUBLIC HEALTH RELEVANCE: This proposal is aimed at elucidating the function of all the genes in Bacillus subtilis, the major model organism for an important class of bacteria, Gram-positive bacteria. The project will generate genome-scale tools; pioneer the development of global phenotypic analysis for bacteria; and establish a web resource that will ensure continuity beyond the funding term. The concerted, community-based effort we propose will have far-reaching impact on research in bacteria in general, and on many Gram-positive pathogens.