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 ofnosocomial infections. To obtain the rstcomprehensive view of transcriptional organi-zations in this bacterium, we used a modi edRNA-seq approach enabling to discriminateprimary from processed 5’RNA ends. We alsovalidated our approach by con rming knownfeatures in Escherichia coli.We mapped 559 transcription start sitesand 352 processing sites in E. faecalis. Ablind motif search retrieved canonical featuresof SigA- and SigN-dependent promoters pre-ceding TSSs mapped. We discovered 95 novelputative regulatory RNAs, small- and anti-sense RNAs, and 72 transcriptional antisenseorganisations.Presented data constitute a signi cant in-sight into bacterial RNA landscapes anda step towards the inference of regula-tory processes at transcriptional and post-transcriptional levels in a comprehensive man-ner. Introduction Enterococcus faecalis is a ubiquitous Gram-positive bacterium and one of the rst colo-nizers of the human gastro-intestinal tract af-ter birth. It belongs to the core-microbiotaand lives in the guts during the entire hu-man life, suggesting a contribution of the bac-terium to intestinal homeostasis [Adlerberthand Wold, 2009, Campeotto et al., 2007, Qinet al., 2010]. In contrast to this potentiallybene cial role, E. faecalis is also the thirdcause of nosocomial infections and may carryand transfer various antibiotic resistances toother bacterial species, making its presencein the medical environment a serious concern[Arias and Murray, 2012]. The opportunismof E. faecalis, i.e. the transition from com-mensalism to pathogenicity in response to en-vironmental cues, underlines its capacity toadapt and survive to harsh conditions. Thus,deciphering the regulatory pathways that en-able E. faecalis to undergo the transition fromcommensalism to pathogeny is a key compo-nent in the understanding the dual lifestyleof this microorganism [Gilmore and Ferretti,2003]. The V583 strain was one of the rstdiscovered vancomycin-resistant clinical iso-lates of E. faecalis [Sahm et al., 1989]. Itsgenome, a circular chromosome (3218 kbp)and three circular plamids pTEF1 (66 kbp),pTEF2 (57.7 kbp) and pTEF3 (18 kbp), con-tains at least 3264 annotated protein-codinggenes [Paulsen et al., 2003]. Although partialtranscriptomic analyses have been performed[Aakra et al., 2010, Opsata et al., 2011, Veboet al., 2009, 2010], a comprehensive and dy-namic view of the RNA landscape of V583 ismissing.Whole-transcriptome studies of prokaryotesvia tiling arrays and RNA sequencing (RNA-seq) have unveiled a plethora of actively tran-scribed RNAs, and highly complex transcrip-tional organizations due to numerous promot-ers nested in open reading frames (ORFs), an-tisense (asRNAs) and small RNAs (sRNAs)genes (among other reviews [Georg and Hess,2011, Toledo-Arana and Solano, 2010]). Al-though these global studies have been ex-tremely valuable, their functional and regu-latory insights remain incomplete as primaryand processed RNAs cannot be distinguishedand hence transcriptional (RNA synthesis)and post-transcriptional processes (RNA pro-cessing and stability) cannot be separated.The use of di erential RNA-seq (dRNA-seq),an astute method that enriches an RNA pop-ulation for primary transcripts, partially over-comes these limitations and gives access tothe primary transcriptome [Albrecht et al.,2010, Bohn et al., 2010, Irnov et al., 2010,Sharma et al., 2010]. Yet, a major limita-tion of dRNA-seq is that all transcripts can-not be detected in a single experiment as theyare degraded by a 5’-phosphate-dependentexonuclease, and thus information on post-transcriptional events is lost [Sharma et al.,2010]. Global scale analysis of RNA stabil-2