Dear Dr. Hernandez Lucas,

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Academic Editor

PLOS ONE

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“We would like to thank M. Dunn for stimulating discussions and critical reading. M. 476 Fernández-Mora, F. J. Santana and A. Vazquez for scientific suggestions. Alcocer A. 477 and Marcos-Duran J. M. for their technical assistance. This work was supported by 23 478 grants from: Dirección General de Asuntos del Personal Académico (DGAPA/UNAM, 479 IN203621-IN202224) and Consejo Nacional de Humanidades Ciencia y Tecnología 480 (CONAHCYT, CF-2023-I-2079) to I.H.L. V. H was supported by Universidad Nacional 481 Autónoma de México Posdoctoral Programs (POSDOC)”

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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This manuscript presents a highly comprehensive and systematic functional characterization of 18 previously uncharacterized LysR-Type Transcriptional Regulators (LTTRs) in Salmonella enterica serovar Typhi. The high-throughput approach, covering key virulence-associated phenotypes such as bile tolerance, antimicrobial resistance, motility, and biofilm formation, represents a significant contribution to S. Typhi pathogenesis research. The methodological rigor, particularly the use of non-polar mutagenesis and host-relevant minimal medium, makes this work suitable for publication in PLOS ONE, pending revisions.

Overall Assessment:

Major Revisions are suggested to enhance the mechanistic depth and structural completeness of the manuscript.

Reviewer #2: 1. The authors should clarify whether the identified LTTRs directly regulate ompC and ompF transcription or whether their effects are indirect. Likewise, for biofilm formation and motility, it would strengthen the manuscript to indicate whether specific virulence- or flagellar-related genes are misregulated in the mutants. Including even preliminary data or a focused discussion on potential regulatory targets would greatly enhance the study’s impact.

2. The assignment of mutants into four functional groups appears somewhat arbitrary and is not consistently aligned with the experimental data. For example, the manuscript states that Group II mutants “produced OmpF but not OmpC,” yet the corresponding figures suggest that some Group II strains express both porins, while others like ΔSTY0341 (also classified as Group II) lack OmpF entirely. The authors should carefully re-evaluate this classification using all available phenotypic evidence and provide a clear, data-driven justification for the final groupings.

3. The introduction would benefit from a brief mention of the clinical significance of typhoid fever and the growing challenge of antibiotic resistance. This context would more effectively underscore the relevance of investigating virulence mechanisms in Salmonella typhi.

4. In the discussion, the authors should either temper or remove the claim regarding the global regulon of the transcription factor, as 1D gel electrophoresis alone is insufficient to support such a broad conclusion. A comprehensive assessment of a regulon would require more robust approaches, such as proteomic or transcriptomic analyses.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Ashraf Kariminik

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Attachments

Attachment

Submitted filename: Rviewer coments.docx

Functional role of 18 LysR-Type transcriptional regulators of Salmonella enterica serovar Typhi.

PONE-D-25-53096R1

Dear Dr. Hernandez Lucas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohammad Faezi Ghasemi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

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Reviewer #2: (No Response)

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes:  Ashraf Kariminik

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