Fig. 1: Nanopore direct RNA sequencing can be used to study bacterial rRNA modifications involved in antibiotic resistance mechanisms.

A 3D structure of the ribosome (gray) depicting the tRNA in the P-site (orange) and A-site (green). The residues surrounding the kasugamycin binding site are shown in blue, and are located near the anticodon region of the tRNA that is located at the P-site, whereas residues surrounding the streptomycin binding site are shown in pink, and are located near the anticodon region of the A-site tRNA. Surrounding residues were defined as those that have at least one of its atoms at less than 10 Å from the antibiotic. In the zoomed regions, modified nucleotides involved in antibiotic resistance are represented as brown surfaces, whereas the antibiotics are shown in black. PDB structure corresponds to 7K00. B Growth curves of E. coli WT (left) and E.coli rsmG KO (right) upon increasing concentrations of streptomycin. Data shown represents the average OD₆₀₀ values of 2 independent biological replicates. Each biological replicate was calculated as the median of 3 technical replicates. Data was collected every 9 minutes, during a time course of 16 h. Source data are provided as a Source Data file. C Growth curves of E. coli WT (left) and E.coli rsmA KO (right) upon increasing kasugamycin concentrations. Data shown represents the average OD₆₀₀ values of 2 independent biological replicates, and each biological replicate was calculated as the median of 3 technical replicates. Data was collected every 9 minutes, during a time course of 16 h. Source data are provided as a Source Data file. D IGV snapshots of E.coli WT and knockout strains illustrating loss of base-calling errors upon enzyme knockout (rsmG and rsmA). Positions with mismatch frequencies greater than 0.1 are colored, whereas positions with mismatch frequencies lower than 0.1 are shown in gray. E Scatterplots of the summed base-calling error frequencies (sum of insertion, deletion and mismatch frequencies) at each nucleotide position in the knockout strain, relative to WT. The rRNA modified sites that are lost upon knockout of the gene are shown in red (position 0); the neighboring positions (±4 nt) to the rRNA modification site (position 0) are shown in blue. Remaining positions are shown in gray.