Comprehensive post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

Post-transcriptional modifications modulate transfer RNA (tRNA) structure, stability, and codon decoding properties, contributing to translation regulation and adaptation across diverse organisms, including bacterial pathogens. We provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of m2A37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and evidence suggesting a dedicated pseudouridine synthase responsible for Ψ32. Besides, heterogeneous modification patterns were observed in tRNALeu(UAA) and tRNALys(UUU), highlighting a complex interplay in anticodon hypermodification. Time-course proteomics revealed dynamic expression of tRNA modifying enzymes during growth. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of S. aureus decoding properties, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework to dissect the role of tRNA modifications in S. aureus physiology and pathogenesis.