Inactivation of ackA and pta Genes Reduces GlpT Expression and Susceptibility to Fosfomycin in Escherichia coli

ABSTRACT Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.


Tables and Figures:
Tables 4 and 5 seem more like they belong as supplemental material. In their place, you could add a table with MICs for the other, FOS-unrelated drugs that were tested as a new table or add that data into Table 1. Figures 2, 4, and 5 could be combined into one figure with three panels since they all deal with intracellular concentrations of small molecules. Figure 3 could be beefed up by the addition of torC transcript level data, or add this data to supplementary material. A potential last figure (Figure 4, if you combine figures 2, 4, and 5 into one) could be the "model" figure with all the proteins and small molecule regulators of fosR, including your new findings, AckA and Pta. I also highly recommend adding a Supplemental Material section that could include, for example: 1) Tables 4 and 5 2) MIC data for Fos-unrelated drugs (if it's not placed in the main manuscript) 3) Sequences of ackA-pta and glpT regions of GU2019-E4 4) The torC transcript data if it doesn't go into Figure 3 Staff Comments:

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Fosfomycin is still effective even for the E. coli strains that are resistant to commonly used antimicrobials. However, several factors have been reported to reduce the susceptibility of E. coli to fosfomycin. In this study, by screening a transposon mutant library of E. coli causing urinary tract infections, the authors found that mutation of the ackA and pta genes rendered E. coli less susceptible to fosfomycin. Further investigation revealed that the deletion of ackA and pta resulted in the down-regulation of the expression of GlpT, a transporter protein of fosfomycin, as the main reason for the resistance of E. coli to fosfomycin.The present study reported novel mutations that reduce the action of fosfomycin, although the frequency of mutations in ackA and pta is low, as the authors pointed out.
Although the authors identified this new phenomenon, they lacked a rigorous and in-depth exploration of the mechanisms behind it. In addition, this work has less data to support some of the assertions and hypotheses made by the authors in this article. From my point of view, this article is of insufficient quality to be published in Microbiology Spectrum.
The reasons for my assessment and the issues I hope the author will study in depth are as follows： 1. The deletion of ackA and pta decreased the expression of GlpT, but the its reason was not well resolved. I think it is important for understanding the resistance of E. coli to fosfomycin.
The results of this study indicated that GlpT was the main protein that transports fosfomycin, compared to UhpT. The authors found that ackA and pta deletion decreased the expression level of GlpT ( Fig. 3), resulting in the resistance of E. coli to fosfomycin. The levels of cAMP and indole did not vary significantly in different strains, and theoretically ackA and pta do not affect GlpT expression by regulating their levels. By comparing MICs of glpR-deficient strains, the authors concluded that ackA and pta also do not act through GlpR. Base on these results, the authors discussed and speculated that "metabolic changes may cause decreased glpT expression via uncharacterized regulatory molecules.
I can hardly agree with this view. First, it is unconvincing to determine from the changes in MICs alone that GlpR is not involved in the regulation of GlpT by ackA and pta. The authors preferably determined the transcript levels of glpR and its regulated genes in ackA and pta-deficient strains. More importantly, there are more factors that regulate glpT expression.
According to the database RegulonDB, the DNA-binding transcriptional dual regulator FNR, factor for inversion stimulation FIS and Integration host factor IHF can also regulate glpT expression, in addition to GlpR and cAMP-regulated CRP, (http://regulondb.ccg.unam.mx/operon?term=ECK120014760&organism=ECK12&format=jsp&t ype=operon). It is worth noting that FNR activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism, and also regulates the genes expression involved in the acid resistance, including ackA, pta and glpT. The authors should examine these existing regulatory factors, so as not to mislead other researchers who are interested in the issue.
2. In the section of Introduction, the authors pointed out "We aimed to obtain further insight into the bacterial genes associated with glpT and uhpT expression that affect the fosfomycin activity" (line 104-105). I do not believe that the authors' strategy of screening fosfomycin-sensitive mutants from the transposon libraries is consistent with the above purpose.
3. There was no clear description of how the transposon library was constructed, nor was there a citation for it. 4. The authors used RT-qPCR to characterize the transcript levels of genes, however, the method of data analysis was not clearly described. The relevant methods are also not clearly described in the cited literature. 5. Some abbreviations was not defined at their first mention, such as MICs (line 125) and the protein MurA(line 83).
6. I think "data not shown" is inappropriate for the testing of other antibiotics (line 146).
Review of: Inactivation of ackA and pta genes reduces GlpT expression and susceptibility to fosfomycin in Escherichia coli by Hirakawa et al.

Manuscript overview
In this manuscript, Hirakawa et al. found that transposon insertions into the ackA or pta genes lead to a 4-fold increase in MIC for fosfomycin in E. coli. They then make these knockouts in CFT073 and in two clinical strains and show that the MICs in the individual knockouts of ackA and pta and the double knockout of both genes also display this increased resistance to fosfomycin. The knockout phenotype is also present when the cells are grown anaerobically. Complementation studies are done showing that replacement of the genes by expression from a plasmid causes reversion to the original fosfomycin sensitive phenotype. Additional genetic studies indicate that when the transporter GlpT is knocked out, knocking out ackA or pta has no effect on fosfomycin MIC, indicating that the effect of knocking out ackA and pta requires the presence of GlpT. In contrast, another transporter, UhpT, the transcriptional regulator GlpR, and the two-component response system proteins CpxAR, are dispensable for the increase in MIC seen with ackA and pta knockouts.
In the molecular biology part of the paper, the authors demonstrate that intracellular accumulation of fosfomycin is impaired in the two knockout strains, and hypothesize that, in light of the genetic results with the GlpT knockout, glpT transcription may be down-regulated in the ackA and pta knockout strains. In Figure 3, they show that indeed glpT transcripts are lower in the ackA and pta deletion strains, though murA mRNA levels (MurA is the target of fosfomycin) are unchanged. They also measure uhpT transcript levels, and although it appears to me that it is quite a bit lower in the pta knockout, the authors conclude that uhpT transcription is also unchanged in the mutants.
The last data looks at cAMP and indole levels in the wt and mutant strains. These molecules represent potential regulators of glpT expression. Both appear to be unchanged in the mutants, supporting the authors' conclusion that mutants' main mechanism for inducing fosfomycin resistance is through downregulation of glpT by an unknown mechanism.
Overall, I think this work has general relevance, particularly in light of the fact that fosfomycin is being investigated for systemic use against MDR strains (Silver, Cold Spring Harb Perspect Med 2017;7:a025262). In this context it will be important to understand the various ways fosfomycin resistance can arise, and these two genes do not appear to have been linked to fos R before. However, the manuscript is a little light on data, and I feel more context is needed to bolster the findings. For example, all the "data not shown" could be included in the main text or in a new supplemental section (which I highly recommend adding). For context, I feel more information is needed about the original transposon screen, and I'd recommend adding a "model" figure. In addition, there are places where the writing should be clarified. I will list my suggestions in order of line numbering below. (Line numbers correspond to those in the merged PDF.) Specific suggestions: Line 72: Replace "used" for "using" Line 74: Rephrase to something like, "there is a shortage of…" Line 80: "reattracted" must be following by something, such as "reattracted attention" Line 85: I read in this excellent review (Silver, Cold Spring Harb Perspect Med 2017;7:a025262), that FosC is actually a version of FomA. Please review this finding and edit this line as necessary. Also, I could not find evidence that fosX is found on plasmids, only on IS elements. Please review this as well and edit wording as necessary (for example, you could say "mobile elements" in place of "plasmids" if necessary). You may need to include updated references.
Line 86-87: Consider grammar changes to read: "In addition to these exogenous mechanisms…" Line 93: Add parenthesis around "(required for cAMP production)" for clarity as was done in line 221, and then omit this phrase from line 221 because it will already have been defined here. :575031) are also involved in fos R but these proteins receive no mention here. They don't have to be included if the authors think this is too much information, but I'm curious as to the reason for omitting some mechanisms while including others.
Lines 123-127. I'd really like to know more about the original transposon screen. Is there a reference for the details of this particular screen from your laboratory? If so, please include the reference. If not, I think at least the following details should be included here: 1) What transposon was used? Does it have an outward promoter as part of the sequence to minimize downstream effects? 2) How saturated do expect this screen of 2200 colonies to be? In other words, is this likely to represent a collection where almost every non-essential gene should be knocked out at least once? 3) You mention that two strains had insertions into ackA and pta. What other insertions were isolated in this screen? Will there be a future paper about them? Did you also get insertions into the expected genes (glpT, cyaA etc)? If no other insertions besides the expected ones and your ackA and pta isolates were observed, that would be a compelling result worth mentioning. 4) I found two other articles that used transposon mutagenesis to look for effects on fosfomycin susceptibility: Turner et al, J Antimicrob Chemother 2020; 75: 3144-3151; and Coward et al, FEMS Microbiology Letters, 367, 2020, fnaa185. These are both fairly recent studies and seem relevant to your work. Were these genes found in those screens? Glancing through those papers it seems they weren't, although I didn't go through all the data and supplemental materials in detail. Why do you think they weren't found in these or other screens? Do you think those studies should be mentioned here?
Line 136: Should say that the survival rates correspond to x hours post-exposure at x-times the MIC of the parent strain (you fill in the x's).
Line 146: I really think the MICs to these other antibiotics should be shown either in Table 1, as another separate table in the paper, or at the very least in the supplemental material. Line 179: Consider adding the word "Also," at the beginning of sentence: "Also, deletion of ackA and pta genes…" Lines 182-191: Is the point of this section to explain why acetyl-phosphate and/or acetyl-CoA was not measured? I found this section somewhat confusing. Perhaps state up-front (good place to start a new paragraph) that the effects probably aren't due to an accumulation of acetyl-phosphate, which can regulate CpxR, and then state your reasoning.
Line 189: Consider replacing "as well" with "to the same extent" for clarity.
Line 191: Put this data into Figure 3, or as a supplemental figure.
Lines 193: Need to reword the title of this section for clarity. For example, "Deletion of ackA and pta also results in increased resistance to fosfomycin in an ESBL-producing strain and in an enterohemorrhagic strain of E. coli." (You don't really mean these deletions result in susceptibility, right?) Lines 203-206: It would beef up the paper a bit if you could include these new, unpublished sequences in supplemental material (that of the ackA-pta and glpT regions of GU2019-E4).
Line 208: This sentence: "The sequences were >96% identical," what does it refer to? Identical to Sakai or to the original CFT073 strain? Or all to each other? Please clarify.
Line 211: Consider adding some kind of closing remark such as, "… multidrug resistant strain and EHEC, providing potential clinical relevance to mutations in these genes." LIne 217: Consider starting the sentence with "The transporters GlpT and UhpT are required…" Line 219: Add "expression levels" in place of "expression" for clarity.
Line 221: Can omit "(require for the cAMP synthesis)" because this was stated earlier.
Line 223: Consider adding "also" as in "… AckA and Pta also contribute to expression…" Line 225: Change "the source" to "a source".
Line 231: Switch word order to "another uncharacterized regulatory molecule".
Line 239: Make this more clear. For example, "…less sensitive to fosfomycin as compared to the wildtype parent strain to the same extent as was the ackA single-deficient strain…" Line 257: Typo: two "that"'s in a row. Consider: "…the possibility that such mutants are generated…" Line 268: Do you think there is a way to check for clinical relevance of mutations in these genes? Has it been done before? It might be worth pointing the reader in a future direction as a parting remark.

Tables and Figures:
Tables 4 and 5 seem more like they belong as supplemental material. In their place, you could add a table with MICs for the other, FOS-unrelated drugs that were tested as a new table or add that data into Table 1.   1. The deletion of ackA and pta decreased the expression of GlpT, but the its reason was not well resolved. I think it is important for understanding the resistance of E. coli to fosfomycin.
The results of this study indicated that GlpT was the main protein that transports fosfomycin, compared to UhpT. The authors found that ackA and pta deletion decreased the expression level of GlpT (Fig. 3), resulting in the resistance of E. coli to fosfomycin. The levels of cAMP and indole did not vary significantly in different strains, and theoretically ackA and pta do not affect GlpT expression by regulating their levels. By comparing MICs of glpR-deficient strains, the authors concluded that ackA and pta also do not act through GlpR. Base on these results, the authors discussed and speculated that "metabolic changes may cause decreased glpT expression via uncharacterized regulatory molecules.
I can hardly agree with this view. First, it is unconvincing to determine from the changes in MICs alone that GlpR is not involved in the regulation of GlpT by ackA and pta. The authors preferably determined the transcript levels of glpR and its regulated genes in ackA and pta-deficient strains. More importantly, there are more factors that regulate glpT expression.
According to the database RegulonDB, the DNA-binding transcriptional dual regulator FNR, factor for inversion stimulation FIS and Integration host factor IHF can also regulate glpT expression, in addition to GlpR and cAMP-regulated CRP,  (Table 1). Furthermore, no reduction in MIC was observed when the fis gene was exogenously expressed on the glpT mutant (Table 1). A decrease in promoter activity of glpT was also observed when the fis gene was deleted ( Figure 4A). Purified Fis protein was also found to bind to the upstream region of glpT ( Figure 4B). These results indicate that Fis activates the expression of glpT, but that the ackA and pta mutations reduce the level of Fis, which in turn reduces the expression of glpT, resulting in reduced fosfomycin activity.
We also mentioned in our discussion that FNR is involved in the regulation of ackA and pta as well as acid resistance genes.
2. In the section of Introduction, the authors pointed out "We aimed to obtain further insight into the bacterial genes associated with glpT and uhpT expression that affect the fosfomycin activity"(line 104-105). I do not believe that the authors' strategy of screening fosfomycin-sensitive mutants from the transposon libraries is consistent with the above purpose.
Response: Our purpose is to identify novel genes that affect fosfomycin activity in UPEC.

Fosfomycin activity is dependent on the GlpT and UhpT transporters. Since GlpT and
UhpT are secondary transporters, their expression is thought to be regulated by various transcriptional regulators, including known regulators. On the other hand, fosfomycin inhibits the function of MurA, which gene is considered to be house-keeping since murA is an essential gene. For this reason, we hypothesize that the screened set of genes contributing to fosfomycin resistance includes a significant number of genes that affect the expression of GlpT and UhpT. As pointed out, the original text may mislead the readers, then we simply changed it to "We aimed to obtain further insight into the bacterial genes that affect fosfomycin activity". 6. I think "data not shown" is inappropriate for the testing of other antibiotics (line 146).
Response: As suggested, the MICs were presented in Supplementary table.

Reviewer2
Overall, I think this work has general relevance, particularly in light of the fact that fosfomycin is being investigated for systemic use against MDR strains (Silver, Cold Spring Harb Perspect Med 2017;7:a025262). In this context it will be important to understand the various ways fosfomycin resistance can arise, and these two genes do not appear to have been linked to fosR before. However, the manuscript is a little light on data, and I feel more context is needed to bolster the findings. For example, all the "data not shown" could be included in the main text Specific suggestions: Line 72: Replace "used" for "using"

Response: Replaced.
Line 74: Rephrase to something like, "there is a shortage of…" Response: We rephrased it to "there is a shortage of new alternative agents".
Line 85: I read in this excellent review (Silver, Cold Spring Harb Perspect Med 2017;7:a025262), that FosC is actually a version of FomA. Please review this finding and edit this line as necessary. Also, I could not find evidence that fosX is found on plasmids, only on IS elements.
Please review this as well and edit wording as necessary (for example, you could say "mobile elements" in place of "plasmids" if necessary). You may need to include updated references.
Response: Thank you for letting us know. We now update our knowledge. We accordingly modified line85 with citing the review. In addition, we deleted "plasmids" for the fosX statement.
Line 86-87: Consider grammar changes to read: "In addition to these exogenous mechanisms…" Response: Changed.
Line 93: Add parenthesis around "(required for cAMP production)" for clarity as was done in line 221, and then omit this phrase from line 221 because it will already have been defined here.
Response: As suggested, we added parenthesis and deleted (Required for the cAMP synthesis) from the sentence in line 221 sentence.
Lines 82-103: This is a very long paragraph. Consider breaking it up, perhaps after the sentence ending in line 94.

Response: As suggested, it was split into two paragraphs.
Lines of the pta gene may be affected. However, we constructed the "clean deletion mutants" for ackA and pta genes. We also confirmed that susceptibility to fofomycin was complemented by exogenous gene expression. 2) We think that this screen of 2200 colonies is not saturated. Originally, we performed transposon mutagenesis with this kit to find out genes that are involved in biofilm formation. In the process, we obtained 2200 clones with transposon inserts. We used this clone sets for this study (We explained it in Introduction and the start of Result). Therefore, our screen has a limitation. However, our aim is not to conduct comprehensive screening to find out genes that is responsible for fosfomycin activity. In addition to searching for new genes that contribute to fosfomycin activity, we are focusing on elucidating the detailed mechanisms of resistance. Line 138: Replace "estimated" with "determined" or "measured".

3) You mention that two strains had insertions into
Response: We replaced "estimated" with "determined".
Line 146: I really think the MICs to these other antibiotics should be shown either in Table 1, as another separate table in the paper, or at the very least in the supplemental material.

Response: As suggested, the MICs to drugs except fosfomycin were presented in
Supplementary table (Table S1).
Lines 148-170: Paragraph too long. Consider breaking it up, possibly after sentence ending in line 161.

Response: As suggested, it was split into two sentences.
Lines 165-170: Flesh this out a little more for those not so well versed in bacterial genetics.
What do you expect if the genes are involved in the same pathway vs. if they act independently of each other? the wildtype parent strain to the same extent as was the ackA single-deficient strain…"

Response: We accordingly changed it.
Line 257: Typo: two "that"'s in a row. Consider: "…the possibility that such mutants are generated…"

Response: Corrected.
Line 268: Do you think there is a way to check for clinical relevance of mutations in these genes? Has it been done before? It might be worth pointing the reader in a future direction as a parting remark.

Response: We agree that that is a good approach for the future direction. No clinically isolated fosfomycin-resistant strains with mutations in ackA and pta have been reported.
There are no reports on the association between ackA/pta and antimicrobial resistance, not only to fosfomycin, and we assume that no one has paid attention to ackA/pta in drug resistance studies. As NGS analysis is becoming more accessible, it is possible to find out the fosfomycin-resistant strains, with ackA and pta SNPs, that have been isolated so far and those that will be isolated in the future if we look for mutations in ackA and pta in those resistant strains with a particular attention. We added it to the end of Discussion.

Tables and Figures:
Tables 4 and 5 seem more like they belong as supplemental material. In their place, you could add a table with MICs for the other, FOS-unrelated drugs that were tested as a new table or add that data into Table 1.
Response: As suggested, we moved Tables4 and 5 to supplemental material (Table S2 and S3), and MIC data for other drugs was provided as a supplementary table (Table S1). Response: As suggested, we combined these figures (Figure 2A-C). Response: As suggested, we provided the model figure (Figure 5).
I also highly recommend adding a Supplemental Material section that could include, for example: 1) Tables 4 and 5 2) MIC data for Fos-unrelated drugs (if it's not placed in the main manuscript) 3) Sequences of ackA-pta and glpT regions of GU2019-E4 4) The torC transcript data if it doesn't go into Figure 3 Response: As suggested, in addition to tables 4 and 5 contexts, MIC data and sequences were provided in supplemental data (Table S1-3). The torC data was included in the qPCR figure ( Figure 3B). Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. I am reviewing the resubmission of this paper after having reviewed the original manuscript. In this resubmission, the authors have addressed many of my concerns and also added experimental information about the involvement of the Fis protein in regulating glpT expression. In particular, I was pleased to see further explanation of the transposon screen, the MICs of other antibiotics in Supplemental Table S1 as well as the addition of the "data not shown" into supplemental material, and the inclusion of a model figure (Figure 5), which adds much clarity to the complex regulatory mechanisms at work in this system. I still have some suggestions (below) many of which are geared toward the clarity of writing. In particular, the Discussion needs some reworking. Also, I feel Figure 5, which is a great addition, needs to be made into one panel and the regulatory network described with additional color coding.
I have the following comments (Line numbers based on clean, resubmitted manuscript): Line 45: Add comma: "nucleoid-associated protein, Fis." Also correct spelling to "nucleoid" here and throughout document. Line 45-47: Awkward sentence structure making it less clear what your conclusion is. Consider changing to something more direct like: "We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta knockout strains to be due to a decrease in Fis levels in these mutants. " Lines 92-93: Consider wording change to "...GlpT, UhpT and MurA on the chromosome alter susceptibility to the drug. In particular, functional deletion..." Lines 96-113. This is a long paragraph with a lot of information in it, which may be hard to digest for a reader not familiar with the field. It would help to walk the reader through it a little, and refer to your new model figure (Fig. 5). Some suggestions: 1. First sentence, consider: "Fosfomycin sensitivity is also attenuated by decreased expression of GlpT and UhpT, which can occur by several mechanisms (Figure 5)." 2. Line 102-103, reduced expression of uhpT presumably comes first (not consequent to) fosR. Consider reversing the order of this sentence to something like, "...were found to result in reduced expression of uhpT, with consequent resistance to fosfomycin." 3. Start new paragraph at line 103 sentence, "In addition 104 to the positive regulators..." Could also reference Figure 5 at the end of this sentence, i.e., "...act as repressors for the glpT and uhpT genes ( Figure 5)." 4. I would add the abbreviation: "... trimethylamine-N-oxide (TMAO)..." since it is labeled that way in the figure.
Line 122: Correct to "acetate" Line 141-142: Were these the only two clones you found with substantial resistance besides the obvious ones? Was your cut-off for "moderate resistance" 4-fold? Again, if these were the only two in your collection that were 4-fold or over, that would be nice to state here. You could say, "In addition to these clones, we found only two other clones..." Also, I feel you should report an estimate for how much coverage you expect your library of 2200 clones to represent, i.e., 50% (or whatever the number is) of expected non-essential genes. Maybe this is reported in the original paper -if so, refer to that number here or elsewhere. (You could consider adding the information about the transposon screen into a small paragraph in the Methods section if it seems like too much to put into the results section. Then you could describe in detail how you screened the transposon library and what cut-offs were etc.) Line 143: Consider starting a new paragraph at "To verify this result..." Line 149-152: Consider keeping this to one sentence: "We also compared the survival of the mutant strains with that of the parent strain at 1 to 3 hours post-exposure at 6.25-times the MIC of the parent strain." Line 182: Split into multiple sentences for clarity: "Based on the qPCR results, we hypothesized that the increased resistance to fosfomycin seen in the ackA and pta mutants is caused mostly by decreased glpT expression. If so, then we would expect to see no further increase in resistance to fosfomycin upon deletion of ackA and pta in the absence of glpT, while the absence uhpT should not impact the effects of these mutations on fosfomycin sensitivity." Lines 188-189: For clarity, use fold-increases here: "...deletion of ackA and pta from the uhpT mutant resulted in a further 4-fold increase in fosfomycin MICs (Table 1)." Line 204: Change to "activates". Line 222: Need to point out this lower than the parent strain: "reduced the MIC to 2 mg/L, 2-fold lower than the parent strain (Table 1)." Lines 225-226 : Would it be fair to add the following?: "These observations suggest that the increased susceptibility to fosfomycin due to fis expression is dependent on glpT, and that AckA and Pta act upstream of Fis in the regulation of glpT." Line 234: I definitely see a shift at 1 pmol with the rhlR control construct, but I agree that it could be non-specific. I would still say the shift can be seen at 1 pmol (not at 2 pmol) with the control as compared to 0.25 with glpT. Lines 235-238: Perhaps soften these statements since the EMSA results are not super-convincing: "These results suggest that Fis binds to the upstream region of glpT, increasing its promoter activity. Altogether, we conclude ackA and pta mutations cause a decrease in...." Line 249-250: Add your Supplemental reference here: "...the sequence of the ackA-pta operon for GU2019-E4 was determined 249 because the complete genome sequence for this strain has not been available ( Figure S1)." Then delete this sentence: "We found orthologous ackA and pta genes in the GU2019-E4 chromosome." Line 254-255: Similarly, add Supplemental ref here: "...for GU2019-E4 were also determined and compared with those for CFT073 and EHEC O157:H7 Sakai strains ( Figure S2, S3)." Then drop ref after next sentence: "...three strains." Also note extra space in line 254. Line 264: This first sentence is awkward. You could say something like, "It is known that chromosomal mutations can affect fosfomycin sensitivity." Line 269: Note extra space. Line 270-274. Needs to be reworded for clarity. How about: "The current study shows that AckA and Pta also contribute to expression of glpT and sensitivity to fosfomycin because deletion of the ackA and pta genes reduced expression of glpT, but not uhpT, and decreased sensitivity to the drug. Therefore, the ackA and pta genes may be a source of fosfomycin resistance." Lines 275-284: While I'm glad you acknowledge these studies here, it's not necessary to say which are better or worse. Better to keep it short and let the reader explore for themselves. Here is a possible re-wording of this section (please use your own judgment/wording here, this is just an example of how to reduce this section to only the key points): "Recently, two comprehensive studies combining transposon mutagenesis with next-generation sequencing (NGS) analysis identified genes contributing to fosfomycin activity [35] [36]. Interestingly, ackA and pta were not identified in these studies. One difference between our study and these is that in the other studies, mutants were selected to survive in the presence of fosfomycin while in our study mutants were obtained in the absence of fosfomycin. These differences could account for the differences in results obtained." Line 284: Start a new paragraph with "Expression..." Line 286: Need to be more clear about what data you are considering to make this statement. For example, you could say: "According to results from our knockout strain fosfomycin sensitivity assays, small molecule (cAMP and indole) measurements, and qPCR gene expression analyses, the decreased expression of glpT due to ackA and pta gene inactivation likely does not involve these regulators." Line 290: End sentence at "...glpT expression." Next sentence, "In a series of experiments including MIC assays of fis deletion mutants, fis expression qPCR analyses, promoter expression assays, and gel shift assays, we showed that Fis increases glpT expression." Line 298: Change to "other regulators" or "additional regulators" rather than "another regulators". Line 306: Extra "t" Lines 311-315: This paragraph seems out of place here. I would put it instead in between the paragraphs in lines 298-299. Lines 314-315: Consider changing to: "...free CoA pool [42], which may cause decreased fis expression." Line 320: Extra space Line 327: Use "disadvantageous" or "a disadvantage" in place of "disadvantage". Line 329: Change "its" to "their". Lines 331-337: These last sentences need to be more clear. Consider something like: "As more NGS data becomes available, it will be possible check if fosfomycin-resistant strains that have been isolated so far and those that will be isolated in the future have mutations in ackA and pta. We believe that this study will aid us to more precisely predict fosfomycin resistance and thereby help to keep fosfomycin treatment effective." Line 376: Did you do your MIC measurements more than once for each strain? If so, please state the minimum number of MIC measurements taken to establish each strain's MIC. I hope this is at least 3 times for each strain, as MICs are known to drift by 2-fold here and there. Lines 676, 679, 682: I would use "MICs" rather than "MIC" in the titles of the tables, but use "MIC" rather than "MICs" in the "Strain" line (line below 676, line below 679, and line below 682). Line 682: You can say, " Table 3. Fosfomycin MICs of other clinical E. coli strains" rather than just "other strains". Line 713: Extra "_" Line 716: Is this supposed to say *, P<=0.05? Line 723: Need to define what "'P" and "TMAO" and "G6P" stand for in the figure. Figure 4A: Label Y-axis as "β-galactosidase activity" not "activities" Figure 5: While I think this figure is a great addition to the paper, it doesn't quite unify all the data as it is now. Would it be possible to also color code the second level regulators, such as TorT/TorS, CpxA, UhpC/B, and all the small molecules, or does this get too confusing? Also, I'd rather see Pta and AckA put into the top part of figure 5 and have only one panel. If you need more room, you could put the regulators all around glpT and uhpT in a circle, rather than having them all above. For the Pta and AckA effect, you could direct an arrow toward Fis, which would then direct toward glpT (all with appropriate colors), and also an arrow with a question mark directly to glpT to represent the unknown additional regulator. You could highlight your contribution in some way (bold, circled etc) to show what you've added to this picture.
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Review of "Inactivation of ackA and pta genes reduces GlpT expression and susceptibility to fosfomycin in Escherichia coli" Resubmission 4/16/2023 I am reviewing the resubmission of this paper after having reviewed the original manuscript. In this resubmission, the authors have addressed many of my concerns and also added experimental information about the involvement of the Fis protein in regulating glpT expression. In particular, I was pleased to see further explanation of the transposon screen, the MICs of other antibiotics in Supplemental Table S1 as well as the addition of the "data not shown" into supplemental material, and the inclusion of a model figure (Figure 5), which adds much clarity to the complex regulatory mechanisms at work in this system. I still have some suggestions (below) many of which are geared toward the clarity of writing. In particular, the Discussion needs some reworking. Also, I feel Figure 5, which is a great addition, needs to be made into one panel and the regulatory network described with additional color coding.
I have the following comments (Line numbers based on clean, resubmitted manuscript): Line 45: Add comma: "nucleoid-associated protein, Fis." Also correct spelling to "nucleoid" here and throughout document.
Line 45-47: Awkward sentence structure making it less clear what your conclusion is. Consider changing to something more direct like: "We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta knockout strains to be due to a decrease in Fis levels in these mutants. " Lines 92-93: Consider wording change to "…GlpT, UhpT and MurA on the chromosome alter susceptibility to the drug. In particular, functional deletion…" Lines 96-113. This is a long paragraph with a lot of information in it, which may be hard to digest for a reader not familiar with the field. It would help to walk the reader through it a little, and refer to your new model figure (Fig. 5). Some suggestions: Line 141-142: Were these the only two clones you found with substantial resistance besides the obvious ones? Was your cut-off for "moderate resistance" 4-fold? Again, if these were the only two in your collection that were 4-fold or over, that would be nice to state here. You could say, "In addition to these clones, we found only two other clones…" Also, I feel you should report an estimate for how much coverage you expect your library of 2200 clones to represent, i.e., 50% (or whatever the number is) of expected non-essential genes. Maybe this is reported in the original paper -if so, refer to that number here or elsewhere. (You could consider adding the information about the transposon screen into a small paragraph in the Methods section if it seems like too much to put into the results section. Then you could describe in detail how you screened the transposon library and what cut-offs were etc.) Line 143: Consider starting a new paragraph at "To verify this result…" Line 149-152: Consider keeping this to one sentence: "We also compared the survival of the mutant strains with that of the parent strain at 1 to 3 hours post-exposure at 6.25-times the MIC of the parent strain." Line 182: Split into multiple sentences for clarity: "Based on the qPCR results, we hypothesized that the increased resistance to fosfomycin seen in the ackA and pta mutants is caused mostly by decreased glpT expression. If so, then we would expect to see no further increase in resistance to fosfomycin upon deletion of ackA and pta in the absence of glpT, while the absence uhpT should not impact the effects of these mutations on fosfomycin sensitivity." Lines 188-189: For clarity, use fold-increases here: "…deletion of ackA and pta from the uhpT mutant resulted in a further 4-fold increase in fosfomycin MICs (Table 1)." Line 204: Change to "activates".
Line 222: Need to point out this lower than the parent strain: "reduced the MIC to 2 mg/L, 2-fold lower than the parent strain (Table 1)." Lines 225-226 : Would it be fair to add the following?: "These observations suggest that the increased susceptibility to fosfomycin due to fis expression is dependent on glpT, and that AckA and Pta act upstream of Fis in the regulation of glpT." Line 234: I definitely see a shift at 1 pmol with the rhlR control construct, but I agree that it could be non-specific. I would still say the shift can be seen at 1 pmol (not at 2 pmol) with the control as compared to 0.25 with glpT.
Lines 235-238: Perhaps soften these statements since the EMSA results are not super-convincing: "These results suggest that Fis binds to the upstream region of glpT, increasing its promoter activity. Altogether, we conclude ackA and pta mutations cause a decrease in…." Line 249-250: Add your Supplemental reference here: "…the sequence of the ackA-pta operon for GU2019-E4 was determined 249 because the complete genome sequence for this strain has not been available ( Figure S1)." Then delete this sentence: "We found orthologous ackA and pta genes in the GU2019-E4 chromosome." Line 254-255: Similarly, add Supplemental ref here: "…for GU2019-E4 were also determined and compared with those for CFT073 and EHEC O157:H7 Sakai strains ( Figure S2, S3)." Then drop ref after next sentence: "…three strains." Also note extra space in line 254.
Line 264: This first sentence is awkward. You could say something like, "It is known that chromosomal mutations can affect fosfomycin sensitivity." Line 269: Note extra space.
Line 270-274. Needs to be reworded for clarity. How about: "The current study shows that AckA and Pta also contribute to expression of glpT and sensitivity to fosfomycin because deletion of the ackA and pta genes reduced expression of glpT, but not uhpT, and decreased sensitivity to the drug. Therefore, the ackA and pta genes may be a source of fosfomycin resistance." Lines 275-284: While I'm glad you acknowledge these studies here, it's not necessary to say which are better or worse. Better to keep it short and let the reader explore for themselves. Here is a possible rewording of this section (please use your own judgment/wording here, this is just an example of how to reduce this section to only the key points): "Recently, two comprehensive studies combining transposon mutagenesis with next-generation sequencing (NGS) analysis identified genes contributing to fosfomycin activity [35] [36]. Interestingly, ackA and pta were not identified in these studies. One difference between our study and these is that in the other studies, mutants were selected to survive in the presence of fosfomycin while in our study mutants were obtained in the absence of fosfomycin. These differences could account for the differences in results obtained." Line 284: Start a new paragraph with "Expression…" Line 286: Need to be more clear about what data you are considering to make this statement. For example, you could say: "According to results from our knockout strain fosfomycin sensitivity assays, small molecule (cAMP and indole) measurements, and qPCR gene expression analyses, the decreased expression of glpT due to ackA and pta gene inactivation likely does not involve these regulators." Line 290: End sentence at "…glpT expression." Next sentence, "In a series of experiments including MIC assays of fis deletion mutants, fis expression qPCR analyses, promoter expression assays, and gel shift assays, we showed that Fis increases glpT expression." Line 298: Change to "other regulators" or "additional regulators" rather than "another regulators".
Line 306: Extra "t" Lines 311-315: This paragraph seems out of place here. I would put it instead in between the paragraphs in lines 298-299.
Lines 331-337: These last sentences need to be more clear. Consider something like: "As more NGS data becomes available, it will be possible check if fosfomycin-resistant strains that have been isolated so far and those that will be isolated in the future have mutations in ackA and pta. We believe that this study will aid us to more precisely predict fosfomycin resistance and thereby help to keep fosfomycin treatment effective." Line 376: Did you do your MIC measurements more than once for each strain? If so, please state the minimum number of MIC measurements taken to establish each strain's MIC. I hope this is at least 3 times for each strain, as MICs are known to drift by 2-fold here and there.
Lines 676, 679, 682: I would use "MICs" rather than "MIC" in the titles of the tables, but use "MIC" rather than "MICs" in the "Strain" line (line below 676, line below 679, and line below 682).
Line 682: You can say, " Table 3. Fosfomycin MICs of other clinical E. coli strains" rather than just "other strains".
Line 723: Need to define what "'P" and "TMAO" and "G6P" stand for in the figure. Figure 4A: Label Y-axis as "β-galactosidase activity" not "activities" Figure 5: While I think this figure is a great addition to the paper, it doesn't quite unify all the data as it is now. Would it be possible to also color code the second level regulators, such as TorT/TorS, CpxA, UhpC/B, and all the small molecules, or does this get too confusing? Also, I'd rather see Pta and AckA put into the top part of figure 5 and have only one panel. If you need more room, you could put the regulators all around glpT and uhpT in a circle, rather than having them all above. For the Pta and AckA effect, you could direct an arrow toward Fis, which would then direct toward glpT (all with appropriate colors), and also an arrow with a question mark directly to glpT to represent the unknown additional regulator. You could highlight your contribution in some way (bold, circled etc) to show what you've added to this picture.
I am reviewing the resubmission of this paper after having reviewed the original manuscript. In this resubmission, the authors have addressed many of my concerns and also added experimental information about the involvement of the Fis protein in regulating glpT expression. In particular, I was pleased to see further explanation of the transposon screen, the MICs of other antibiotics in Supplemental Table S1 as well as the addition of the "data not shown" into supplemental material, and the inclusion of a model figure (Figure 5), which adds much clarity to the complex regulatory mechanisms at work in this system. I still have some suggestions (below) many of which are geared toward the clarity of writing. In particular, the Discussion needs some reworking. Also, I feel Figure 5, which is a great addition, needs to be made into one panel and the regulatory network described with additional color coding. Figure 4A: Label Y-axis as "β-galactosidase activity" not "activities" Response: Corrected.