نبذة مختصرة : Efficient and highly organized regulation of transcription is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.
Author summary The canonical relationship between a bacterial operon and the mRNA transcript produced from the operon has become significantly more complex as numerous regulatory mechanisms that impact the stability, translational efficiency, and early termination rates for mRNA transcripts have been described. With the rise of antibiotic resistance, these mechanisms offer new potential targets for antibiotic development. In this study we used a combination of high-throughput sequencing technologies to assess genome-wide transcription start and stop sites, as well as determine condition specific global transcription patterns in the human pathogen Streptococcus pneumoniae. We find that the majority of multi-gene operons have alternative start and stop sites enabling condition specific regulation of genes within the same operon. Furthermore, we identified many putative RNA regulators that are widespread in the S. pneumoniae pan-genome. Finally, we show that separately collected RNA-Seq data enables identification of conditional triggers for regulatory RNAs, and experimentally demonstrate that our approach may be used to identify drug-able RNA targets by establishing that pyrR RNA functionality is critical for successful S. pneumoniae mouse colonization and infection. Thus, our study not only uses genome-wide high-throughput approaches to identify putative RNA regulators, but also establishes the importance of such regulators in S. pneumoniae virulence.
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