Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 2/20/2009
Publication Date: 5/17/2009
Citation: Filiatrault, M.J., Stodghill, P., Wang, W., Schweitzer, P., Grills, G., Cartinhour, S.W., Schneider, D.J. 2009. High-throughput identification of transcriptional start sites in Pseudomonas syringae. American Society for Microbiology. p. 251.
Technical Abstract: Pseudomonas syringae pv. tomato strain DC3000 is a bacterial plant pathogen capable of causing disease in tomatoes and Arabidopsis. The genome of this bacterium has been sequenced. However, little information is available regarding the transcriptional activity and regulation involved when this organism responds to various surroundings. To decipher the mechanisms used to control gene expression, it is necessary to determine the genes expressed under a particular condition, transcription initiation and termination sites, and where overlapping transcription occurs. We have developed a new method to capture the 5’ends of transcripts and to sequence the transcripts using the Illumina Genome Analyzer’s high-throughput sequencing technology to evaluate bacterial transcriptional start sites (TSS) on a global scale. Total RNA was isolated from P. syringae and then processed and sequenced using the Illumina GA small RNA protocol with several modifications. Computational methods were developed to filter the large volume of sequence data generated and to compare the strand-specific sequences with the genomic sequence. This high-throughput sequencing approach provided us with the 5’ ends of a number of transcripts. A large percentage of the TSS obtained by the new method were consistent with results identified by 5’RACE, demonstrating that this approach works efficiently at capturing TSS. In addition, our high-throughput method for experimental identification of TSS revealed transcriptional activity within intergenic regions where no neighboring genes have been annotated, providing evidence for the expression of un-annotated genes and/or small RNAs. In addition, TSS were discovered within annotated genes, suggesting that the annotation of these genes should be revisited. Our technique provides a very useful and efficient way of evaluating TSS and promoter regions in bacteria on a genome-wide scale and can be adapted for use in the study of other organisms. Furthermore, this method allows for the rapid, large-scale discovery of specific areas in the genome where promoters reside, thus enabling targeted searches for regulatory motifs.