Identification of small proteins in Staphylococcus aureus
To identify novel small open reading frames (sORFs) and validate the production of the sORF-encoded small proteins in Staphylococcus aureus, we combined multi-omic approaches, including total RNA sequencing (RNA-seq), ribosome profiling (RIBO-seq) and shotgun proteomics analyses.The S. aureus 15981 strain, a clinical isolate from an otitis media infection, was grown in a rich medium and incubated at environmental- and host-related temperatures (22 and 37*C, respectively). Total RNAs, polysomes and protein extracts were obtained from the bacterial cultures and subjected to RNA-seq, RIBO-seq and mass spectrometry analyses, respectively.The results from each analysis were converted into signal track files and integrated into a web server (available at http://rnamaps.unavarra.es), using the genome browser JBrowse application.To facilitate sORFs identification, the browser was loaded with three different genome annotation files for the S. aureus NCTC 8325 strain, including those generated by the NCBI staff (https://www.ncbi.nlm.nih.gov/nuccore/NC_007795.1/), the Bacterial and Viral Bioinformatics Resource Center (BV_BRC, https://www.bv-brc.org/view/Genome/93061.5), and the Bakta web application (https://bakta.computational.bio). Additionally, the web browser was enhanced with supplementary tracks compiling data from previous studies, including transcriptomic sequencing data of S. aureus 15981 strain (Lasa et al., 2011), transcriptional start sites (TSSs) mapping of an S. aureus Sc-01 derivative strain (strain O) (Koch et al., 2014), RIBO-seq analysis of S. aureus JE2 strain grown in rich and minimal medium (Basu and Yap, 2016), and shotgun proteomic data of extracellular vesicles purified from S. aureus CICC 10384 strain (Wei et al., 2022).Altogether, the combination of multi-omic analyses using both in-house and publicly available data contributed to validating the expression of 330 small proteins shorter than 100 amino acids and revealed 31 novel small proteins from the S. aureus dark proteome.