An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The "Staphylococcal Regulatory RNA Database" (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNAseq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature.

The human pathogen Staphylococcus aureus expresses a set of transcriptional factors and small RNAs (sRNAs) to adapt to environmental variations. Recent harmonization of staphylococcal sRNA data allowed us to search for novel sRNAs using DETR’PROK, a computational pipeline for identifying sRNA in prokaryotes. We performed RNA-Seq on Newman strain and identified a set of 48 sRNA candidates. To avoid bioinformatic artefacts, we applied a series of cut-offs and tested experimentally each selected intergenic region. This narrowed the field to 24 expressed sRNAs, of which 21 were new and designated with Srn identifiers. Further examination of these loci revealed that one exhibited an unusual condensed sRNA cluster of about 650 nucleotides. We determined the transcriptional start sites within this region and demonstrated the presence of three contiguous sRNA genes (srn_9342, srn_9344 and srn_9345) expressed from the positive strand, and two others (srn_9343 and srn_9346) transcribed from the opposite one. Using comparative genomics, we showed that genetic organization of the srn_9342-9346 locus is specific to Newman and that its expression is growth-phase dependent and subjected to nutrient deprivation and oxidative stress. Finally, we demonstrated that srn_9343 encodes a secreted peptide that could belong to a novel S. aureus toxin-antitoxin system.

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.

Staphylococcus aureus , a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs.