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开发及其对全转录组RNA测序（RNA-seq）的应用提供了一个机会，可以以非常高的分辨率分析细菌转录本，并提供使用更传统的方法（如DNA微阵列）无法获得的额外信息。RNA-seq允许准确测定相对转录水平、映射操纵子并鉴定来自基因间区域的反义和转录物，这些区域通常编码调节性小RNA（sRNA）。此外，使用新的cDNA制备技术，现在可以找到反义转录本，并确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物的全面的单核苷酸分辨率图谱。这项工作的目的不仅是准确地量化三种不同的铜绿假单胞菌菌株中的转录水平，而且要识别所有表达的调节sRNAs，反义转录物和顺式作用核糖核酸调节子，并生成高分辨率操纵子图谱。使用用于产生用于测序的链特异性文库的新方法，将映射所有mRNA和sRNA的5 '末端。对于一组选定的受调控sRNA，将鉴定其靶标。这项工作的结果将编入一个可自由取用的交互式因特网网址，它将作为研究界今后从信号传导和基因调节的基本机制到抗感染疗法新目标的发展等专题项目的资源。 公共卫生相关性：拟议的项目将利用一种新的分子工具，RNA的深度测序，以增加我们对原核生物基因调控的理解。在定义不同条件下生长的铜绿假单胞菌菌株的转录组时，所提出的工作的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，这项工作将导致发现铜绿假单胞菌调节性小RNA的重要部分，并评估它们在控制毒力因子表达中的作用。

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.