A simple and efficient method to search for selected primary transcripts: non-coding and antisense RNAs in the human pathogen Enterococcus faecalis

Abstract

Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5'RACE-derivative method coined '5'tagRACE', to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5'tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5'tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin-antitoxin modules.

Figure 1.

Principle of the 5′tagRACE method. 5′ monophosphate RNA extremities generated by processing or degradation are ligated to an excess of a 5′ RNA adaptor (‘Short 5′ adaptor’), resulting in inactive 5′-ends for subsequent ligation steps. The excess of 5′ RNA adaptor is eliminated via exclusion chromatography. 5′ triphosphate ends of primary transcripts (i.e. ‘+1’ or ‘TSS’) are then transformed into 5′ monophosphate by the TAP enzyme and ligated to a second 5′ RNA adaptor (‘5′tag adaptor’). Reverse transcription is performed using random primers to generate a cDNA library (5′tag-cDNA library) containing two types of 5′tagged single-stranded-DNA molecules: those ligated to the first 5′ adaptor (Short 5′ adaptor), i.e. processed ends, and those ligated to the 5′ tag adaptor, i.e. TSSs. The use of a specific primer to the 5′ tag adaptor and an oligonucleotide specific to the selected RNA (transformed into cDNA) will allow the specific amplification by PCR of the cDNA synthesized only from the primary RNA. An untreated TAP 5′tag-cDNA library is used as negative control (hash sign).

Figure 2.

Probing for the housekeeping ncRNAs by 5′tag RACE. Names of RNAs probed are indicated at the top of each panel, antisense RNAs are denoted by ‘as’. Amplicons were separated on 2.5% agarose gels and stained with ethidium bromide. At the top of each lane, ‘−’ and ‘+’ refer to untreated and treated TAP RNA samples, respectively. The arrow heads show amplicons that were sequenced and their estimated lengths (nt) on gel. The amount of template used in these set of experiments was 0.05 ng Eq.RNA. With this amount of template, amplicons obtained for RnpB and SsrA with treated and untreated TAP samples were in equivalent amounts.

Figure 3.

Primary transcripts detected by 5′tagRACE in IGR candidates. Names of oligonucleotides used to probe each RNA candidate are indicated at the top of each panel. Signs ‘+’ and ‘−’ refer to treated or untreated TAP RNA samples, respectively. Numbers on the right indicate lengths estimated for 5′tagRACE PCR products observed on 2.5% agarose gels. Amplicons below ∼80 bp were considered as aberrant products due to the average length of the specific primer (∼25–30 nt) and the 5′tag RNA adaptor (38 nt), (Supplementary Table S1). Asterisk: signals not sequenced or corresponding to an unrelated primary transcript as determined by sequencing. 5′tagRACE data obtained for rejected IGR candidates are shown in Supplementary Figure S2.

Figure 4.

ref loci and features of Ref RNAs. Squares are: ‘red filled’ for ncRNAs; ‘red empty’ for ncRNAs or riboswitches; ‘black empty’ for potential 5′-UTRs; and ‘gray’ for antisense organization. Red arrows symbolize characterized transcripts, dotted lines represent putative 5′-UTRs. Red and gray lollipops represent putative transcription terminators. Annotated ORFs in the V583 genome are shown by gray horizontal arrows, and predicted ORFs in Ref RNA sequences are symbolized by diagonally striped bar arrows. Representation is not at scale; sequences are provided as supplementary data (Supplementary Figure S3).

Figure 5.

Distribution of Ref RNAs along the chromosome of V583 E. faecalis strain. ncRNAs characterized are shown in white; potential mRNA and 5′-UTRs are in gray. Arrows indicate transcription orientation. PAI: pathogenicity island.

Figure 6.

Detection of Ref RNAs by northern blot. Names of detected transcripts are indicated at the top of each panel. Probes used were oligonucleotides designed for 5′tagRACE characterization (Supplementary Table S1). About 10 µg of total RNA was loaded in each lane: on left, static growth conditions (S), and on the right, respiratory (R) growth conditions. Arrow-heads show transcripts with an estimated length agreeing with sizes deduced from end-mapping of primary RNAs, asterisks indicate processed forms. Only 1 µg of total RNA was loaded for SsrS, SsrA and Ffs RNAs. (^a): see comments and legend in Supplementary Table S2.

Figure 7.

Characterization of three ref loci. (A) Expression of SsrS and its antisense Ref44. The genome organization of ssrS and ref44 genes deduced from 5′tag- and 3′-end mapping is shown in the upper part of the panel. Arrowheads indicate oligonucleotide used as probes for northern blots. ‘pS-44’ : plasmid construct based solely on mapping data and containing the genomic region represented by the black line. The existence of the Ref44 RNA is verified by overexpression-based 5′- and 3′-end mapping (lower part of panel 7A). About 5 µg total RNA was loaded in each lane when probing for Ref44 (right panel), and 1 µg was loaded for SsrS (left panel), respectively. Lanes noted: ‘W’, wild-type V583 strain; ‘V’, V583 strain harboring the plasmid vector; ‘pS-44’, V583 strain transformed by the plasmid pS-44; ‘L’, 50 base DNA ladder. Lengths indicated for Ref44 and SsrS correspond to the most abundant forms detected. Note that DNA (L) runs slightly faster than RNA. Exposure times were optimized for each panel and signal intensities do not indicate relative abundance between Ref44 and SsrS. Legend is otherwise as in Figure 6. (B) Oxygen-dependent expression of Ref25C ncRNA. Samples were taken at OD₆₀₀ between 0.80 and 0.90. Growth conditions are indicated at the top of each lane: ‘S’ and ‘Sh’ correspond to static growth without and with hemin, respectively; ‘A’ and ‘R’ correspond to aeration growth without and with hemin, respectively. About 10 µg total RNA was loaded in each lane and the oligonucleotide FR.25C was used as probe for Ref25C. Ribosomal RNAs were used as loading controls (1 µg per lane). (C) Transcriptional organization of the overlapping group I and II TA modules. Location and orientation of specific oligonucleotides used for 5′tag- and 3′RACE analysis of mazEF1-2, Ref45 and Ref46 transcripts are indicated by arrowheads. Direct repeat motifs drm1 and drm2 are shown by gray boxes. A single and common transcription terminator for mazEF1-2, Ref45 and Ref46 RNAs was predicted (shown in black) and corresponds to the bidirectional terminator predicted for folP (in gray). Putative transcription termination ends for primary transcripts mazEF1-2, Ref45 and Ref46 RNAs are shown by dashed lines; numbers at the end of those transcripts are estimated length (see Supplementary Figure S3). Genome coordinates of TSSs mapped for mazEF1-2, Ref45 and Ref46 are given at the bottom of the panel.