Codon usage determines tRNA-modification requirements for bacterial virulence

Bacterial virulence genes exhibit distinctive codon usage that may impose tRNA-modification requirements for efficient translation, but a comprehensive understanding is lacking. Here, a curated codon-tRNA modification map for Salmonella predicted six modifications that could preferentially support virulence gene translation. Mutant phenotypes in infected mice confirmed requirements for i⁶A ₃₇ and C ⁵ modification of U ₃₄ , whereas essential k ² C ₃₄ could not be tested. Q ₃₄ , Cm ₃₂ /Um ₃₂ , and Cm ₃₄ had limited impact consistent with weak decoding effects in other systems. Non-predicted tRNA modifications were not required. Synonymous reporter variants, proteomics, and targeted recoding demonstrated that virulence-associated codons directly conferred modification dependence. Additionally, four Leu-TTA codons in ssrB , the master regulator of Salmonella pathogenicity island 2 (SPI-2), contributed to MiaA-dependent SPI-2 gene expression. Extending our analysis to other pathogens predicted shared and divergent virulence dependencies consistent with published mutant phenotypes. Thus, codon usage explains tRNA-modification requirements for bacterial virulence.