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.

为鉴定金黄色葡萄球菌（Staphylococcus aureus）中新的小型开放阅读框（small open reading frames, sORFs）并验证sORF编码的小型蛋白的表达，我们结合了多组学研究方法，涵盖总RNA测序（total RNA sequencing, RNA-seq）、核糖体印迹测序（ribosome profiling, RIBO-seq）与鸟枪法蛋白质组学分析。本研究使用的金黄色葡萄球菌15981菌株为中耳炎感染临床分离株，将其接种于丰富培养基中，分别在环境相关温度（22℃）与宿主相关温度（37℃）下培养。分别从细菌培养物中提取总RNA、多聚核糖体与蛋白质提取物，依次开展RNA-seq、RIBO-seq及质谱分析。
将各分析所得结果转换为信号轨迹文件，并整合至基于基因组浏览器JBrowse搭建的网页服务器（访问地址：http://rnamaps.unavarra.es）。为便于sORFs的鉴定，该基因组浏览器加载了金黄色葡萄球菌NCTC 8325菌株的三套基因组注释文件，分别由NCBI团队（https://www.ncbi.nlm.nih.gov/nuccore/NC_007795.1/）、细菌与病毒生物信息学资源中心（Bacterial and Viral Bioinformatics Resource Center, BV-BRC，https://www.bv-brc.org/view/Genome/93061.5）以及Bakta网页应用（https://bakta.computational.bio）生成。
此外，该基因组浏览器还补充了源自既往研究的附加轨迹数据集，包括金黄色葡萄球菌15981菌株的转录组测序数据（Lasa等，2011）、金黄色葡萄球菌Sc-01衍生菌株（O菌株）的转录起始位点（transcriptional start sites, TSSs）定位结果（Koch等，2014）、在丰富培养基与基本培养基中培养的金黄色葡萄球菌JE2菌株的RIBO-seq分析数据（Basu与Yap，2016），以及从金黄色葡萄球菌CICC 10384菌株纯化的细胞外囊泡的鸟枪法蛋白质组数据（Wei等，2022）。
综上，结合本研究内部数据与公开可用数据集开展的多组学联合分析，共验证了330个长度小于100个氨基酸的小型蛋白的表达，并从金黄色葡萄球菌暗蛋白质组（dark proteome）中发现了31种新型小型蛋白。