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

Reviewer #1 (Public review):

Summary:

Cotton et al. investigated the role of tusB in antibiotic tolerance in Yersinia pseudotuberculosis. They used the IP2226 strain and introduced appropriate mutations and complementation constructs. Assays were performed to measure growth rates, antibiotic tolerance, tRNA modification, gene expression and proteomic profiles. In addition, experiments to measure ribosome pausing and bioinformatic analysis of codon usage in ribosomal proteins provided in-depth mechanistic support for the conclusions.

Strengths:

The findings are consistent with the authors having uncovered new mechanistic insights into bacterial antibiotic tolerance mediated by reducing ribosomal protein abundance.

Weaknesses:

Since the WT strain grows faster than the tusB mutant, there is a question of how growth rate, per se, impacts some of the analysis done. The authors should address this issue. In addition, it may not be essential, but would analysis of another slow-growing mutant (in some other antibiotic tolerance pathway if available) serve as a good control in this context?

Reviewer #2 (Public review):

Summary:

This study addresses a critical clinical challenge-bacterial antibiotic tolerance (a key driver of treatment failure distinct from genetic resistance)-by uncovering a novel regulatory role of the conserved s2U tRNA modification in Yersinia pseudotuberculosis. Its strengths are notable and lay a solid foundation for understanding phenotypic drug tolerance. The study is the first to link s2U tRNA modification loss to antibiotic tolerance, specifically targeting translation/transcription-inhibiting antibiotics (doxycycline, gentamicin, rifampicin). By establishing a causal chain - s2U deficiency → codon-specific ribosome pausing (at AAA/CAA/GAA) → reduced ribosomal protein translation → global translational suppression → tolerance - it expands the functional landscape of tRNA modifications beyond canonical translation fidelity, filling a gap in how RNA epigenetics shapes bacterial stress adaptation.

Strengths:

This study makes a valuable contribution to understanding tRNA modification-mediated antibiotic tolerance.

Weaknesses:

There are several limitations that weaken the robustness of the study's mechanistic conclusions. Addressing these gaps would significantly enhance its impact and translational potential.

Reviewer #3 (Public review):

Summary:

In the manuscript of Cotten et al., the authors study the 2-thiolation of tRNA in bacterial antibiotic resistance. The wildtype organism, Yersinia pseudotuberculosis, downregulates 2-thiolation as a response to antibiotics targeting the ribosome. In this manuscript, the authors show that a knockout of tusB causes slower translation. They provide evidence on the mechanisms of the slowing by determining transcription and translation, ribosome profiling and performing codon-usage analysis. They successfully determined that 2 codons are drivers of the translation slowdown, and the data is highly conclusive. Technically, I have nothing to criticize.

Strengths:

All in all, the study is very well made, and the writing is clear and concise. It covers a wide array of state-of-the-art analyses to unravel the interplay of tRNA modifications in translation.

Weaknesses:

The only question that remains to be asked is why the slowed translation leads to a better survival of the bacteria under antibiotic stress. In my opinion, the mechanism itself remains unclear. Thus, the statement that "We expect that this reduction in ribosomal proteins is globally reducing the translational capacity of the cell and is responsible for inducing tolerance to ribosome and RNA polymerase-targeting antibiotics" does not truly emphasize the remaining open question of why slowed translation favors survival. Therefore, I would recommend a minor text revision.

Author response:

Reviewer #1 (Public review):

Summary:

Cotton et al. investigated the role of tusB in antibiotic tolerance in Yersinia pseudotuberculosis. They used the IP2226 strain and introduced appropriate mutations and complementation constructs. Assays were performed to measure growth rates, antibiotic tolerance, tRNA modification, gene expression and proteomic profiles. In addition, experiments to measure ribosome pausing and bioinformatic analysis of codon usage in ribosomal proteins provided in-depth mechanistic support for the conclusions.

Strengths:

The findings are consistent with the authors having uncovered new mechanistic insights into bacterial antibiotic tolerance mediated by reducing ribosomal protein abundance.

Weaknesses:

Since the WT strain grows faster than the tusB mutant, there is a question of how growth rate, per se, impacts some of the analysis done. The authors should address this issue. In addition, it may not be essential, but would analysis of another slow-growing mutant (in some other antibiotic tolerance pathway if available) serve as a good control in this context?

We would like to thank the reviewer for their time spent reviewing our manuscript and for their positive review. We plan to address their comment as to how growth rate impacts the analyses and plan to incorporate another slow-growing mutant in the revised version of the manuscript.

Reviewer #2 (Public review):

Summary:

This study addresses a critical clinical challenge-bacterial antibiotic tolerance (a key driver of treatment failure distinct from genetic resistance)-by uncovering a novel regulatory role of the conserved s2U tRNA modification in Yersinia pseudotuberculosis. Its strengths are notable and lay a solid foundation for understanding phenotypic drug tolerance. The study is the first to link s2U tRNA modification loss to antibiotic tolerance, specifically targeting translation/transcription-inhibiting antibiotics (doxycycline, gentamicin, rifampicin). By establishing a causal chain - s2U deficiency → codon-specific ribosome pausing (at AAA/CAA/GAA) → reduced ribosomal protein translation → global translational suppression → tolerance - it expands the functional landscape of tRNA modifications beyond canonical translation fidelity, filling a gap in how RNA epigenetics shapes bacterial stress adaptation.

Strengths:

This study makes a valuable contribution to understanding tRNA modification-mediated antibiotic tolerance.

Weaknesses:

There are several limitations that weaken the robustness of the study's mechanistic conclusions. Addressing these gaps would significantly enhance its impact and translational potential.

We would like to thank the reviewer for their time spent reviewing our manuscript, and for both their positive comments about the significance and novelty of this work as well as their critiques. We plan to address their specific recommendations in the revised manuscript by focusing on the contribution of specific ribosomal proteins (i.e. the 30S subunit protein, S13) through overexpression, codon replacement, and stability experiments. We also plan to design experiments to assess in vivo relevance and assess possible impacts on other pathways involved in antibiotic tolerance.

Reviewer #3 (Public review):

Summary:

In the manuscript of Cotten et al., the authors study the 2-thiolation of tRNA in bacterial antibiotic resistance. The wildtype organism, Yersinia pseudotuberculosis, downregulates 2-thiolation as a response to antibiotics targeting the ribosome. In this manuscript, the authors show that a knockout of tusB causes slower translation. They provide evidence on the mechanisms of the slowing by determining transcription and translation, ribosome profiling and performing codon-usage analysis. They successfully determined that 2 codons are drivers of the translation slowdown, and the data is highly conclusive. Technically, I have nothing to criticize.

Strengths:

All in all, the study is very well made, and the writing is clear and concise. It covers a wide array of state-of-the-art analyses to unravel the interplay of tRNA modifications in translation.

Weaknesses:

The only question that remains to be asked is why the slowed translation leads to a better survival of the bacteria under antibiotic stress. In my opinion, the mechanism itself remains unclear. Thus, the statement that "We expect that this reduction in ribosomal proteins is globally reducing the translational capacity of the cell and is responsible for inducing tolerance to ribosome and RNA polymerase-targeting antibiotics" does not truly emphasize the remaining open question of why slowed translation favors survival. Therefore, I would recommend a minor text revision.

We would like to thank the reviewer for their time spent reviewing our manuscript and for their positive review of the technical aspects, experimental design, and writing. We will incorporate their suggested text revision into the revised manuscript, and will add to this statement if additional planned experiments shed light on this remaining question.