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The manuscript by Azam et al. examines the role of EzrA as a regulator of cell division. The authors explored the role of EzrA in cell wall homeostasis, demonstrating that EzrA mutants produced excess LTA, WTA and peptidoglycan when compared to wild-type controls. In addition to changes in the synthesis of different membrane components, the data presented shows that lack of EzrA leads to abnormal cell morphology, specifically relating to aberrance in the localization of membrane components during division. Furthermore, assays presented here suggest that lack of EzrA can result in DNA damage and other functional consequences for the cell as it divides. These findings suggest that EzrA may be involved in partitioning essential cellular components, such as Noc, along the dividing septum. However, the exact nature of EzrAs role in regulating septal division remains unclear. We provide below several suggestions to strengthen the establishment of a direct and causative relationship between EzrA function and dysregulation of organization during membrane synthesis. To directly implicate EzrA as an essential component in organizing cell division by constricting the necessary molecular components to the septum, we suggest the inclusion of additional controls.

Major comments

-We suggest including more experiments to strengthen the claim that Δezra mutants exhibit DNA damage and guillotining due to aberrance in their ability to segregate their DNA from the dividing septum. For instance, please provide a form of quantification, such as a qPCR of the undamaged fragments in wild-type versus Δezra presented in Figure 5D. Additionally, please include quantification of NoC in Figure 5 as this data is significant in showing that ΔezrA mutants fail to maintain proper nucleoid occlusion. Similarly, it would be helpful to include a quantification of the difference in fluorescence between the wild-type, positive control and Δezra in Figure 6. Figure 5 suggests that absence of EzrA caused GFP-labeled Noc foci to be spatially distributed around the dividing septum, suggesting that nucleoid occlusion is compromised in the mutant cells. Please consider including data from complementation strain for Figure 5A to support your claim. The work presented in Figure 5, including the quantification of DNA damage in Δezra mutants using fluorescently labeled dUTP, is interpreted to indicate that DNA damage is occurring in lines without EzrA; a quantification and normalization is missing to assess whether that’s the case. To support the claim that DNA damage results in reduced cell viability (Line 213), the study should incorporate a growth or survival assay comparing the viability of wild-type, Ezra, and complementation strains.

Taken together, data from these experiments implies that Noc function is compromised in EzrA mutants, resulting in downstream functional consequences. However, the discussion section (265-280) calls into question whether EzrA and Noc localization are related. Please discuss Veiga et al. Molecular microbiology (2011) and Adrian et al. Nature Communications (2025), which demonstrate an interaction between EzrA and Noc.

- It may be that the observed phenotype is due to an indirect effect from other proteins that depend on EzrA for localization/ function, and/or could be due to the larger cell size of an EzrA mutant. In the introduction, you have mentioned GpsB as another contributor that connects cell division to cell wall synthesis. The interaction between EzrA and GpsB has been reported in B. subtilis, and studies in pneumococcus report that depletion of GpsB mislocalize the PBP responsible for septal PG synthesis (Costa et al. Bacteriology 2024). Please rephrase lines 151-153 to include this possibility or provide further experiments to test that Ezra is directly involved in localized display of proteins.

In the discussion section (and line 103), the possibility is raised that the dysregulation and spatial disorganization observed in the membranes of EzrA mutants could be because EzrA-deficient cells are larger, leading to a diffusion of synthetic activity across cells. To characterize this morphological change, please quantify cell size. Figures 1, 2 and 4 include both wild-type and EzrA mutant cells with vancomycin-labelled cell walls, which could potentially be quantified and compared.

- The intensity of the fluorescent signal labelling SpA, especially compared with wild-type (Figure 1,3), would suggest that SpA synthesis is highly upregulated in Δezra mutants. SpA does not seem to be excluded from the actively dividing septum in Δezra mutants, rather, it seems to be present across the entire cell including the actively dividing septum (Figure 4). The increased SpA raises the concern that the lack of confinement of SpA to the septal region could be a result of its magnitude compared to wild-type controls, not necessarily due to defects in its partitioning within the septum. Please address this concern by using an immunoblot to quantify SpA between wild-type and Δezra to see whether the levels of protein expression are comparable. If increased, a line expressing a similar amount of SpA to the Δezra mutant could be generated to test that the organization defects observed are not simply due to increased abundance of SpA.

- Based on Figure 4, it is concluded that PBPs are mislocalized in Δezra mutants, resulting in impaired formation of the septum. However, to fully support this conclusion, please clarify if the images shown in Figure 4D depict live cells or fixed cells. The way this figure is presented implies that these images show a time lapse of a single cell undergoing septum formation and division. If, rather, several different fixed cells at different points of division have been used to draw the conclusion that septal division is altered due to mislocalization of PBPs, the data presented does not provide a conclusive representation of how septum formation and division are altered in mutant cells. Additionally, it would provide further context to indicate which cell or cells from the larger images are being focused on in the subsequent panels. To further increase clarity, consider including which PBPs are mislocalized (PBP2 or PBP4?) and discussing how these results fill the gap on EzrA and cell division.

Minor comments

Figure content

- Some indication of sample size (biological replicates) should be added to the figure descriptions.

- Figure 1C, lines 81-82: To ensure that the increased SpA-specific signal phenotype associated with the knockout of ezrA is restored in the complemented strain, it would be helpful to show an indication of the statistical significance (or lack thereof) between WT and the complemented strain.

- Figure 2C,D: To provide more context regarding the significance of the data displayed, please include how many cells were quantified. Do the data points in Mander’s co-efficient represent the average of cells from one replicate, or individual cells from a single replicate?

- Figure 2E: Please consider including the complementation strain in this figure, as in previous figures, to determine whether PG content was restored back to WT levels or not.

- Figure 3D: This immunoblot, which illustrates that LTA content is increased in Δezra mutants over wild-type and complemented strains, shows bands at both 15kDa and 23 kDa. It is unclear which band is relevant for the findings presented. It would increase the clarity of this figure to put a tick mark at the relevant band(s) with what they are predicted to represent. Please consider using a loading control for assays determining the amount of LTA and WTA in WT vs mutant cells.

We noticed that WT results from Figure 3 and Figure 5 showing wheat germ agglutinin contradict each other. In Figure 3B, the WT cells show low intensity for WGA-AF488 as compared to ΔezrA mutant, while in Figure 5A WT cells display higher intensity for WGA-AF647. Please also include images from the complementation strain in Figure 5.

- Figure 4D: ΔezrA cells presented in this figure have pronounced differences in morphology as compared to figure 4A. Is it just an artifact of magnification?

- Please mention the strain/accession number of strains used to generate the phylogeny analysis in Figure 7.

Further explanation needed

- Please consider including an explanation in the introduction that in S. aureus EzrA is not essential, unlike in other gram-positive bacteria, and a rationale for using S. aureus strain RN4220 in this study.

- The term “molecular organizer” is not very specific, as in this context, it could describe many types of proteins with different functions. Please consider providing a more specific definition of what a molecular organizer is, and what characteristics determine whether a protein is considered a molecular organizer within the scope of this study or not.

- Line 33: Consider providing a more exhaustive explanation of the Sec secretion pathway, particularly relating to its relevance to Spa.

- Lines 39, 73: The abbreviation “PBP” is used in line 39 to describe work where PepV was shown to interact with EzrA to modulate “EzrA-PBP interactions”. It would increase clarity to define the abbreviation here, since this is the first time the abbreviation has been used. Also, since this interaction between EzrA and PBP2 is important to understanding how EzrA is related to peptidoglycan wall synthesis, it would be helpful if the specific nature of this interaction was expounded upon. There are several PBPs in S. aureus, it would be nice to distinguish if ΔezrA is impacting PBP2 or PBP4 in Figure 4?

- Line 41: Consider explaining Min system.

- Line 75: Please add more context to the line EzrA itself modulates “septal protein display” by specifying how EzrA modulates septal protein display.

- Line 82: To contextualize the significance of the finding that SpA-specific fluorescent signal is increased in ezrA knockout lines, please provide a summary sentence of the implications of that finding here addressing the potential functional consequences of enhanced SpA synthesis for wall assembly.

- Line 88: Since this passage introduces EzrA as a molecular organizer due to its structural homology with eukaryotic spectrins, it would validate this choice to include a figure showing a structural model of these two proteins side by side.

- Line 97: Please provide further context regarding the use of RADA in labelling dividing cells. While the figure description (Figure 2B) does specify that RADA is a fluorescent D-alanine analog, it is not explained why it is used or what it is labelling. Furthermore, for readability and enhanced clarity for broader audience, please consider explaining the use of Bocillin-FL vs vancomycin labeling vs RADA labeling.

- Line 109: Further context regarding the meaning of the Mander’s coefficient, and how it is calculated, is required.

- Figure 4C: Please consider discussing circumferential PBP movement and PBPs ingressing at the septum in the results and discussion. This background information would enhance the reader’s understanding of the model proposed here.

- Line 254: Please specify the envelope components (LTA and WTA). Also, please consider including a reference to Figure 3 for clarity and readability.

- Line 257-258: an alternative hypothesis that EzrA may regulate envelope synthesis through “direct interaction” is put forward. Please consider elaborating the nature of this interaction, specifically, which components are interacting and how they are interacting.

- While the current title of the manuscript captures the broader significance of the study, we suggest an alternative title that is more specific to the results of this study “EzrA in S. aureus is required to prevent the guillotining of the chromosome by the newly forming cell wall”.

References

- References 8 and 26 are duplicates.

- Lines 70, 80: There seems to be a typo when referring to reference 5, as the formatting of this citation does not match the formatting of other citations in the manuscript.

- Line 87: This should be cited.

- Line 251: This needs a reference

- Line 258: A reference is missing here.

- Line 277: This should be cited

Figure formatting

- For figures that include a schematic of the experimental design (Figures 2 and 4), consider giving the panel featuring the experimental schematic its own letter rather than including it with a microscopy image.

- Please consider including phase-contrast images for all the cell morphology images.

- Figure 1B: The title for the “Overlay” images is off-center.

- Figure 2A: Consider including color coding for the labels like other microscopy images.

- Figure 2E: There is no information for panel 2E in the figure description. Please describe the method used here for increased clarity.

- Figure 3A: Please include an explanation in the figure to specify that the assay aims to determine the amount of WTA.

- Figure 5A: Consider including an explanation that wild type which does not have Noc labelled with GFP is used as a negative control here

- The magnification of images is different, but the scale bar is not completely visible. If possible, please add a scale bar to every image.

Typos/Miscellaneous

- Line 39: There is an extra space after reference 8.

- Line 44: Since, Noc is an abbreviation for “nucleoid occlusion”, please consider rephrasing the sentence.

- Line 253: This is a typo. It should be “In addition to dispersion...”

- In the discussion section, Δezra mutants are referred to as ezra mutants. Please consider keeping the naming of these mutants consistent across sections.

- Line 296: It seems like there is a reference to a paper or figure missing here.

- Figure 4D: “Magnified” is spelled incorrectly here.

Lily Pumphrey and Tahreem Zaheer (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Carter Collins, Camy Guenther, and Josy Joseph. We are part of the Dept. of Biology where Xindan Wang’s group is located. Xindan is a contributing author of a recent publication on ‘chromosome segregation dynamics during the cell cycle of Staphylococcus aureus’ and did not influence the choice of this preprint for our class.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.