Kouvela Adamantia, Jaramillo Ponce Jose R, Giarimoglou Nikoleta, Chicher Johana, Marzi Stefano, Stathopoulos Constantinos, Stamatopoulou Vassiliki
Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece.
Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, 67000 Strasbourg, France.
Nucleic Acids Res. 2025 Jul 8;53(13). doi: 10.1093/nar/gkaf599.
Redundancy in transfer RNA (tRNA) gene copies across species remains poorly understood and, in many cases, largely unexplored. In Staphylococcus aureus, multiple tRNAGly genes encode isoacceptors involved in protein synthesis and cell wall formation, aminoacylated by a sole glycyl-tRNA synthetase (GlyRS) which is under the transcription regulation of a species-specific glyS T-box riboswitch. The T-box can interact with all tRNAGly isoacceptors to adopt species-specific conformations and affect both pathways. Using CRISPR/Cas9 editing, we ablated a gene copy corresponding to the proteinogenic P1 tRNAGlyGCC. Surprisingly, the growth and the overall translational activity of the edited strain were found unaffected, suggesting functional compensation by the remaining tRNAGly genes. On the other hand, transcriptomics and proteomics analyses combined with functional assays revealed nutrient-dependent stress responses with surprisingly impaired cell wall integrity and increased susceptibility to cell wall-targeting antibiotics. Additionally, the edited strain displayed reduced biofilm formation but retained the ability to invade human cells in vitro. Overall, the present study underscores the critical role of tRNA gene redundancy in the physiology of S. aureus and highlights tRNAs as regulators of metabolic homeostasis in pathogenic bacteria.
不同物种间转运RNA（tRNA）基因拷贝的冗余情况仍未得到充分理解，在许多情况下，基本上还未被探索。在金黄色葡萄球菌中，多个tRNAGly基因编码参与蛋白质合成和细胞壁形成的同功受体，它们由唯一的甘氨酰-tRNA合成酶（GlyRS）进行氨酰化，而该合成酶受物种特异性glyS T-盒核糖开关的转录调控。T-盒可与所有tRNAGly同功受体相互作用，以形成物种特异性构象，并影响这两条途径。利用CRISPR/Cas9编辑技术，我们敲除了与蛋白质合成用P1 tRNAGlyGCC相对应的一个基因拷贝。令人惊讶的是，发现编辑菌株的生长和整体翻译活性未受影响，这表明其余tRNAGly基因进行了功能补偿。另一方面，转录组学和蛋白质组学分析结合功能测定揭示了营养依赖性应激反应，其细胞壁完整性令人惊讶地受损，且对靶向细胞壁的抗生素敏感性增加。此外，编辑菌株的生物膜形成减少，但在体外仍保留侵入人类细胞的能力。总体而言，本研究强调了tRNA基因冗余在金黄色葡萄球菌生理学中的关键作用，并突出了tRNA作为病原菌代谢稳态调节因子的作用。
