Alanine Uptake Is Required to Maintain Staphylococcus aureus Cell Envelope Stability Under Magnesium and Calcium Limitation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
I reviewed the manuscript entitled “Alanine Uptake Is Required to Maintain Staphylococcus aureus Cell Envelope Stability Under Magnesium and Calcium Limitation”. This is a strong, well-written, and highly cohesive manuscript. The study is carefully executed, and the findings are presented in a clear, straightforward, and logical manner, making it a valuable addition to the field. The Introduction is very well-written, coherent, and scientifically accurate. It perfectly follows the classic “funnel” approach, starting with a broad context and elegantly narrowing down to the specific objective of your research.
Figure 1: Ambiguity in X-axis Labels (Panel A): The use of (-) and (+) beneath the bars could be slightly misleading. According to the caption, the base medium is not entirely devoid of these components (it contains 1 mM L-alanine and 0.04 mM Mg2+, Ca2+). Therefore, (-) does not mean zero concentration, but rather “unsupplemented basal medium. It is recommended to clarify this explicitly in the figure legend to prevent reader confusion. Y-axis Formatting (Panel B): The scientific notation values on the y-axis (2×108, 4×108 etc.) are a bit crowded. A cleaner visual approach would be to label the ticks as 2, 4, 6 and write (×108) at the top of the axis next to the unit label.
In the second paragraph of discussion (lines 342-344), it is mentioned that upon the depletion of Mg2 and Ca2+, the stabilization of the cell wall relies on processes such as teichoic acid modification. It is highly recommended to add a brief sentence explicitly explaining the underlying mechanism.
Author Response
Figure 1: Ambiguity in X-axis Labels (Panel A): The use of (-) and (+) beneath the bars could be slightly misleading. According to the caption, the base medium is not entirely devoid of these components (it contains 1 mM L-alanine and 0.04 mM Mg2+, Ca2+). Therefore, (-) does not mean zero concentration, but rather “unsupplemented basal medium. It is recommended to clarify this explicitly in the figure legend to prevent reader confusion. Y-axis Formatting (Panel B): The scientific notation values on the y-axis (2×108, 4×108 etc.) are a bit crowded. A cleaner visual approach would be to label the ticks as 2, 4, 6 and write (×108) at the top of the axis next to the unit label.
Response: Thank you to the reviewer for their attention to detail. The meaning of the (-) and (+) in Figure 1A has now been specified in the caption. The Y-axis in Figure 1B has also been changed to reflect the reviewer’s comments.
In the second paragraph of discussion (lines 342-344), it is mentioned that upon the depletion of Mg2 and Ca2+, the stabilization of the cell wall relies on processes such as teichoic acid modification. It is highly recommended to add a brief sentence explicitly explaining the underlying mechanism.
Response: More detail and additional references have been added to the paragraph to give more information and context to the proposed mechanism as well as the mechanisms of teichoic acid modification.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript studies an interesting phenomenon when Staphylococcus aureus bacteria have a higher need for alanine while Mg2+ and Ca2+ ions are not available to maintain their growth and stability.
In general, the data are well presented, the conclusions are sound well supported by the data. There are some issues mainly in the introduction and methods description that need to be addressed before publication.
Comments/concerns:
In the introduction, page 2, line 50, the statement is not supported by the cited literature. Ref 14 identifies the Ca2+ and Mg2+ binding sites, but does not say anything about lysis or antibiotic stress. Using this reference here is wrong. Ref. 15 is a study on Gram-negative bacteria Pseudomonas, but the authors state this for Gram-positive Staphylococcus. There needs to be a link provided, why the authors think the same phenomenon is valid for Gram-positive bacteria. Similarly, ref. 16 is a study on Micrococcus luteus and Escherichia coli. These are again, different than S. aureus. Is there really no evidence for this in S. aureus? If not, discuss why. If yes, add the reference.
Moreover, too much Ca2+ ions in S. aureus can also be bactericidal! See Xie and Yong Scientific Reports 2016. This was shown to be specific for S. aureus, and is not true for other Gram-positive bacteria. So stating that Ca2+ and Mg2+ ions enhance resistance to lysis and to antimicrobial stress in S. aureus based on references to studies that were done on other bacteria, is not correct. Please, discuss in more detail.
In the introduction, page 1, lines 36–39, reference is missing for the statement “In addition to the role…”.
The strains are identified only in the SI table. I suggest moving this table to the methods section. Without it, the manuscript cannot be properly assessed and understood, and many readers do not download the SI. Mutants description should be improved. I suggest adding a column in the SI table S1 (after moving it to the methods) with description of what the mutant lacks or has extra and what are the consequences for alanine uptake or whatever. Now it’s a bit of a guessing game.
Methods 2.5 Daptomycin susceptibility testing: Add the line that this was done only at high Ca2+ condition. It is later written in the main text, but from the methods it seems like you want to test daptomycin activity in low and high Ca2+ and Mg2+ presence, which does not make sense as daptomycin needs Ca2+ for its activity.
Methods 2.7 Infection of RAW 264.7 cells, page 4, line 140: How do you know you killed the extracellular bacteria by gentamicin? How much gentamicin was added? Some extracellular bacteria were probably left alive in the sample, which also contribute to the counting. Can you estimate the % of error from this?
Methods 2.7. Infection of RAW 264.7 cells, page 4, line 146: The competition index is not explained or described anywhere in the manuscript. Please, provide the equation and explain the exact meaning. Otherwise it’s hard to understand and interpret the Figure 5C. In Figure 5B–C, on top of this, the meaning of P=0.28 is not clear. Are these p values from the t-test? Add clear description to the legend.
Results 3.1. page 4, line 169–175: The authors talk about preliminary experiments. Could you show the data? Maybe in the SI? Otherwise these are statements without proof.
Page 5, line 182 “…well-documented role…” : Reference is missing here.
The results and figures are good and clearly presented, and good to go for publishing. I applaud the authors for good work in experimental design and their description. No comments there.
Author Response
In the introduction, page 2, line 50, the statement is not supported by the cited literature. Ref 14 identifies the Ca2+ and Mg2+ binding sites, but does not say anything about lysis or antibiotic stress. Using this reference here is wrong. Ref. 15 is a study on Gram-negative bacteria Pseudomonas, but the authors state this for Gram-positive Staphylococcus. There needs to be a link provided, why the authors think the same phenomenon is valid for Gram-positive bacteria. Similarly, ref. 16 is a study on Micrococcus luteus and Escherichia coli. These are again, different than S. aureus. Is there really no evidence for this in S. aureus? If not, discuss why. If yes, add the reference. Moreover, too much Ca2+ ions in S. aureus can also be bactericidal! See Xie and Yong Scientific Reports 2016. This was shown to be specific for S. aureus, and is not true for other Gram-positive bacteria. So stating that Ca2+ and Mg2+ ions enhance resistance to lysis and to antimicrobial stress in S. aureus based on references to studies that were done on other bacteria, is not correct. Please, discuss in more detail.
Response: Thank you to the reviewer for this thoughtful input. The text was changed to reflect what is known regarding S. aureus, and references were changed to articles more relevant to this work (with more focus on gram-positives). The reviewer will note changes throughout the manuscript to make this shift more cohesive.
In the introduction, page 1, lines 36–39, reference is missing for the statement “In addition to the role…”.
Response: References are now included that review the structure and role of alanine in the cell wall.
The strains are identified only in the SI table. I suggest moving this table to the methods section. Without it, the manuscript cannot be properly assessed and understood, and many readers do not download the SI. Mutants description should be improved. I suggest adding a column in the SI table S1 (after moving it to the methods) with description of what the mutant lacks or has extra and what are the consequences for alanine uptake or whatever. Now it’s a bit of a guessing game.
Response: The table is now included in the methods section, and has been edited to provide more specifics and additional information, including an additional column within the table to direct the reader to references that have done relevant work with these strains.
Methods 2.5 Daptomycin susceptibility testing: Add the line that this was done only at high Ca2+ condition. It is later written in the main text, but from the methods it seems like you want to test daptomycin activity in low and high Ca2+ and Mg2+ presence, which does not make sense as daptomycin needs Ca2+ for its activity.
Response: This information is now included in the specified section.
Methods 2.7 Infection of RAW 264.7 cells, page 4, line 140: How do you know you killed the extracellular bacteria by gentamicin? How much gentamicin was added? Some extracellular bacteria were probably left alive in the sample, which also contribute to the counting. Can you estimate the % of error from this?
Response: The killing capacity of gentamicin was confirmed in control experiments, which showed no surviving bacteria when the inoculum was incubated in 100 ug/mL gentamicin for 30 minutes. The additional information is now included in the Methods section as requested.
Methods 2.7. Infection of RAW 264.7 cells, page 4, line 146: The competition index is not explained or described anywhere in the manuscript. Please, provide the equation and explain the exact meaning. Otherwise it’s hard to understand and interpret the Figure 5C. In Figure 5B–C, on top of this, the meaning of P=0.28 is not clear. Are these p values from the t-test? Add clear description to the legend.
Response: The calculation for the CI is now included in the methods. The P=0.28 means that there is not a significant difference in fitness between the mutant and wild type. The legend has been modified to make this clearer.
Results 3.1. page 4, line 169–175: The authors talk about preliminary experiments. Could you show the data? Maybe in the SI? Otherwise these are statements without proof.
Response: The introductory paragraph of the results section has now been changed to remove mention of the preliminary data. This removal was necessary because introducing this preliminary data would require tangential context and detract from the flow of the results section. Motivation for investigating the role of alanine uptake and Mg/Ca is apparent based on existing literature (now mentioned in the new text), as well as the role of Mg/Ca in cell envelope stability not being established in S. aureus, as pointed out by the reviewer.
Page 5, line 182 “…well-documented role…” : Reference is missing here.
Response: This text was changed to “Given the role of the divalent cations Mg2+ and Ca2+ in cell envelope stability in other species [16-19], we hypothesized that the growth defect observed in the aapA mutant when cultured in low Mg2+/Ca2+ conditions was due to increased cell lysis.” This also aims to address the issue raised in other comments from the reviewer regarding the unclear role of Mg2+ and Ca2+ in S. aureus.
