Vibrio cholerae tRNA epitranscriptome

Transfer RNAs (tRNAs) are central to protein synthesis, ensuring precise decoding of the genetic code by delivering aminoacids to the ribosome. Among all RNA species, tRNAs are the most heavily and diversely modified, with modifications playing critical roles in stability, folding, and function. Here, we present a comprehensive, isodecoder-level map of tRNA modifications in the human pathogen Vibrio cholerae. This map was generated by chemical-based sequencing methods, comparing wild-type and deletion strains. By linking specific modifications and corresponding enzymes, we established a detailed modification landscape, and uncovered species-specific features, such as the presence of a functional TrmK enzyme, previously considered restricted to Gram-positive bacteria. To assess the biological significance of these modifications, we evaluated fitness under both standard conditions and subinhibitory antibiotic stress, and examined how modifications in the anticodon stem-loop region influence codon decoding efficiency and accuracy. Based on a comparative analysis of E. coli and V. cholerae, we discuss how species-specific differences in tRNA isodecoder gene repertoires may influence the functional impact and biological importance of tRNA modifications. This work provides the first experimentally validated, genome-wide map of tRNA modifications in V. cholerae, serving as a reference for future research into RNA modifications, translation regulation, and pathogen biology.