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Review of the Preprint "Variability in Intrinsic Promoter Strength Underlies the Temporal Hierarchy of the Caulobacter SOS Response Induction"

This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

Paper reviewers: Beatriz Kopel, Erik Rios, Guilherme Goes

"Variability in Intrinsic Promoter Strength Underlies the Temporal Hierarchy of the Caulobacter SOS Response Induction"

Aditya Kamat, Asha M. Joseph, Deeksha Rathour, Anjana Badrinarayanan

This review is structured into major comments addressing key areas for improvement in the article and minor comments concerning specific corrections and clarifications.

Major Comments

The research question posed by the article is highly relevant, as it investigates whether the SOS response induction in Caulobacter crescentus follows a hierarchical activation pattern similar to Escherichia coli, where fidelity-prone DNA repair mechanisms are activated earlier in response to mutagenic stress than error-prone mechanisms.

• The rationale for choosing C. crescentus as a model organism should be explicitly stated, especially considering the observed dependence of the SOS response on cell cycle regulation and sigma factor activity during different growth phases.

• The introduction lacks specificity in certain areas, employing vague descriptions such as "a wide range of DNA-damaging agents" and "diverse mechanisms that repair or reverse DNA damage" without specifying these agents and mechanisms.

The experimental approach is well-designed to investigate the temporal hierarchy of SOS response gene expression, with a strong emphasis on bioinformatics for gene, SOS box, and promoter strength comparisons across models.

• While the study successfully characterizes genes involved in Caulobacter's SOS response to mitomycin-C (MMC), additional experiments with other genotoxic agents are required to confirm that the identified genes are solely responsible for the SOS response. MMC induces intra-strand crosslinks and DNA mono-adducts (line 89), but a broader range of DNA lesions that trigger SOS activation should be examined [1]. In E. coli, gene recruitment varies depending on lesion type [2], suggesting that the six genes previously reported as unresponsive to MMC (lines 119-120) could be involved in responses to other DNA damage types.

• The methodology lacks descriptions of key experimental procedures, including the generation of Caulobacter mutants with gene deletions and the construction of yfp reporter strains.

• The unavailability of supplementary tables significantly hinders data interpretation. Tables S1-S7 are listed on page 24 but are not provided, restricting access to crucial details on strains, plasmids, and oligonucleotides (Tables S1-3).

• Although experimental controls are generally well-applied, the study does not include non-alkylating DNA-damaging agents, which are necessary to determine whether the observed response is specific to alkylation damage or extends to other types, such as oxidative DNA damage.

• There is no explanation regarding the selection of MMC doses and time points used in the experiments. Details on trial runs and optimization should be included. Furthermore, alternative stressors should be tested to determine whether the observed responses are specific to alkylation-induced DNA damage.

• Establishing correlations between gene function and SOS response dynamics would enhance result interpretation. The association between early and late gene expression should be explored further, as functional insights could facilitate comparisons with prior research on fidelity and temporal hierarchy. The criteria for classifying upregulated genes as "early" or "late" require clarification. Why were the 20- and 40-minute time points chosen? Does gene expression plateau after 40 minutes?

• The study should examine whether the same genes are differentially expressed under different stress conditions. Additionally, a more detailed discussion on the identified genes' roles in the SOS response is needed.

• The analysis of downregulated genes in C. crescentus should be expanded. Many of these genes appear to be repressed early in the response—clarifying their cellular functions could provide valuable mechanistic insights.

• RNA-seq data should be validated using qPCR for at least 10 upregulated genes to confirm transcriptomic findings.

• The order of sections should be revised; presenting Methods before Results and Discussion would improve readability.

Minor Comments

• Introduction

  • Line 56: "The SOS is inherently leaky, and pulses of SOS gene expression occur throughout the population even in the absence of genotoxic stress." Evidence linking SOS system leakiness to endogenous DNA damage should be referenced.

• Results

  • Line 113: "We found that 48 of these genes exhibited LexA enrichment in their 114 promoter sequence." The basis for this claim should be specified. What criteria were used to determine that these genes are regulated by LexA?

  • Lines 141-143: "This phenomenon was independent of damage concentration (…) over a wide range of MMC concentrations." This claim is not well-supported, as only two additional MMC concentrations were tested, with no late promoter induction at 0.125 µg/mL. More concentration points are needed to substantiate this assertion.

  • Line 155: "Using MEME analysis (…)." The full name of the analysis should be provided upon first mention.

  • Line 175: "(…) our time-course RNA-seq experiment revealed that the induction kinetics of these two genes under MMC exposure were significantly different." While fold-change values differ, both genes appear to be induced at 20 minutes, with increased expression at 40 minutes. The claim of distinct kinetics should be justified. Furthermore, two time points do not constitute a kinetic study; additional time points would be required to support this conclusion.

• Discussion

  • Line 243: "Error-prone SOS genes seem to peak later as compared to error-free genes." Were similar observations made in this study? Discussing this would strengthen the argument.

  • Lines 258-259: "Some of the late genes did also exhibit regulation by the alternative sigma factor (RpoH)." Is there evidence that genotoxic stress triggers RpoH activity? Citing relevant literature would enhance this point.

References:

1.       Maslowska KH, Makiela‐Dzbenska K, Fijalkowska IJ. The SOS system: A complex and tightly regulated response to DNA damage. Environ and Mol Mutagen. 2019 May;60(4):368–84.

2.       Serment-Guerrero J, Dominguez-Monroy V, Davila-Becerril J, Morales-Avila E, Fuentes-Lorenzo JL. Induction of the SOS response of Escherichia coli in repair-defective strains by several genotoxic agents. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2020 Jun;854–855:503196. 

Competing interests

The authors declare that they have no competing interests.