5-Methyluridine is Ubiquitous in Pseudomonas aeruginosa tRNA and Modulates Antimicrobial Resistance and Virulence.

Building on decades of work in characterizing the dozens of RNA modifications in the microbial epitranscriptome, recent advances in analytical technology and genetics have revealed systems-level functions for many tRNA modifications. The tRNA (uracil-5-)-methyltransferase TrmA and its product, 5-methyl uridine (m5U) at position 54 in the T-loop, however, has not been linked to a specific phenotype. Here, we defined the functional and biological roles of TrmA in Pseudomonas aeruginosa (PA14), a major multidrug-resistant pathogen. Surprisingly, though TrmA was found to site-specifically catalyze m5U54 on all PA14 tRNAs, loss of TrmA had no effect on the levels of any of 36 tRNA modifications except m5U and had minimal effects on multiple phenotypic parameters, including growth rate, morphology, motility, and biofilm formation. However, loss of TrmA conferred a striking polymyxin antibiotic resistance. mRNA and tRNA profiling and proteomics analyses revealed that TrmA regulates the expression of codon-biased gene families at the level of translation, including components of a type III secretion system (T3SS). Loss of TrmA upregulated T3SS, leading to increased macrophage IL-1β in bacterial challenge tests. Altogether, these results revealed novel biological functions of TrmA and its roles in modulating gene expression at multiple levels in P. aeruginosa.