PONE-D-19-16979

Protein:Protein Interactions in the Cytoplasmic Membrane Influencing Sugar Transport and Phosphorylation Activities of the E. coli Phosphotransferase System.

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The present manuscript presents experiments that show that modification of protein expression of the fructose-specific phosphotransferase transport (PTS) system has a positive or negative effect on the apparent activity of other sugar-specific PTS systems in Escherichia coli. Based on previously published global interactome data, which show that soluble and integral membrane elements of multicomponent PTS systems interact with each other, with the the fructose PTS FruA and FruB components forming the most extensive PTS interactome network, they further analyze the basis of this phenomenon.

They use different physiological conditions related to induction of the fructose specific PTS and different strains of E. coli, genetically deleted for FruA or/and FurB, as well as, plasmid-driven overexpression of the Fur components, to investigate how other PTS systems are affected. The authors use basically two functional methods to draw their conclusions; in vivo measurements of radiolabeled sugar accumulation in intact cells, and in vitro enzymatic assays measuring sugar-specific transphosphorylation apparent activities. They additionally use β-galactosidase transcriptional fusions to test whether the de novo synthesis of the affected PTS components is altered in a FurA/B PTS-dependent manner. Based on these assays they conclude that the observed positive or negative effect of FurA/B PTS expression on the activity of other sugar-specific PTS systems take places via protein-protein interaction within the PM, and go on speculating that this is a physiologically meaningful cellular mechanism by which prokaryotes finely regulate PTS-dependent sugar uptake in complex and constantly changing environments.

Main conclusion

This is a very interesting scientific story, which happens to be familiar to me, as in my laboratory we have also come across phenomena of dominant negative or positive effects related to apparent transport activities, caused by the overexpression or absence of a specific transporter. Our observations concern fungal transporters, which makes the whole story broader and probably concern the fine and dynamic balancing of PM transporter composition in all kinds of cells. So, the findings described worth publication in PlosOne, but only after some important issues are clarified, conclusion partly modified and some extra experiments are made. In conclusion the text/figures should be partly re-written, conclusions modified, and a western blot analysis performed. Please see details below.

Points to addressed

1. The tile of the manuscript is not justified by the results presented. (Protein:Protein Interactions in the Cytoplasmic Membrane Influencing Sugar Transport and Phosphorylation Activities of the E. coli Phosphotransferase System). Protein-protein interactions are apparently the reason of the dominant negative or positive effects observed but this is not formally shown.

2. Introduce a cartoon showing the components of different PTS systems in the introduction. The existing figure is not friendly to the reader. Please use full names when abbreviations appear for first time in the text. What is PEP for example?

3. The first two paragraphs of Results introduction recapitulate published results from the same group. It can be significantly reduced with reference in Table 2 or entirely deleted.

4. Confirmation of the interactome results using a bacterial two hybrid system: I cannot see any figure related to this, except S2. It is not clear whether the 2-hybrid system is partly performed here or partly previous publication? Why S2 appears before S1?

5. Having the figure legends interrupting the text in annoying. Either you need both figure/figure legends within the text, or both at the end.

6. In Table S1 explain better what WT and WT-Fr means. I guess the latter means induction by fructose while the former stand for non0induced conditions. One also guess that each experiment is carried out twice, as for each substrate two values are given in all cases, no?

Please, explain abbreviations in all figure legends.

7. Uptakes shown describe ‘apparent’ transporter activity or better ‘apparent accumulation of radiolabelled substrate’, not transport activities per se. Moreover, they are carried out for 5-10 min which definitively describes steady state accumulation and not rate of uptake, despite the results given per min. To my knowledge uptake rates are liner of much shorter times than 5-10 min.,

8. The accumulation of some sugars (e.g. Trehalose and Galactinol) increases significantly (up to 9-fold) in the TM compared to WT upon fructose addition (Table S3). However, the addition of fructose makes a 2-fold difference (Table 2). Please explain this point better in the text.

9. There are some significant deviations in different experiments (S1-S3). Please comment on those.

10. Why S Tables are labelled S4, S5 and S4’, S5’, and not simply with consequent numbers?

11. Most of the data appear in 26 supplementary tables! This makes the manuscript difficult to read and little elegant. I suggest that more clear and composite main figures (with several panels) should highlight the most important results. These should appear in the main text. I am in general against very long supplementary material. The main article should be read without the need for supplements, which are only there to reinforce main findings.

12. A main experiment missing is a western for measuring steady state levels of all components that are affected. Modification of o expression in specific PTS could have an effect on the translocation of PTS in the PM via the translocon complex or in the stability of PTS due to displacement in non-physiological PM microdomains. These possibilities should be discussed.

13. The in vitro PEP-dependent phosphorylation assays are not convincing as they do not lead to a general conclusion. Moreover, I cannot see how crude extracts of membrane proteins can have an effect. My doubts are further enhanced by the fact sugar-P:sugar transphosphorylation assays had no effect by fruA overexpression or purified fruB.

14. “The effects appear to be on the transport and PEP-dependent phosphorylation

activities of these enzyme transporters, not on their syntheses or sugar-P:sugar

transphosphorylation activities” No.

The effect is on apparent activities which is most easily explained by imbalances in protein-protein interactions, including modification in translocation in the PM, stability and/or localization in specific PM microdomains.

15. The first part of the discussion should be deleted, is a repetition of the introduction and results. Also lines 478-507 should be deleted, as they are a repetition of information given ealrler.

Reviewer #2: The manuscript of Aboulwafa et al. addresses the biochemical and physiological significance of interactions of the fructose PTS transporter, consisting of FruA and FruB, with other PTS transporters, including glucose, mannitol, N-acetylglucosamine, galactitol and trehalose transporters. The authors show that: 1) the previous interactome results of FruA and FruB (Nat Biotechnol. 2018; 36:103-112) can be reproduced using a bacterial two-hybrid system, 2) through FruA and FruB, fructose stimulates transport of glucose, mannitol, and N-acetylglucosamine but inhibits transport of galactitol and trehalose, 3) overproduction of FruA and/or FruB (in the absence of fructose) also enhances transport activities of glucose, mannitol, and N-acetylglucosamine (but does not affect significantly those of galactitol and treharose), 4) either induction with fructose or overexpression of FruAB increases the PEP-dependent phosphorylation activities of mannitol and N-acetylglucosamine but decreases that activity of galactiol, and 5) such conditions do not affect transcription of the mannitol, galactitol, and mannose operons. On the basis of the present and previous results, the authors conclude that the fructose PTS transporter regulates the activities of several different PTS transporters through protein-protein interactions. Although mechanistic aspects remain for the future, the findings presented here provide a new perspective on the regulation of sugar transport in bacteria. Overall, this manuscript is clearly written and presents good experimental data to support the major claims of this study. However, I make some minor comments and questions that the authors will address the following issues, and suggest additional experiments, which will hopefully improve the manuscript.

1. Although the authors often describe about mannose transport activity (Lines 33, 251, and 280), the data is not presented anywhere.

2. Add sugar substrate(s) of each transport system in Table 1. This would be helpful for readers.

3. Why does overproduction of FruA and/or FruB not decrease treharose and galactitol transport activities? This is incompatible with the result obtained from mutational analysis of fruA and/or fruB (Fig. 1).

4. Lines 350-351, “FruB activation is not due to the activity of the FPr domain of FruB”. I do not agree with this claim because the authors do not analyze effect of this domain on sugar phosphorylations.

5. Line 372, galP. Add a brief description of galP.

6. Lines 41-42, “the rates of synthesis and protein levels in the membrane of the target PTS permeases were not altered”, Line 288, “the enzyme II activities and not their synthesis”, or Line 475, “only on the activities, not the synthesis or turnover”. To rule out the possibility that the fructose system affects synthesis or turnover of the target PTS proteins, the authors should check expression levels of one or more of these proteins in crude extracts and membrane pellets used for in vitro experiments as well as bacterial culture used for in vivo experiments. Only testing transcriptional activities (Table 3) is insufficient to support the authors’ claim.

Reviewer #3: In their manuscript “protein:protein interactions in the cytoplasmatic membrane influencing sugar transport and phosphorylation activities of the E. coli phosphotransferase system” the authors carefully investigate new roles of FruA and FruB, which are components of the PTS for fructose uptake and phosphorylation. In a previous study among many other results potential interactions of FruA and FruB with further EII-proteins for uptake of other sugars were observed. In this manuscript the authors now follow up and carefully investigate by various approaches the role of FruA and FruB for the control of PTS mediated transport and phosphorylation of sugars via the previously identified potentially interacting EII proteins. The authors here demonstrate that the EII permeases for glucose, mannose, mannitol, and N-acetylglucosamine show a FruA and FruB dependent enhancement of transport activity, while for trehalose and galacitol EII-protein FruA and FruB mediated transport inhibition is observed. The here observed novel effects on the PTS mediated uptake of sugars are shown to exclusively depended on the presence of FruA and FruB and do not depend on fructose transport itself. The authors demonstrate that the positive effects by FruA and FruB on mannitol, galacitol, and mannose uptake are neither based on increased of transcription of the operons encoding the EII components for the uptake of these sugars nor altered transphosphorylation activities, but a stimulation of PEP-dependent phosphorylating activity mediated via protein:protein interactions. Taken together, the authors in this article provide novel insights into a new mode for the control of sugar uptake via the PTS in E. coli and thereby provide further good indications, that this control occurs via protein:protein interactions. The authors finally discuss based on these additional role of FruA and FruB the evolution of PTS systems in bacteria.

The article is very interesting and well written, the experiments fully support the conclusions by the authors. Just some minor concerns might be addressed before publication.

Line 41: change to fructose

Line 58: Maybe instead of putting this scheme in the text an additional graph would be usefull, as some legend might be helpful. Thereby also the situation and names of genes/proteins for the uptake of the other PTS substrates investigated in this manuscript e.g. trehalose can be introduced, facilitationg the understanding of names e.g. used in table 2.

Line 189/Table 2: why not include the results from the bacterial 2 hybrid assay now presented in the supplementary data file also in table 2?

Table S1 and S2: For some sugars 2 and for galactose 3 series of measurements are provided. For the latter the uptake rates between the first two and the third series a highly different – maybe some explanation would be helpful.

Table S1 – S5: Addition of Fructose is abbreviated by writing e.g. “WT-Fru”, I suggest to replace this by “LB + Fru” (and accordingly for the control by “LB”).

Fig S2 (page 31 of supplementary data file) change “mtlA” to “MtlA” and “nagE” to “NagE”

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Protein:Protein Interactions in the Cytoplasmic Membrane Apparently Influencing Sugar Transport and Phosphorylation Activities of the E. coli Phosphotransferase System.

PONE-D-19-16979R1

Dear Dr. Saier Jr.,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Hernâni Gerós, PhD

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All my points were satisfactorily addressed and the revised manuscript can be accepted fro publication

Reviewer #3: (No Response)

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Reviewer #1: Yes: George Diallinas

Reviewer #3: No