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

Analysis of prophages harbored by the human-adapted subpopulation of Staphylococcus aureus CC398

“…4). This is consistent with the general increase in gene expression during stress, particularly during infection (Beaume et al, 2010). Virulence-related mRNAs were similarly abundant during stationary and exponential growth phases in both S94 and S89, two isolates that only harbour StauST398-4pro; however, these mRNAs were more abundant in S81, the isolate carrying StauST398-4pro and Fig.…”

“…DNA of pCN38 was prepared from Escherichia coli DH5a and Staphylococcus RN4220 (Lee, 1995;Gizard et al, 2013). DNA of pMW401 was prepared from Enterococcus faecalis as previously described (Beaume et al, 2010). The media used for overnight culture and subsequent mini-preps (Qiaprep Spin Miniprep Kit, Qiagen) included Luria Bertani Broth (LB Broth, Miller, BD Difco) for E. coli DH5a, supplemented with 100 mg/L of ampicillin; Mueller-Hinton broth (MHB; BD Difco) for S. aureus RN4220; and Tryptic soy Broth for E. faecalis (Soyban-Casein Digest Medium, BD Bacto).…”

“…Chloramphenicol (10 mg/L) was included as appropriate. Preparation of competent cells and transformation of RN4220, S100, S123, S124, S124/S100-u, S92, S93 and S94 were as described previously (Beaume et al, 2010). Following electroporation, transformants were plated on Mueller-Hinton Agar, supplemented with chloramphenicol: 10 mg/L for RN4220; 15 mg/L for S100, S123, S124, S124/S100-/; and 20 mg/L for S92, S93, S94.…”

“…File Table 2. Oligonucleotide primers and probes were used at final concentrations of 0.2 and 0.1 lM, respectively, in a final volume of 10 lL of one-step RT-PCR enzymatic mixture (Invitrogen, Carlsbad, Germany); a Mx3005P system (Agilent) was used as described previously (Beaume et al, 2010). Results were normalized to those for the hu gene, as described previously.…”

Genome-wide antisense transcription drives mRNA processing in bacteria

“…In the past 10 years, the development of new approaches based on high-resolution tiling arrays and RNA deep sequencing (RNA-seq) has uncovered that a significant proportion (depending on the study, varying between 3% and >50%) of protein coding genes are also transcribed from the reverse complementary strand (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In most cases, overlapping transcription generates a noncoding antisense transcript whose size can vary between various tens of nucleotides (cisencoded small RNAs) to thousands of nucleotides (antisense RNAs).…”