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 cutoffs 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. Staphylococcus aureus is an opportunistic pathogen responsible for a large spectrum of human and animal infections 1. The bacterium finely modulates gene expression to efficiently adapt its growth and physiology to the local environment. Besides global transcriptional regulators, small RNAs (sRNAs, also cited as regulatory RNAs when a regulatory function was demonstrated) have emerged as key players in a wide range of biological processes, from central metabolism to virulence and antibiotic resistance 2–4. In S. aureus, sRNAs, are typically 50-to 500-nucleotides (nt) long. Some of them regulate mRNA expression and/or stability without the need of Hfq chaperone 5, 6. sRNAs with regulatory functions were first discovered by Mizuno in E. coli 7 and then by Novick in S. aureus 8. They can modulate target mRNA expression by cis-or transacting mechanisms 2. Cis-encoded sRNAs are transcribed from the opposite strand of an mRNA or another sRNA. Accordingly, they display perfect complementarity with their target sequences, although their ability to bind other RNAs cannot be excluded. Trans-encoded sRNAs are transcribed apart from their targets, and usually display only partial complementarity with them. Over the last decade, several studies have focused on the identification of S. aureus sRNAs via bioinformat-ics, next-generation sequencing (NGS), and other experimental approaches 9–21. This has resulted in the publication of hundreds of RNA sequences, but their functions are mostly unknown. Most of these studies were done using methicillin-resistant strain N315 11, 18. In others, newly discovered sRNAs were described based on their genomic location in that particular strain 9, 12, 13, 16. S. aureus sRNA identification has been hampered by the lack of naming consensus and the absence of dedicated annotation files. Recently, we collected all of the sequences published so far, proposed a simple sRNA gene identifier (srn) to avoid redundancies, and provided Inserm U1230 Biochimie Pharmaceutique, Université de Rennes 1, Rennes, France. Correspondence and requests for materials should be addressed to Y.A.