tRNA hydroxylation is an epitranscriptomic modulator of metabolic states affecting Pseudomonas aeruginosa pathogenicity.

Post-transcriptional modification of transfer RNAs (tRNAs) represents an essential layer of translational regulation critical for bacterial adaptation to environmental changes. Increasing evidence links the tRNA epitranscriptome to pivotal roles in the regulation of gene expression and various cellular processes, including stress responses and establishment of virulence. In this study, we used mass spectrometry and nanopore sequencing to quantify and identify the sites of TrhPO-dependent tRNA hydroxylation in total and purified Pseudomonas aeruginosa tRNAs. Furthermore, transcriptome, ribosome profiling, and proteome data were integrated to demonstrate the post-transcriptional consequences of the absence of xo5U34 modifications at the wobble position of selected tRNAs. We suggest that the impaired ability to infect host cells and attenuated virulence in Galleria mellonella are driven by changes in metabolic fluxes. In the absence of TrhPO-mediated tRNA modification, chorismate, the precursor for the biosynthesis of xo5U modifications, is funneled into alternative pathways, including the production of aromatic amino acids and phenazines. Our findings that metabolic rerouting, rather than changes in proteome profiles, attenuates P. aeruginosa virulence highlight the multifunctional roles of tRNA-modifying enzymes and suggest an underexplored role for these enzymes in monitoring and modulating metabolic fitness. These insights open new avenues for combatting the pathogenicity of this challenging opportunistic pathogen.