PPATHOGENS-D-26-01235

The two-component system ColRS discriminates metal signals to regulate virulence and resistance in Pseudomonas aeruginosa

PLOS Pathogens

Dear Dr. Bao,

Thank you for submitting your revised manuscript to PLOS Pathogens. Your manuscript was evaluated by members of the editorial board and two external experts. We feel that it has merit but Reviewer 2 has a few mostly minor points that should be addressed, including concerns about the order of the figures. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Matthew A Mulvey, PhD

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Ninglin Zhao, Ziqi Zhu, Xiyu Wu, Jianping Ying, Dan Fan, Cheng Nong, Yingjie Song, Tonggen Liu, Chenxi Duan, Yaping Zheng, Shuang Zou, Xingyu Mou, and Rui Bao. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The reviewer has read the responses and the revised version of the manuscript and found that the concerns raised have been satisfactorily addressed. Therefore, the reviewer has no further requests.

Reviewer #2: The authors describe a novel role for the P. aeruginosa ColRS two-component system in this manuscript. In general, it is well written and most of the data support the conclusions drawn by the authors. The manuscript uses genetics, proteomics and biophysics to identify the genes controlled by ColRS and the mechanisms used by ColRS to respond to zinc and copper. The study is quite novel as it shows the diversity of ColRS responses and shows that copper and zinc are driving the responses. The biophysics (MST) and genetics are convincing in showing a novel Zn/Cu binding site in ColS. The weaknesses of the manuscript are minor The authors have done an exceptional job in responding to the previous reviewer’s concerns.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: None

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: 1. Pg. 5, line 135. Fig. S2A is mentioned in the text before Fig. S1. The order of the figures should be changed.

2. Pg. 10, line 252; pg. 12, line 297; pg. 20, line 494; pg. 23, line 545; Figure S8, line 95; “qRT-PCR” should be “RT-qPCR”. Reverse transcriptase isn’t quantitative.

3. Pg. 16, line 372. Figs. S6G,H are mentioned before S6 panels A-F.

4. Pg. 27, figure 8; What is the significance for the green and blue ColR and ColS associated arrows? Is the orange “arnB” gene supposed to be blue?

5. The fig 8 legend says “ColR~P selectively associates with the promoter regions of amrZ,eptA, arnB, oprQ and czcR, thereby promoting their transcription.” Doesn’t the data in Fig. 2I show that arnB was repressed?

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Reviewer #1: No

Reviewer #2: No

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Figure resubmission:

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Dear Professor Bao,

We are pleased to inform you that your manuscript 'The two-component system ColRS discriminates metal signals to regulate virulence and resistance in Pseudomonas aeruginosa' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Matthew A Mulvey, PhD

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):