TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

Abstract

BackgroundBacteria rely on efficient gene regulatory mechanisms to switch between genetic programs when they are facing new environments. Although this regulation can occur at many different levels, one of the key steps is the initiation of transcription. Identification of the first nucleotide transcribed by the RNA polymerase is therefore essential to understand the underlying regulatory processes, since this provides insight on promoter strength and binding sites for transcriptional regulators, and additionally reveals the exact 5’ untranslated region of the transcripts, which often contains elements that regulate translation.ResultsHere we present data from a novel TSS-EMOTE assay (Transcription Start Specific Exact Mapping Of Transcriptome Ends) to precisely map the transcription initiation sites of four entire transcriptomes. TSS-EMOTE is a variation of the dRNA-seq method, which has been combined with the EMOTE protocol, in order to increase detection of longer transcripts and limit biases introduced by PCR amplification of the Illumina sequencing library. Using TSS-EMOTE, 2018 promoters were detected in the opportunistic pathogen Staphylococcus aureus, and detailed consensus sequences could be obtained for the RNA polymerase recognition elements (e.g. sigma factor binding sites). The data also revealed a 94 nt median length of the 5’ untranslated region in S. aureus, giving important insights for future work on translational regulation. Additionally, the transcriptomes of three other opportunistic pathogens, Staphylococcus epidermidis, Acinetobacter baumannii and Enterobacter aerogenes, were examined, and the identified promoter locations were then used to generate a map of the operon structure for each of the four organisms.ConclusionsMapping transcription start sites, and subsequent correlation with the genomic sequence, provides a multitude of important information about the regulation of gene expression, both at the transcriptional and translational level, by defining 5’ untranslated regions and sigma-factor binding sites. We have here mapped transcription start sites in four important pathogens using TSS-EMOTE, a method that works with both long and 3’-phosphorylated RNA molecules, and which incorporates Unique Molecular Identifiers (UMIs) to allow unbiased quantification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3211-3) contains supplementary material, which is available to authorized users.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus

“…The identified TSS confirmed our RNA‐Seq data and the TSS prediction of Prados et al (). In addition, 5′ RACE revealed one clone with a putative TSS at position −128 bp upstream of the capA start codon, which was also predicted by Prados et al (). A conserved SigA consensus sequence was identified in front of this TSS containing canonical −35 and −10 regions.…”

“…CP serotypes 5 and 8 are the two main CP serotypes produced by S. aureus strains (Arbeit et al, 1984;Hochkeppel et al, 1987;Roghmann et al, 2005;Verdier et al, 2007). Their structure is highly similar due to the closely related cap5 and cap8 gene clusters.…”

“…Their structure is highly similar due to the closely related cap5 and cap8 gene clusters. These allelic operons consist of 16 genes, cap5/8A to cap5/8P, whose gene products are involved in CP biosynthesis, O-acetylation, transport and regulation (O'Riordan and Lee, 2004;Weidenmaier and Lee, 2017;Rausch et al, 2019). The cap operon is thought to be mainly transcribed as a single large 17 kb transcript driven by one principal promoter element (P cap ), located upstream of capA (Sau et al, 1997;Ouyang et al, 1999).…”

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster inStaphylococcus aureus

“…Previous primer extension analysis revealed a TSS −17 bp upstream of the ATG start codon of capA (12). However, RNA-Seq data indicate an alternative TSS further upstream, at position −41 from the capA coding region (data not shown), consistent with recent whole genome analyses of TSSs in S. aureus (49, 50). To solve this ambiguity, we employed 5’ Rapid Amplification of cDNA Endings (5’ RACE) (Figure 1A).…”

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in α-Proteobacteria

“…The TSS-EMOTE protocol and analyses were performed according to the scheme in S3 Fig and the detailed protocol described in [75]. We used a Worst-Case (i.e.)…”