Antibiotic resistance is a growing threat, underscoring the need to understand the underlying mechanisms. Aminoglycosides kill bacteria by disrupting translation fidelity, leading to the synthesis of aberrant proteins. Surprisingly, mutations in fusA, a gene encoding translation elongation factor G (EF-G), frequently confer resistance, even though EF-G neither participates in mRNA decoding nor blocks aminoglycoside binding. Here, we show that EF-G resistance variants selectively slow ribosome movement along mRNA when aminoglycoside is bound. This delay increases the chance that the drug dissociates before misreading occurs. Over several elongation cycles, this selective silencing of drug-bound ribosomes prevents error clusters formation, preserving proteome and membrane integrity. As a result, fusA mutations confer resistance early in treatment by preventing self-promoted aminoglycoside uptake. Translation on drug-free ribosomes remains sufficiently rapid to sustain near-normal bacterial growth. This previously unrecognized resistance mechanism—selective silencing of corrupted targets—reveals a novel antibiotic resistance strategy with potential therapeutic implications.

Figure 4B/C/S7B: Aminoglycoside (AGA)-induced missense peptides were targeted by label free Parallel Reaction Monitoring (PRM). Missense peptides with individual amino acid substitutions were identified by their close to one dot product derived from DDA data. Figure 5A/C: AGA-induced missense peptides were targeted by label-free PRM. Stable isotope labeled reference (SIL) peptides were used to confidently identify missense peptides with single and consecutive amino acid substitutions (not used for quantification). Figure 5D/E/F: AGA-induced missense peptides with consecutive amino acid substitutions were targeted by label-free PRM. SIL peptides were used for confident identification of missense peptides. Figure 6B: 2-3 peptides of proteins of the unfolded stress response (IbpA, IbpB, RpoH) were targeted by label-free PRM using SIL peptides for identification. Peptides of constitutively expressed proteins uL10 and EF-Tu were quantified as loading controls. Figure S8B: Missense peptides were detected by data-dependent acquisition, identified as dependent peptides in MaxQuant and subsequent analysis pipelines (PMID: 31353208) and quantified by MS1 filtering in Skyline.

Figure 4B: Escherichia coli strains (carrying mutations in the translation elongation factor G (EF-G): F593L, A608E, P610L, wt, and parental strain MG1655) were grown in LB medium in the presence and absence of apramycin (16 µM). Cells were lysed and proteins were separated using Criterion TGX SDS PAGE gels. The EF-Tu (most prominent) band was excised and in-gel proteolysed with trypsin. Figure 4C: Cells were grown as in Figure 4B. After 1 h of Apr treatment, cells were harvested and inverted inner membrane vesicles were prepared to quantify missense peptides in inner membrane proteins. Proteins were separated using Criterion TGX SDS PAGE gels. Prominent protein bands were cut from the gel and proteoylzed with trypsin. Figure 5A/C: Same strains were grown as described above. Apramycin was added at different concentrations and strains were incubated for indicated time intervals. Missense peptides were quantified in EF-Tu. Figure 5D: Strains (P610L and wt) were grown as described above (-/+ 16µM Apr). Missense peptides with consecutive amino acid substitutions were quantified in EF-Tu. Figure 5E: Strains (F593L, A608E, P610L, wt) treated as in Figure 5D. Figure 5F: Strains (F593L, A608E, P610L, wt) were treated with various AGAs. For each AGA, concentrations that provoke maximal misreading were chosen and missense peptides were quantified in EF-Tu. Figure 6B: Strains (F593L, A608E, P610L, wt) grown in the absence and presence of Apr (16 µM, 180 min) were harvested. Proteins were desalted by SDS-PAGE and in-gel proteolyzed with trypsin. Figure S7B: Biological replicates of cells treated as in Figure 4B (16 µM Apr, 60 min incubation). Figure S8B: Strains (P610L and wt) were grown in the absence and presence of different concentrations Apr. Missense peptides with consecutive amino acid substitutions were quantified in EF-Tu.