Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis

Abstract

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5 ′ RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small-and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

“…Our previous 3 0 RNA end mappings have shown the antisense organization of mRNAs encoding the MazF and TxpA toxins. 4,40 Two additional transcripts, Ref5J and Ref45 were previously reported. 4 Because none of them was detected by Northern blot in the diverse strains and growth conditions used here, they will be not considered.…”

Regulation of Bottromycin Biosynthesis Involves an Internal Transcriptional Start Site and a Cluster-Situated Modulator

“…This evidence for btmC-M co-transcription did not exclude the possibility of alternative operons, and the specific increase in btmD transcription following btmL overexpression hinted toward an alternative transcription start site (TSS) for btmD that would explain how it is differentially regulated from its surrounding genes. In order to map the TSSs for the whole cluster we employed 5 -tag-RNA-seq (also called tagRNAseq) (Innocenti et al, 2015) that enables the identification of TSSs in an untargeted, genome-wide fashion. This powerful technique has the advantage of differentially distinguishing primary transcripts (those generated by an RNA polymerase and therefore coming from true TSSs) from processed transcripts, which arise upon degradation or RNase mediated cleavage of the original transcripts at specific processing sites (PSs).…”

“…Total RNA samples of S. scabies WT and btmL were extracted as previously described and, following quality control assays to ensure their integrity, they were submitted to Vertis Biotechnologie AG (Germany) for the construction and sequencing of tagRNA-seq libraries in a protocol adapted from the technique described in Innocenti et al (2015). Prior to library construction, rRNA was depleted in the samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicenter).…”

RNA-seq – Revealing Biological Insights in Bacteria

“…Consequently, they are eliminated by the dRNA-seq technique. A new methodology was created to overcome this problem by tagging and clustering the two groups together in an RNA-seq-derived approach named tagRNA-seq [31]. This technique also considers the difference between processed and primary transcripts, but instead of degrading the processed ones, two different ligation reactions are implemented with two different markers: PSS-tag (processed start site) and TSS-tag (transcription start site).…”

“…Figure 1 exhibits briefly the methodology, considering the three main steps: (1) the first reaction tags (PSS-tag) on the processed transcripts; (2) treatment with tobacco alkaline phosphatase (TAP), where the 5'PPP loses two phosphates, which allows the third step; (3) the second ligation reaction (TSS-tag) on the primary transcripts. After those steps are completed, the transcripts are sequenced and, due to the different markers, they can be distinguished and compared [31]. This methodology was first described for Enterococcus faecalis [31] and was based on another technique, 5'tagRACE [32], a 5'RACE derived method.…”