Enhancement of growth media for extreme iron limitation in Escherichia coli
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A corrigendum of this article has been published full details can be found at https://doi.org/10.1099/acmi.0.000887
Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli , we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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Thank you for your resubmission. In your manuscript, please clarify the following point regarding the number of experiments performed. Have you performed your experiments three times (biological replicates) in triplicate (technical replicates), or once (biological replicate) in triplicate (technical replicates)? I assumed the former, but in response to reviewer 1 I realised I might have misunderstood this.
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Comments to Author
This manuscript is an important addition to the literature as a stand-alone piece, and a valuable supplement to previous work from this group. A major strength of this manuscript is the exquisite detail in which the methods are reported. Readers will be able to reproduce this work easily. I commend the authors for this. I don't fully agree with the statements made in relation to microaerobic conditions enhance iron-limiting processes, and I would expect many additional stresses would have a similar impact, but this is beyond the scope of this work and I think the authors present their findings simply and clearly enough, that any reader will be able to take their data at face value. I support acceptance with the following points taken into consideration. Line 74: could the authors include evidence or …
Comments to Author
This manuscript is an important addition to the literature as a stand-alone piece, and a valuable supplement to previous work from this group. A major strength of this manuscript is the exquisite detail in which the methods are reported. Readers will be able to reproduce this work easily. I commend the authors for this. I don't fully agree with the statements made in relation to microaerobic conditions enhance iron-limiting processes, and I would expect many additional stresses would have a similar impact, but this is beyond the scope of this work and I think the authors present their findings simply and clearly enough, that any reader will be able to take their data at face value. I support acceptance with the following points taken into consideration. Line 74: could the authors include evidence or speculate about the possible sources of iron - this might be informative to readers Line 78: consider introducing the concept of formation constant - it took me a few reads to understand what this sentence was telling me and perhaps a conceptual introduction leading into this point would add to the justification of the study. Line 86: consider use of "later". Line 97: consider including the company alongside the specific reagents rather than a stand alone list Line 110 (and further examples): the sentence structure starting "was added x,y, z…" The meaning here is clear and will be understood, but please consider "To make a 10 mL stock, the following were combined…" Line 190: please check if you mean Sanderson 2020, not 2021 Figure 2: apologies if I missed it, but id there an explanation for why data are collected over different time scales? Line 249: reference error
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Please accept my apologies for the prolonged review period your manuscript experienced. I have no received two reviews for this manuscript and both positively evaluated your work. Please consider the points raised by the reviews for your resubmission. Having read your manuscript, I wondered if "augmentation" is the best description for how you've altered the media for your experiments. Would enhancement (as used in your manuscript) or improvement better capture the essence of the work?
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Comments to Author
This manuscript clearly outlines the method used to obtain a "zero-iron" media for iron limiting conditions for E. coli. The authors show the suitability of the media and of additional conditions e.g. microaerobic environment and addition of siderophores with well thought-out experiments. They also describe how this media can be used for applications such as ricapitulating in vivo conditions e.g. the gut as well as investigating antibacterial siderophore compounds. These results would be helpful to microbiologists interested in studying antimicrobial compounds or bacterial phenotypes in iron-limiting conditions. The following revisions are recommended, mostly to improve formatting - The introduction could contain a more in depth summary of iron acquisition via siderophores in E. coli which could help …
Comments to Author
This manuscript clearly outlines the method used to obtain a "zero-iron" media for iron limiting conditions for E. coli. The authors show the suitability of the media and of additional conditions e.g. microaerobic environment and addition of siderophores with well thought-out experiments. They also describe how this media can be used for applications such as ricapitulating in vivo conditions e.g. the gut as well as investigating antibacterial siderophore compounds. These results would be helpful to microbiologists interested in studying antimicrobial compounds or bacterial phenotypes in iron-limiting conditions. The following revisions are recommended, mostly to improve formatting - The introduction could contain a more in depth summary of iron acquisition via siderophores in E. coli which could help the reader to better understand the results in 7.3 - Line 53 and throughout the text: add punctuation after the reference in text e.g. "selectivity for Fe(III) (1-3)." rather than "selectivity for Fe(III).(1-3)" - Line 55: Fur is a master iron regulator not only in Gram negative but also in Gram positive pathogens such as S. aureus. Adding this can highlight an even wider impact and applicability of your work - Line 167: were the experiments performed also in biological replicates i.e. on different days? - Line 178 and throughout reference figures in text as (Fig. 1A), makes it more concise - Line 179, 181,224: add the publication year of the paper you're referencing e.g Soma et al. (2023) - Figure in text should be referenced in order e.g. figure 1D after 1A ,1B and 1C - change the text or the figure accordingly - Line 193: used rather than "using"? - Figure 1C should also be referenced in the text - Line 202-3 suggested rephrasing "which might increase requirement for iron-binding proteins" -Line 249 -250: reference has formatting issue -Line 254: use the same units in the figure and in the text for clarity - Line 267: (32) not in italics - Line 274: and perhaps best mimicked - Line 283: inhibit rather than inhibited - Line 287: to utilise - Investigating the growth dynamics of E. coli mutants with deletions in genes for siderophores or iron receptors could further support the use of exogenous siderophores to induce iron limitation, the choice of appropriate siderophore and the concept of competitive iron binding. If any of these experiments had already been performed, they'd be a nice addition to the manuscript. Equally the authors could perhaps comment on this as potential further work.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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