Towards a Yersinia pestis lipid A recreated in an Escherichia coli scaffold genome
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Synthetic biology and genome engineering capabilities have facilitated the utilization of bacteria for a myriad of applications, ranging from medical treatments to biomanufacturing of complex molecules. The bacterial outer membrane, specifically the lipopolysaccharide (LPS), plays an integral role in the physiology, pathogenesis, and serves as a main target of existing detection assays for Gram-negative bacteria. Here we use CRISPR/Cas9 recombineering to insert Yersinia pestis lipid A biosynthesis genes into the genome of an Escherichia coli strain expressing the lipid IV a subunit. We successfully inserted three genes: kdsD , lpxM , and lpxP into the E. coli genome and demonstrated their expression via reverse transcription PCR (RT-PCR). Despite observing expression of these genes, analytical characterization of the engineered strain’s lipid A structure via MALDI-TOF mass spectrometry indicated that the Y. pestis lipid A was not recapitulated in the E. coli background. As synthetic biology and genome engineering technologies advance, novel applications and utilities for the detection and treatments of dangerous pathogens like Yersinia pestis will continue to be developed.
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Thank you for your resubmission, we are pleased that your response has addressed all our concerns and are happy to accept the manuscript. Congratulations, if you have any questions, please don't hesitate to ask and we hope you will consider ACMI for future submissions. This study would be a valuable contribution to the existing literature.
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Comments to Author
The authors have applied all my recommendations. Thank you very much for the efforts in doing this even after shutting down this research line, hopefully this work will set a precedent for future directions in the field.
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, …
Comments to Author
The authors have applied all my recommendations. Thank you very much for the efforts in doing this even after shutting down this research line, hopefully this work will set a precedent for future directions in the field.
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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Comments to Author
The authors have addressed the concerns diligently.
Please rate the manuscript for methodological rigour
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?
Partially 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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Comments to Author
In this manuscript, the authors describe their attempts to engineer an E. coli strain to produce Y. pestis lipid A by introducing 3 genes. Overall, the text reads well and is easy to read, however, there are some parts of it in which the rationale is not easy to follow and the final aim of the study is not clearly described (why would you want to produce Y. pestis lipid A in E. coli at all?). Also, given that the final result of the study is negative, the manuscript would greatly benefit from presenting a limitations and future directions section. Here below there are some specific comments: -L67-68: "during Y. pestis". This sentence sounds incomplete. -Figure 1A: a legend stating what each of the symbols in the LPS schematic are would make ti a lot clearer. -L76-81: At this point, the aim of producing …
Comments to Author
In this manuscript, the authors describe their attempts to engineer an E. coli strain to produce Y. pestis lipid A by introducing 3 genes. Overall, the text reads well and is easy to read, however, there are some parts of it in which the rationale is not easy to follow and the final aim of the study is not clearly described (why would you want to produce Y. pestis lipid A in E. coli at all?). Also, given that the final result of the study is negative, the manuscript would greatly benefit from presenting a limitations and future directions section. Here below there are some specific comments: -L67-68: "during Y. pestis". This sentence sounds incomplete. -Figure 1A: a legend stating what each of the symbols in the LPS schematic are would make ti a lot clearer. -L76-81: At this point, the aim of producing Y. pestis lipid A in an E. coli scaffold should be clearly explained. -L90: which expression plasmids? -Abbreviations (e.g. kan, spec) should be defined throughout the text. Also, the "et al" contraction and the gene names need to be in italics throughout the text and figures. -L124-129: does the pEcCas plasmid not need curation after the editing? -L130: RT-PCR needs to be define. In this work, the authors do reverse transcription PCR, which is not quantitative. However, RT-PCR can also stand for real-time PCR, which is quantitative. -L162-164: how do you determine at this point that kdo needs restoring and why this carbohydrate and not other? It would be better to introduce the explanation given in L231-233 here instead of later. -L170-176: it is not clear why the authors pick the gutQ locus instead of the kdsD locus. If the only reason is that the kdsD locus does not have a PAM sequence, then it can just be said that the paralogous gene is selected. This fragment can be simplified. -Fig2C lacks controls. -L204: change "promotor" to "promoter" throughout the text. -L235-236: is (kdo)2-lipidIVa being produced at all? As shown in figure 1 and later mentioned, the order of the reactions is KdsD>LpxP>LpxM, but they are intorduced in the opposite order. That makes it impossible to know which reaction is the bottleneck, i.e. is specifically one of the reactions is now working. -A possible control would be to reintroduce the E. coli native genes to see if lipid A production can be restored with this approach at all. Another control could be complementing the respective Y. pestis mutants in kdsD, lpxM and lpxP. -L260-261: mutations happen in genes, not in proteins. -L270-277: Still there is no clear sentence here stating the purpose of the study, only the separate concepts. -L276-277: this sentence is a bit too overselling given that this work presents a negative result. This need to be put in the context of the results and the final aim that was set. I hope the authors find my comments useful for improving the manuscript.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially 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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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by three experts in the field whose comments are attached below. Please address their concerns thoroughly, specially those involving clarifications that are needed in the aims and structure of the study and the experimental controls and methodologies. As some of the reviewers mention, I also believe a section explaining limitations and future directions would be beneficial for the manuscript. If any of the proposed experiments and controls are not feasible (within reason), please discuss them clearly in the text.
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Comments to Author
In this work McDonald & Antoshak attempt to reconstruct the Y. pestis Lipid A by inserting three genes (lpxP, lpxM, and kdsD) into E. coli ClearColi genome using CRISPR/Cas 9. Successful mutants and their expression are confirmed using RT-PCR. The authors observe no modifications to the ClearColi lipid IVa using MALDI-TOF mass spectrometry on extracts of the newly engineered strains. They propose that this may be due to issues in the kdo biosynthesis or due to a compensating mutation in the transporter gene msbA. The manuscript's research objective is clearly stated, and all the methods used are clearly outlined. The manuscript is easy to follow and although the authors are unsuccessful in modifying the Lipid IVAa solid explanations are provided for the results observed and potential future steps …
Comments to Author
In this work McDonald & Antoshak attempt to reconstruct the Y. pestis Lipid A by inserting three genes (lpxP, lpxM, and kdsD) into E. coli ClearColi genome using CRISPR/Cas 9. Successful mutants and their expression are confirmed using RT-PCR. The authors observe no modifications to the ClearColi lipid IVa using MALDI-TOF mass spectrometry on extracts of the newly engineered strains. They propose that this may be due to issues in the kdo biosynthesis or due to a compensating mutation in the transporter gene msbA. The manuscript's research objective is clearly stated, and all the methods used are clearly outlined. The manuscript is easy to follow and although the authors are unsuccessful in modifying the Lipid IVAa solid explanations are provided for the results observed and potential future steps that may resolve the negative phenotype are briefly outlined. Appropriate controls are included for MALDI-TOF and for the RT-PCR but the authors may wish to consider including a negative control for the gene insertion screening as well (Figure 2.) I have the following suggestions on the experimental data/design: 1. There is no indication of the number of biological replicates for the RT-PCR or the MALDI-TOF experiments. This information should be included either in the methods or the legends. 2. The kdsD gene is inserted into the gutQ genomic locus and the reasoning for this decision is sound. However, kdsD expression from the native gutQ promoter may not be sufficient to achieve kdo modification. Further, the presumption that Y. pestis kdsD can complement the E. coli gene appears unfounded. The authors should include a ClearColi strain with E. coli kdsD expressed from the gutQ locus to show that the use of gutQ locus for kdsD expression is valid. This will also help delineate the proposed negative effects of the compensating msbA mutation on ClearColi. 3. It is widely accepted that the Kdo2-Lipid IVa is the substrate for LpxP in E. coli. This palmitoylation reaction is then rapidly followed by generation of hexa-acylated lipid by LpxM. However, the acylation of the Kdo2-Lipid IVa using LpxP/LpxM has been shown to require cold temperatures in E. coli (PMID: 10092655). This temperature dependence appears to also correlate with the Y. pestis lipid A variations described by the authors (lines 64-70). However, the growth conditions (37°C) used for the MALDI-TOF experiments in this manuscript do not appear to be permissive to this reaction pathway (in E. coli or in Y. pestis); this discrepancy should be addressed by the authors. Minor edits: 4. Figure 1.: The Y. pestis Lipid A and its unique variations are well described in the introduction. However, the supporting Figure 1. shows only a generic Y. pestis lipooligosaccharide structure compared to E. coli K-12 and the ClearColi. The figure would benefit from the inclusion of schematics for the Y. pestis lipid A at different temperatures (as described in lines 64-70), a clear indication of the target structure, and from a legend describing the different components of the lipooligosaccharide structures shown (Fig.1 A). 5. Line 80-81: Missing reference for the link between antimicrobial resistance and the two enzymes investigated. 6. The authors are attempting to recapitulate the wild type Y. pestis Lipid A (line 157), yet do not clarify if the target structure is that of Y. pestis at 37°C or that of Y. pestis at 20-28°C. This should be explicitly stated. 7. The authors should consider including a brief schematic of the Kdo2-Lipid IVA biosynthesis to support their discussion points on the sequence of steps for KdsD, LpxP and LpxM and the reasoning behind the expected/unexpected observations from the MALDI-TOFF data. 8. Line 276: please remove one of the two verbs "this work enables sets the stage for the development of novel detection assays and medical countermeasures."
Please rate the manuscript for methodological rigour
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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Comments to Author
The study by McDonald and Antoshak employs the use of genetic engineering to express Yersinia pestis lipid A into an E. coli strain depleted of lipid IV. The study is well written but seems at an early stage in its current status. It is not clear what was really the purpose of the study. The authors should identify and justify the need to produce an E. coli strain expressing Y. pestis lipid A. As well, gene expression is not a sufficient measure to determine the success of the presence of recombinant enzymes. The authors need to confirm the presence and stability of Yersinia enzymes a well their regulation by Western-Blot and/or proteomics analysis. Overall, a study with limited interest that requires substantial experiments to make a compelling case.
Please rate the manuscript for …
Comments to Author
The study by McDonald and Antoshak employs the use of genetic engineering to express Yersinia pestis lipid A into an E. coli strain depleted of lipid IV. The study is well written but seems at an early stage in its current status. It is not clear what was really the purpose of the study. The authors should identify and justify the need to produce an E. coli strain expressing Y. pestis lipid A. As well, gene expression is not a sufficient measure to determine the success of the presence of recombinant enzymes. The authors need to confirm the presence and stability of Yersinia enzymes a well their regulation by Western-Blot and/or proteomics analysis. Overall, a study with limited interest that requires substantial experiments to make a compelling case.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Not at all
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?
No: noon of the above involved
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