Loss of the s²U tRNA modification induces antibiotic tolerance and is linked to changes in ribosomal protein expression

Stress promotes phenotypic changes in bacteria that allow them to survive antibiotic treatment. This phenomenon, termed antibiotic tolerance, can cause treatment failure, highlighting a need to define bacterial pathways that promote survival. Previously, we found Yersinia pseudotuberculosis downregulates tusB, a gene involved in modifying tRNAs with s²U, in response to doxycycline. Here we find that deletion of tusB results in loss of s²U and induces antibiotic tolerance. Using a combination of sequencing-based approaches and analysis of gene codon usage, our data show that loss of s²U decreases translation of ribosomal proteins. Ribosomal proteins are highly enriched in codons that require s²U-modified tRNAs for efficient translation, and loss of s²U results in ribosome pausing at these codons. Our results highlight a previously unknown mechanism of antibiotic tolerance where reduction in ribosomal protein abundance can globally reduce translation, and describes a novel strategy bacteria use to slow growth by modulating s²U levels.