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Review of: White, M, et al.. February 10, 2022. “MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis.” bioRxiv. DOI: https://doi.org/10.1101/2022.02.09.479790]

This review was written collaboratively by undergraduates at Mount Holyoke College (MA, USA) who selected this preprint for an assignment in a course on peer review taught by Dr. Rebeccah S. Lijek, Assistant Professor of Biological Sciences. Student-reviewers who give their permission to list their names are: Madison Dresler, Valentina Shrum, Rossi Wang, Siyu Yin

Disclosures: The review authors declare no conflict of interest and have no personal or financial relationship with the study’s authors. The reviewers acknowledge a limitation of their review is that some of the bacteriology methodology was beyond the scope of their expertise, being undergraduate students and not practicing bacteriologists. We thank the authors for sharing their manuscript as a preprint.

Summary

The manuscript investigates the role of DNA binding protein MraZ in Bacillus subtilis. Microscopy was used to image and analyze B. subtilis expressing mraZ-GFP and other constructs for structural differences, localization, and FtsZ ring formation. RNA-seq technology was used to analyze differential gene regulation in conditions such as the overexpression or deletion of mraZ. The authors found that increased levels of MraZ cause filamentation that results in cell lysis caused by repression of the mra operon. They were also able to see de-condensation of the Z-ring in response to the overexpression of MraZ. This dissolution is attributed to the depletion of FtsL. The authors use these findings to suggest that FtsL plays a role in the maturation and treadmilling of the Z-ring. This study’s findings have potential implications for the role of MraZ and the mra operon in areas such as DNA replication arrest. Similar studies have involved E. coli, and the authors use B. subtilis as a comparison and to separate MraZ’s effects. Overall, we believe this is a well-researched manuscript that may be of interest to the field of bacteriology.

Strengths:

1. Some of the hypotheses proposed in the preprint depend on the previous work in E.coli and Mycoplasma, which gives the potentiality of having a generalizable conclusion and broad implications.
2. The cartoon figures in this preprint elucidate the mechanism or process unambiguously which is beneficial for the reader’s understanding.
3. The study’s results were clear and interpretable thanks to the use of proper controls, eg. WT B. subtilis and MraZ
4. Throughout the discussion section, the authors bring forward areas of research and findings that are yet to be explained and could be evaluated in the future. Pointing out these next steps establishes the boundary of the manuscript and contextualizes the data. 

Concerns

1. Firmly stating the hypothesis in the introduction would help to clarify the manuscript. We noticed that the introduction moves from the background shown in previous investigations to the authors’ findings (beginning of line 51). Explicitly stating the study’s hypothesis can help the narrative of the manuscript and enhance the readership’s understanding of the work.
2. This study contained many different constructs. Please include a table or specific description of what each variant was and how it was studied, which may help make the results more straightforward - similar to how the genes were described in the supplementary tables. 
3. Are there any specific criteria for the mutation of the R15A and R86A to prevent lethality? Are there other variants that could be tested to better conclude that lethality relies on the presence of the motifs?
4. Some more brief background information on DXXXR motifs would be useful.
5. In line 179, the authors describe MraZ-GFP appearing to bind “promiscuously” - please clarify. The observation is directed to a micrograph of tagged cells in the presence or absence of IPTG. Does this mean it binds indiscriminately? If so, further elaboration and an interpretation of what they are seeing and its potential meaning could be helpful. 
6. It may improve understanding of the manuscript if there was an elaboration on the function of ftsEX and how it is involved in the cell division process. 
7. We would be interested to see a statement regarding the applications for this research. This could further contextualize the manuscript and inspire additional research or connections between findings.