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.