Dynamic tRNA modification programs align codon usage with growth-phase-specific gene expression in Pseudomonas aeruginosa

Post-transcriptional transfer RNA (tRNA) modifications dynamically regulate translational capacity, yet whether the tRNA epitranscriptome is temporally coordinated with gene expression programs during bacterial growth remains unknown. Here, we combine time-resolved nanopore tRNA sequencing, liquid chromatography–tandem mass spectrometry modification profiling, transcriptomics, and proteomics across the Pseudomonas aeruginosa PA14 growth cycle to show that tRNA-modifying enzymes, the corresponding tRNA modifications, and modification-dependent genes are expressed in a coordinated temporal cascade. GidA-dependent 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) modifications exemplify this program: GidA expression peaks during early growth, followed by rising mnm 5 s 2 U levels coinciding with maximal expression of genes enriched in rare AGA and UUA codons, with a concomitant enrichment of virulence-associated functions. This sequential pattern is consistent with a feedforward architecture in which the modification machinery acts in advance of translational demands. This mechanism is especially relevant for a set of horizontally acquired genes, suggesting that temporally programmed tRNA epitranscriptomic remodeling may help bridge local codon usage deviations between horizontally acquired genes and the core translational context.