Phylogenetic distribution of Mtb tRNA modifying enzyme homologs.

Heat map of log10 E-values from BLAST search results. BLAST searches were conducted against 120 manually picked organisms using Mtb tRNA modifying enzymes as queries. When one organism has multiple hits, the lowest log10(Eval) values among hits is shown. iTol[34] was used to depict the results.

Heat map of misincorporation and early termination frequency in sequencing of tRNAs from wild type Mtb.

(A, B) Heatmaps show misincorporation (A) and termination (B) frequencies at all positions across tRNAs (read 5’ to 3’). Predicted modifications are labeled based on similarity to known modifications in other organisms and the presence of the tRNA modifying enzyme homologs (Supplementary Table 3). The positions with more than 10% misincorporation in Mtb but not in E. coli are depicted in white in A. (C) M. tuberculosis tRNA modifications predicted in this study. Schematic tRNA secondary structure with sites of modifications identified either by the presence of modifying enzymes and/or tRNA-seq. Modifications and tRNA species that are not observed in E. coli are shown in red. Modifications and positions that are predicted by both RT-derived signature and the presence of the homologs of tRNA modifying enzymes are shown in yellow (without chemical treatment) and green (with chemical treatment), whereas modifications that are only predicted by the presence of the homologs are shown in light blue. Genes reported to be essential in Mtb are shown in bold.

IAA treatment promotes detection of sulfur modifications on tRNAs by enhancing termination signals.

(A, B) Heatmaps of the termination signals of E. coli tRNAs treated with (A) or without (B) IAA. Known modification sites, including sulfur modifications (s4U, s2C, s2U in white) are shown. (C) Termination frequency at s4U, s2C, and s2U sites of tRNAs treated with or without IAA.

Heat map and plot of early termination frequency from sequencing of tRNAs from wild type and MtbΔmnmA with and without RNA alkylation.

(A, B) Heat map of early termination frequencies across tRNA molecules and positions for WT (A) and MtbΔmnmA (B). Sulfur modification is shown in white. (C) Plot of termination frequencies at position 37 in wild type (WT) Mtb and MtbΔmnmA for lysine_UUU, glutamate_UUG, and glutamine_UUC isoacceptors. IAA; iodoacetamide.

CMC and alkali treatment facilitate detection of Ψ, D, and m7G modifications in E. coli.

(A, B) Heatmaps of the misincorporation signals of E. coli tRNAs treated with (A) or without (B) CMC. In both conditions, tRNAs are incubated in alkali condition. Known Ψ, D, and m7G sites are shown. Ψ is shown in white. (C) Misincorporation frequency at known Ψ, D, m7G sites of tRNAs treated with CMC+/alkali+ or CMC-/alkali+, and tRNAs without treatment.

Heat map of early termination frequency from sequencing of tRNAs isolated from WT and MtbΔtruB following CMC treatment.

Heat map of early termination frequencies across tRNA molecules and positions for WT (left) and MtbΔtruB (right). Termination signals derived from position 55 are shown in white.

MtbΔmnmA is attenuated in a macrophage infection model.

Top: Wild type and MtbΔmnmA do not display growth differences in 7H9 medium. Bottom: Auto-luminescent wild type and ΔmnmA Mtb strains were diluted to a multiplicity of infection of 2 bacteria per mouse bone marrow-derived macrophage with or without all-trans retinoic acid (ATRA). Survival was measured by luminescence and normalized to luminescence at time 0.