tRNA hydroxylation links epitranscriptomic modification to metabolic regulation in Pseudomonas aeruginosa

Post-transcriptional modification of 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 the establishment of virulence phenotypes. In this study, we used mass-spectrometry and nanopore sequencing to systematically identify and quantify sites of TrhPO-dependent tRNA hydroxylation in total and purified tRNAs in the opportunistic human pathogen Pseudomonas aeruginosa. Furthermore, transcriptomic, ribosome profiling, and proteomic data were integrated to demonstrate the post-transcriptional consequences of the absence of xo5U modifications at wobble positions in selected tRNAs. We show that changes in metabolic fluxes drive the impaired ability to infect host cells and strongly attenuated virulence in the Galleria mellonella model. 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 highlights the multifunctional roles of tRNA-modifying enzymes and suggests 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.