Single nucleotide resolution of the Pseudomonas aeruginosa transcriptome

铜绿假单胞菌转录组的单核苷酸分辨率

• 批准号: 8041028
• 负责人: STEPHEN LORY
• 金额: $ 17.22万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8041028
• 关键词: Acute; Anti-Infective Agents; Biology; Cataloging; Catalogs; Chronic; Code; Communities; Complementary DNA; Complex; DNA Microarray Chip; Detection; Development; Environment; Escherichia coli; Functional RNA; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Host Defense; Human; Individual; Infection; Intercistronic Region; Internet; Laboratories; Lead; Libraries; Maps; Messenger RNA; Methodology; Methods; Molecular; Mucous body substance; Nucleotides; Operon; Organism; Pattern; Play; Post-Transcriptional Regulation; Precipitation; Preparation; Pseudomonas aeruginosa; RNA; RNA Sequences; Regulatory Pathway; Relative (related person); Research; Research Personnel; Resolution; Resources; Role; Signal Transduction; Signal Transduction Pathway; Site; Small RNA; Stimulus; Structure; Testing; Tissues; Transcript; Transduction Gene; Use of New Techniques; Variant; Virulence; Virulence Factors; Work; base; environmental adaptation; functional genomics; genetic regulatory protein; microorganism; novel; pathogen; public health relevance; respiratory; response; tool

DESCRIPTION (provided by applicant): The development of new deep sequencing tools and methodologies and their application towards RNA sequencing of whole transcriptomes (RNA-seq) presents an opportunity to analyze bacterial transcripts with a very high resolution and provide additional information that was not accessible using more traditional methods such as DNA microarrays. RNA-seq allows an accurate determination of relative transcript levels, map operons and to identify anti-sense and transcripts from intergenic regions that very often encode regulatory small RNAs (sRNAs). Moreover, using new techniques for cDNA preparation it is now possible to find anti-sense transcripts and to identify all transcriptional start sites in the genome. In this project, RNA-seq will be used to develop comprehensive, single nucleotide resolution maps of P. aeruginosa transcripts grown under laboratory conditions and in human respiratory mucus. The work aims to not only accurately quantify transcript levels in three different P. aeruginosa strains, but to identify all expressed regulatory sRNAs, anti-sense transcripts and cis-acting riboregulators as well as generate a high resolution operon map. Using a novel method for generating strand-specific libraries for sequencing, 5' ends of all mRNA and sRNAs will be mapped. For a group of selected regulated sRNAs, their targets will be identified. The results of this work will be organized into a freely-accessible interactive Internet site and it should serve the research community as the resource for future projects on topics ranging from basic mechanisms of signal transduction and gene regulation to the development of new targets for anti-infective therapy. PUBLIC HEALTH RELEVANCE: The proposed project will utilize a new molecular tool, deep sequencing of RNAs, to increase our understanding of gene regulation in prokaryotic organisms. In defining the transcriptome of different strains of Pseudomonas aeruginosa grown under a variety of conditions, the results of the proposed work should not only describe all regulated transcripts but also identify several novel features of transcriptional and post-transcriptional regulation. Moreover, this work should lead to the discovery of a significant fraction of P. aeruginosa regulatory small RNAs and assess their role in controlling the expression of virulence factors.

描述（由申请人提供）：新的深度测序工具和方法的开发及其在全转录组 RNA 测序（RNA-seq）中的应用提供了以非常高的分辨率分析细菌转录本的机会，并提供使用更传统的方法（例如 DNA 微阵列）无法获得的附加信息。 RNA-seq 可以准确确定相对转录物水平、图谱操纵子，并识别通常编码调节性小 RNA (sRNA) 的基因间区域的反义转录物和转录物。此外，利用新技术制备 cDNA，现在可以找到反义转录本并鉴定基因组中的所有转录起始位点。在该项目中，RNA-seq 将用于开发在实验室条件下和人类呼吸道粘液中生长的铜绿假单胞菌转录本的全面、单核苷酸分辨率图谱。这项工作的目的不仅是准确量化三种不同铜绿假单胞菌菌株的转录水平，而且还鉴定所有表达的调节 sRNA、反义转录本和顺式作用核糖调节子，并生成高分辨率操纵子图谱。使用生成用于测序的链特异性文库的新方法，将绘制所有 mRNA 和 sRNA 的 5' 末端。对于一组选定的受调控 sRNA，将确定它们的靶标。这项工作的结果将被组织成一个可免费访问的交互式互联网站点，它应该为研究界提供服务，作为未来项目的资源，其主题范围从信号转导和基因调控的基本机制到抗感染治疗新靶点的开发。 公共健康相关性：拟议的项目将利用一种新的分子工具，即 RNA 深度测序，来加深我们对原核生物基因调控的理解。在定义在各种条件下生长的不同铜绿假单胞菌菌株的转录组时，所提出的工作结果不仅应该描述所有受调节的转录本，而且还应该确定转录和转录后调节的几个新特征。此外，这项工作应该会导致铜绿假单胞菌调节小RNA的重要部分的发现，并评估它们在控制毒力因子表达中的作用。

项目成果

A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.

使用转座子文库的高通量测序对铜绿假单胞菌的体外和体内遗传适应性进行综合分析。

• DOI: 10.1371/journal.ppat.1003582
• 发表时间: 2013
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Skurnik,David;Roux,Damien;Aschard,Hugues;Cattoir,Vincent;Yoder-Himes,Deborah;Lory,Stephen;Pier,GeraldB
• 通讯作者: Pier,GeraldB

The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature.

• DOI: 10.1371/journal.ppat.1002945
• 发表时间: 2012-09
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Wurtzel O;Yoder-Himes DR;Han K;Dandekar AA;Edelheit S;Greenberg EP;Sorek R;Lory S
• 通讯作者: Lory S