Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus

In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome anno- tation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions.

在金黄色葡萄球菌中，已鉴定出数百种小型调控或小型核糖核酸（sRNAs），然而此类分子仍处于理解不足且研究匮乏的状态。sRNA基因通常不包含在基因组注释文件中，因此其存在往往被忽视，尤其是在全球转录组研究中。为促进金黄色葡萄球菌中sRNAs的检测与分析，我们针对三种常用的金黄色葡萄球菌菌株（MRSA252、NCTC 8325和USA300）生成了更新的GenBank文件，并在其中为超过260种已鉴定的sRNA添加了注释。这些文件是首次在金黄色葡萄球菌中进行全基因组sRNA注释的文件，随后被用作发现社区相关耐甲氧西林菌株USA300中新sRNA的基础。该分析导致了39种之前未识别sRNA的发现。研究这些新识别sRNA的基因组位点揭示了金黄色葡萄球菌基因组注释中存在令人惊讶的不一致性，这可能阻碍了这些元素的分析与功能探索。最后，以我们新创建的注释文件为参照，我们对金黄色葡萄球菌中sRNA基因的表达进行了全局分析，并证实新发现的tsr25是人类血清中最高度上调的sRNA。本研究以我们新生成的注释文件的形式，为金黄色葡萄球菌研究界提供了宝贵的资源，同时首次展示了在病理生理相关条件下的sRNA表达差异分析。