Single nucleotide resolution of the Pseudomonas aeruginosa transcriptome

描述（由申请人提供）：新的深度测序工具和方法的发展及其在全转录组RNA测序（RNA-seq）中的应用为以非常高的分辨率分析细菌转录本提供了机会，并提供了使用DNA微阵列等更传统方法无法获得的额外信息。RNA-seq可以准确测定相对转录水平，绘制操作子，并从通常编码调控小rna （sRNAs）的基因间区域识别反义转录本和转录本。此外，利用新的cDNA制备技术，现在可以找到反义转录物，并确定基因组中所有的转录起始位点。在这个项目中，RNA-seq将用于开发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物的综合单核苷酸分辨率图。这项工作不仅旨在准确量化三种不同铜绿假单胞菌菌株的转录物水平，而且还旨在鉴定所有表达的调控rna、反义转录物和顺式作用核糖体调控物，并生成高分辨率的操纵子图谱。使用一种新的方法来生成用于测序的链特异性文库，所有mRNA和sRNAs的5'端将被映射。对于一组选定的受调控的srna，它们的靶标将被确定。这项工作的结果将被组织成一个可自由访问的交互式互联网站点，它将作为研究社区未来项目的资源，从信号转导和基因调控的基本机制到开发抗感染治疗的新靶点。

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.