Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility, and virulence potential.

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.