Cartography of methicillin-resistant S. aureus transcripts: detection, orientation and temporal expression during growth phase and stress conditions

Abstract

Background: Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. The severity of these infections is highly variable and depends on multiple parameters including the genome content of the bacterium as well as the condition of the infected host. Clinically and epidemiologically, S. aureus shows a particular capacity to survive and adapt to drastic environmental changes including the presence of numerous antimicrobial agents. Mechanisms triggering this adaptation remain largely unknown despite important research efforts. Most studies evaluating gene content have so far neglected to analyze the so-called intergenic regions as well as potential antisense RNA molecules.

Principal findings: Using high-throughput sequencing technology, we performed an inventory of the whole transcriptome of S. aureus strain N315. In addition to the annotated transcription units, we identified more than 195 small transcribed regions, in the chromosome and the plasmid of S. aureus strain N315. The coding strand of each transcript was identified and structural analysis enabled classification of all discovered transcripts. RNA purified at four time-points during the growth phase of the bacterium allowed us to define the temporal expression of such transcripts. A selection of 26 transcripts of interest dispersed along the intergenic regions was assessed for expression changes in the presence of various stress conditions including pH, temperature, oxidative shocks and growth in a stringent medium. Most of these transcripts showed expression patterns specific for the defined stress conditions that we tested.

Conclusions: These RNA molecules potentially represent important effectors of S. aureus adaptation and more generally could support some of the epidemiological characteristics of the bacterium.

Figure 1. Visualization of different categories of transcripts detected and oriented by HTS RNA-Seq visualized in Artemis genome analyzer browser.

The top panels (A–D) display the shape of signals obtained in the detection experiment. The lower panels (A′–D′) shows the corresponding signals obtained during the orientation experiment. The X-axis corresponds to N315 genomic coordinate and Y-axis symbolizes the normalized coverage. Transcripts expressed on the forward strand are depicted in blue whereas transcripts expressed on the reverse strand appear in red. The vertical axis indicates local sequencing coverage. The arrows represent the position of annotated ORFs deduced from N315 genome sequence and the position of Teg identified in our study. Panels A–C presented various intergenic signals and D shows a typical antisense signal.

Figure 2. Secondary structures of some riboswitches and bona fide small RNAs based on in silico analysis.

(A, B, C) Examples of the secondary structure of the 5′ untranslated regulatory regions of S. aureus mRNAs encoding histidyl-tRNA synthetase (T-box 1), glycine-tRNA synthetase (T-box 2) and leucyl-tRNA synthetase (T-box 3). The models were derived from sequence alignment with the regulatory region of the Bacillus subtilis mRNA encoding tyrosyl-tRNA synthetase as derived by Green (2009). Only the specifier hairpin and the expression platform are folded. The T-Box specifier codon are given although the T-box 3 leucine codon is questionable. The predicted residues in the specifier and in the antiterminator loops that participate in pairing with the tRNA are colored in green. The start codon (pink) and the Shine-Dalgarno (SD) binding site (underlined) are represented when located near the end of the T-box. T-box 3 appears to regulate initiation of translation by sequestering the SD binding site of the downstream coding region whereas T-box 1 and T-box 2 regulate the expression of their downstream coding region by a mechanism of termination-antitermination. The structural model of the antiterminator conformation is shown. Residues that participate to the alternate structure (terminator conformation or hairpin sequestering the SD binding site) are colored in blue. (D, E, F) Predicted secondary structure of three bona fide small RNAs. The UCCCU sequence motif in red in Teg130 was shown to be conserved in several sRNAs from S. aureus. This sequence is well appropriate to bind to the ribosome binding site of target mRNAs .

Figure 3. Schematic representation of antisense transcripts in the tnp region.

Among the transposases annotated in the genome of S. aureus N315, antisense RNAs were identified 8 times at the extremities of the tnp transcripts, suggesting an important functional role. This example shows Teg24as and Teg17 (pink italic font) and their complementary tnp gene (green). In the sequences detailed below, the potential promoter sequences are underlined and the potential Shine-Dalgarno sequences appear in bold fonts.

Figure 4. Temporal expression of selected RNAs obtained by RNA-Seq Illumina-HTS.

Transcript expression levels are shown at 2 hours (red), 4h (green), 6h (blue), and 8h (black). The X-axis corresponds to N315 genomic coordinate and Y-axis symbolizes the normalized coverage. We smoothed the coverage profiles by using a sliding average window of size 41. The expression profile of control genes has been used for experimental validation. General profiles are consistent with published studies with respect to the temporal expression of these transcripts. The experiment shows that the expression of most small transcripts identified in this study are regulated during the growth phase.

Figure 5. Temporal expression of selected transcripts in rich medium and under various stress conditions.

Analysis of the expression of 26 selected transcripts by RT-qPCR in rich medium (MHB) and under four stress conditions: oxidative stress, pH stress, heat and cold shocks, changing of carbon source with stringent medium. The data have been normalized against the hu reference gene. We used m-e for mid-exponential phase and s for stationary phase to map the kinetics observed in MHB. Transcript induction or repression has been expressed as fold change against the MHB reference condition. The color scale reflecting the intensity of expression changes: dark red: fold change>100, light red: 1.3<fold change<100, yellow: −1.4<fold change<1.3, light green: −100<fold change<−1.4, and dark green: fold change>−100. ND corresponds to “Not Determined” value, whenever no expression of tested RNA was visualized before the 40 PCR cycles. The symbol “>” is applied whenever signal was detected for the target but no in the MHB reference. Note that Teg17 and Teg18 are found in multicopy. The expression observed in RT-qPCR is therefore the addition of transcription level from different coding sequences or belongs to another genomic location.