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RNA全转录测序(RNA-SEQ)中的应用为以非常高的分辨率分析细菌转录并提供使用诸如DNA微阵列等更传统方法无法获得的额外信息提供了机会。RNA-SEQ可以准确地确定相对转录水平和MAP操纵子，并从经常编码调控小RNA(SRNAs)的基因间隔区识别反义和转录本。此外，使用新的cDNA制备技术，现在有可能找到反义转录本，并识别基因组中所有的转录起始点。在这个项目中，RNA-seq将被用来开发在实验室条件下生长的铜绿假单胞菌转录本和人类呼吸道粘液中的全面的单核苷酸分辨图谱。这项工作的目的不仅是准确地定量三个不同铜绿假单胞菌菌株的转录水平，而且识别所有表达的调控sRNA、反义转录和顺式作用的核糖体调节因子，以及产生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sRNA的5‘端都将被映射。对于一组选定的受调控的sRNA，将确定它们的目标。这项工作的结果将被组织成一个可自由访问的互动因特网网站，它应该为研究界提供资源，供今后的项目使用，主题包括信号转导和基因调控的基本机制，以及抗感染治疗新靶点的开发。 与公共卫生相关：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中基因调控的理解。在确定不同条件下生长的铜绿假单胞菌不同菌株的转录组时，拟议的工作结果不仅应该描述所有受调控的转录本，还应该确定转录和转录后调控的几个新特征。此外，这项工作应该导致发现很大一部分铜绿假单胞菌调控小RNA，并评估它们在控制毒力因子表达方面的作用。

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.