Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs inStaphylococcus aureus

Abstract

The opportunistic pathogen Staphylococcus aureus expresses transcription factors (TFs) and regulatory small RNAs (sRNAs) which are essential for bacterial adaptation and infectivity. Until recently, the study of S. aureus sRNA gene expression regulation was under investigated, but it is now an expanding field. Here we address the regulation of Srn_3610_SprC sRNA, an attenuator of S. aureus virulence. We demonstrate that SarA TF represses srn_3610_sprC transcription. DNase I footprinting and deletion analyses show that the SarA binding site on srn_3610_sprC belongs to an essential 22 bp DNA region. Comparative analysis also revealed another possible site, this time in the srn_9340 promoter. SarA specifically binds these two sRNA promoters with high affinity in vitro and also represses their transcription in vivo. Chromatin immunoprecipitation (ChIP) assays confirmed SarA attachment to both promoters. ChIP and electrophoretic mobility shift assays targeting σA RNA polymerase subunit or using bacterial RNA polymerase holoenzyme suggested that SarA and the σA bind srn_3610_sprC and srn_9340 promoters in a mutually exclusive way. Beyond the mechanistic study of SarA repression of these two sRNAs, this work also suggests that some S. aureus sRNAs belong to the same regulon and act jointly in responding to environmental changes.

Assessment of Bona Fide sRNAs in Staphylococcus aureus

“…Besides the Agr system, as an example, SarA is a transcriptional factor belonging to the core genome, which is implicated in infectivity and biofilm formation. SarA represses sRNA287 and SprC, two sRNAs located on the same pathogenicity island (Mauro et al, 2016 ).…”

A novelStaphylococcus aureus cis–transtype I toxin–antitoxin module with dual effects on bacteria and host cells

“…Therefore, we monitored the transcript levels of another type I TA module (SprG1/SprF1) and of two regulatory RNAs, Srn_9340 and Srn_3610_SprC (Supplementary Figure S9). SprF1 and these regulatory RNAs have half-lives of 10, 5 and 20 min, respectively (25,30), and they all showed reduced transcript levels upon SprA1/2 induction. Conversely, the RNA levels of SprG1, which has a half-life of ∼2 h (20), did not decrease after SprA1/2 induction.…”

“…Recombinant plasmids (Table 1) were transformed in S. aureus as previously described (25). S. aureus was cultured in BHI broth supplemented with the appropriate antibiotics at 37°C and under agitation until reaching an appropriate OD 600nm (see Results).…”

Noncoding RNA

“…The staphylococcal accessory regulator SarA is synthesised from three distinct promoters (P1, P2, P3) and binds DNA or RNA (Bischoff and Romby, 2016). As a transcription factor, it regulates many genes involved in virulence, autolysis, biofilm formation, stress response, antibiotic resistance, or metabolism, but also two sRNAs, SprC and Srn_9340 located on the same pathogenicity island (Mauro et al, 2016). SprC prevented ribosomes to bind the SD of the atl mRNA coding for an autolysin, then reducing internalization by macrophages and attenuating virulence (Le Pabic et al, 2015).…”

“…SprC prevented ribosomes to bind the SD of the atl mRNA coding for an autolysin, then reducing internalization by macrophages and attenuating virulence (Le Pabic et al, 2015). SarA repressed both sprC and srn_9340 transcription and required an ATTTTAT sequence in its binding site (Mauro et al, 2016). However, while SarA level remains relatively constant along the bacterial growth, the expression of SprC fluctuated, which suggest that additional factors might control its synthesis and that a mechanism of derepression should co-exist under specific conditions.…”