Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs
- Equal contributors
1 Department of Food Science, Cornell University, Ithaca, NY, USA
2 Computational Biology Service Unit, Cornell University, Ithaca, NY, USA
3 Department of Computer Science, Cornell University, Ithaca, NY, USA
4 School of Mathematics and Statistics, University of Sydney, NSW, Australia
5 Life Sciences Core Laboratories Center, Cornell University, Ithaca, NY, USA
6 United States Department of Agriculture-Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA
7 Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, USA
BMC Genomics 2009, 10:641 doi:10.1186/1471-2164-10-641Published: 30 December 2009
Identification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many σB-dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ΔsigB mutant, which does not express the alternative σ factor σB, a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase.
Overall, 83% of all L. monocytogenes genes were transcribed in stationary phase cells; 42% of currently annotated L. monocytogenes genes showed medium to high transcript levels under these conditions. A total of 96 genes had significantly higher transcript levels in 10403S than in ΔsigB, indicating σB-dependent transcription of these genes. RNA-Seq analyses indicate that a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs. Application of a dynamically trained Hidden Markov Model, in combination with RNA-Seq data, identified 65 putative σB promoters upstream of 82 of the 96 σB-dependent genes and upstream of the one σB-dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5'- and 3'-UTR regions.
The results from these studies provide powerful evidence that RNA-Seq data combined with appropriate bioinformatics tools allow quantitative characterization of prokaryotic transcriptomes, thus providing exciting new strategies for exploring transcriptional regulatory networks in bacteria.