tRNA Modification Landscapes in Streptococci: Shared Losses and Clade-Specific Adaptations

tRNA modifications are central to bacterial translational control. Here, we integrated genetics, mass spectrometry, epitranscriptomics, and comparative genomics to map the tRNA modification genes of the Gram-positive pathogens Streptococcus mutans and Streptococcus pneumoniae. Both species show a marked loss of modifications dependent on Fe-S enzymes, consistent with a broader trend of Fe-S enzyme reduction in Streptococcus central metabolism. In addition, the D, m¹A, m⁷G, t⁶A, and i⁶A modifications were mapped in S. pneumoniae tRNAs, and we confirmed that a unique DusB1 enzyme is responsible for the insertion of all the detectable D modifications. We uncovered differences in queuosine (Q) metabolism: while S. mutans synthesizes Q de novo, S. pneumoniae instead salvages preQ₁ and accumulates the epoxy-Q precursor, a strategy shared with multiple other Streptococci as revealed by analysis of Q pathways in 1,599 sequenced streptococcal genomes. Comparative essentiality profiling of modification genes revealed notable differences, including the essentiality of the N -threonylcarbamoyladenosine (t A) synthesis enzyme TsaE in S. pneumoniae but not in S. mutans, which was confirmed by genetic studies. We found that suppressor mutations in asnS encoding asparaginyl-tRNA synthetase (AsnRS) restored viability to ΔtsaE mutants, albeit with reduced growth. Our finding highlights the functional importance of modifications in the recognition of tRNAs by aminoacyl-tRNA synthetases.