Protein Export via the Type III Secretion System of the Bacterial Flagellum
Round 1
Reviewer 1 Report
Halte et al clearly summarized their recent findings of the structure and function of a type-III secretion system, especially the flagellar secretion system. This research field is flourishing now. Authors accurately follow the past important results and latest findings, so I think this review provides fruitful information to the readers. The writing is also clear and will draw most readers easily into the story. I have some minor comments listed below. Since the lane number was not displayed on the paper that I received, I will also include the page number.
Page2 Lane5, (Figure1: Schematic …
The description for Figure1 is the same as the figure legend, so it should be deleted or corrected.
Page3 Lane7, (Figure2: Step-wise …
The description for Figure2 is the same as the figure legend, so it should be deleted or corrected.
Page5 Lane15, (a pentamer of …
It is only a model that MotA rotates around MotB, and there is no evidence by direct observation yet. I think it would be better to have a sentence that includes a little more possibility.
Page11 Figure3 legend,
It is better to correct the stoichiometry and domain name as a subscript, like FliH12FliI6FliJ and FlhACD2.
Page15 References [9],
Please write the author’s name correctly.
Author Response
Thank you for your kind words and constructive review. Please find below our point-to-point responses to the raised issues.
Page2 Lane5, (Figure1: Schematic ...The description for Figure1 is the same as the figure legend, so it should be deleted or corrected.
Page3 Lane7, (Figure2: Step-wise ...The description for Figure2 is the same as the figure legend, so it should be deleted or corrected.
RE: We apologize, this has been a mistake. For some reason, during generation of the PDF, the complete figure legend has been referenced in the text and not only the figure numbering. This has been corrected.
Page5 Lane15, (a pentamer of ...It is only a model that MotA rotates around MotB, and there is no evidence by direct observation yet. I think it would be better to have a sentence that includes a little more possibility.
RE: The sentence has been modified as suggested and now reads: “a pentamer of MotA, that has been proposed to rotate around…”
Page11 Figure3 legend, It is better to correct the stoichiometry and domain name as a subscript, like FliHFliIFliJ and FlhA.
RE: Modified as suggested.
Page15 References [9],Please write the author’s name correctly.
RE: The authors names have been corrected, thank you.
Reviewer 2 Report
The present study is a very interesting review that focuses on recent advances in the structure and assembly of the core flagellar type III secretion system (fT3SS), as well as in substrate secretion mechanisms and energization processes. The authors also compare the protein export mechanisms with other secretion systems, mostly with the virulence (vT3SS).
This is a well-written work of high quality that makes an important contribution to the fT3SS field. The authors reviewed and summarized the most recent literature on the topic. The information is clear, concise and well-organized. They also provide possible explanations for contradictory published observations.
Overall, this is a well-balanced review and I only have minor suggestions:
Introduction, page 2, first paragraph: Figure 1 “and rod composed of the P-ring” it is confusing since the rod is made of the 5 proteins previously mentioned in the text. The same comment for figure 1 legend.
In this same paragraph “from early-type substrates (e.g. components of the basal body…” it is also confusing since not all components of the basal body are early substrates secreted via the T3SS.
Figure 1. It will be nice to also point out FlgI and FlgH that are included in the figure legend.
Section 3. first paragraph “complex AT THE AT THE base of the export…”
Page 7 first paragraph “C-ring via FliH interactions and thereby would FUNCTION AS the”. In this same paragraph I suggest to discuss the following references:
The structure of the Type III secretion system export gate with CdsO, an ATPase lever arm. Jaime L. Jensen, Shavait Yamini, Arne Rietsch, Benjamin W. Spiller. 2020
Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry. Dorothy D. Majewski, Liam J. Worrall, Chuan Hong, Claire E. Atkinson, Marija Vuckovic, Nobuhiko Watanabe, Zhiheng Yu & Natalie C. J. Strynadka. 2019
Section 4.1, page 8, last paragraph. In relation to the multiple conformations of FlhA, the following reference could be included:
Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus. Inoue, Y et al. 2019
Section 4.2. The FlhA linker also has a role in the oligomerization (reference 29).
Page 9, last paragraph. “Thermal stability experiments showed….” Also include the reference of Inoue, Y et al. 2019 shown above.
Page 10, second paragraph. Regarding the relation between FlhA and FlhB, I suggest to discuss data on reference:
FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus. Tohru Minamino, Yumi Inoue, Miki Kinoshita, Keiichi Namba
Some other references that might be considered:
Chaperone release and unfolding of substrates in type III secretion. Yukihiro Akeda & Jorge E. Galán
A chain mechanism for flagellum growth. Lewis D. B. Evans*, Simon Poulter*, Eugene M. Terentjev**, Colin Hughes*, and Gillian M. Fraser*
At the end of the manuscript there is a Box 1 that I did not see referenced in the text.
Author Response
Thank you for your kind words and constructive review. Please find below our point-to-point responses to the raised issues.
Introduction, page 2, first paragraph: Figure 1 “and rod composed of the P-ring” it is confusing since the rod is made of the 5 proteins previously mentioned in the text. The same comment for figure 1 legend.
RE: The legend of figure 1 was improved. The legend was accidently duplicated in the text in the original version of the manuscript. This has been corrected (see also our response to the relevant comment of reviewer 1 above).
In this same paragraph “from early-type substrates (e.g.components of the basal body...” it is also confusing since not all components of the basal body are early substrates secreted via the T3SS.
RE: Thank you for this suggested. The sentence has been improved and now reads: “from early-type substrates (e.g. components of the rod or the hook subunits) to late-type substrates (e.g. flagellin or the filament cap)…”.
Figure 1. It will be nice to also point out FlgI and FlgH that are included in the figure legend
RE: We have modified the figure legend and decided to only show labels of T3SS proteins for simplicity reasons. The protein names of the P- and L-rings are mentioned in the manuscript text.
Section 3. first paragraph “complex AT THE AT THE base of the export...”
RE: Corrected, thank you.
Page 7 first paragraph “C-ring via FliH interactions and thereby would FUNCTION AS the”.
RE: Corrected, thank you.
In this same paragraph I suggest to discuss the following references:
- The structure of the Type III secretion system export gate with CdsO, an ATPase lever arm. Jaime L. Jensen, Shavait Yamini, Arne Rietsch, Benjamin W. Spiller. 2020
- Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry. Dorothy D. Majewski, Liam J. Worrall, Chuan Hong, Claire E. Atkinson, Marija Vuckovic, Nobuhiko Watanabe, Zhiheng Yu & Natalie C. J. Strynadka. 2019
RE: Thank you for this suggestion. The references have been added and are now discussed in the text (Chapter 3, first paragraph on pages 5-6 and third paragraph on page 7).
Section 4.1, page 8, last paragraph. In relation to the multiple conformations of FlhA, the following reference could be included : Structural Insights into the Substrate Specificity SwitchMechanism of the Type III Protein Export Apparatus. Inoue,Y et al. 2019
RE: The reference was added as suggested.
Section 4.2. The FlhA linker also has a role in the oligomerization (reference 29).
RE: Thank you for pointing this out. The relevant section now reads: “The flexible linker FlhAL is further required for FlhAC oligomerisation as alanine substitutions in the D1 domain (interacting with FlhAL) and in FlhAL inhibit the formation of the FlhAC ring. These mutations reduce the efficiency filament assembly after completion of the hook, as well as the binding affinity of FlgN, the chaperone of FlgKL”.
Page 9, last paragraph. “Thermal stability experiments showed....” Also include the reference of Inoue, Y et al.2019 shown above.
RE: The reference was added as suggested.
Page 10, second paragraph. Regarding the relation between FlhA and FlhB, I suggest to discuss data on reference: FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus. Tohru Minamino, Yumi Inoue, Miki Kinoshita, Keiichi Namba
RE: The reference was added as suggested and is now discussed on page 10/11.
Some other references that might be considered:
Chaperone release and unfolding of substrates in type III secretion. Yukihiro Akeda & Jorge E. Galán
A chain mechanism for flagellum growth. Lewis D. B.Evans*, Simon Poulter*, Eugene M. Terentjev**, Colin Hughes*, and Gillian M. Fraser*
RE: The reference was added as suggested.
At the end of the manuscript there is a Box 1 that I did not see referenced in the text
RE: Thank you for noticing that. Box 1 now referenced at the appropriate place in the Introduction.
Reviewer 3 Report
This manuscript concisely written by Halte and Erhardt reviews the Type Three Secretion System of the bacterial flagellum including the recent advance of this scientific topic. Principally, I could not find any concerns throughout the manuscript. I think this will be accepted after revising minor points described later and be worthwhile to be published in Biomolecules.
In Figure 1;
It is discernable for general readers to include the labels of the protein names for the stator unit (MotA and MotB) in the figure if you describe it in the main text (for CheY if possible).
In Figure 3;
This schematic model explains the late flagellar substrate/chaperone complexes are docked and exported through the export gate by the ATPase complex (FliH12FliI6FliJ). However, I feel there may be a discrepancy. The assembled hook is depicted only in state IV but the completion of the hook assembly should occur before export of such late flagellar proteins. The order of assembly of the bacterial flagellum must be reflected in the figure.
Minor concerns;
There found mixed expression of the number for subunit compositions as subscriptions throughout the manuscript. Please correct them properly.
The names of authors are not properly cited in “Reference #9”.
Author Response
Thank you for your kind words and constructive review. Please find below our point-to-point responses to the raised issues.
In Figure 1;It is discernable for general readers to include the labels of the protein names for the stator unit (MotA and MotB) in the figure if you describe it in the main text (for CheY if possible).
RE: Thank you for this suggestion. We have modified the legend of figure 1. However, we decided to only show labels of T3SS proteins for simplicity reasons. The protein names of stator units are mentioned in the manuscript text on page 5.
In Figure 3;This schematic model explains the late flagellar substrate/chaperone complexes are docked and exported through the export gate by the ATPase complex(FliHFliIFliJ). However, I feel there may be a discrepancy. The assembled hook is depicted only in state IV but the completion of the hook assembly should occur before export of such late flagellar proteins. The order of assembly of the bacterial flagellum must be reflected in the figure.
RE: This is of course correct. Thank you for noticing this. We have modified the figure accordingly and now show the completed hook structure.
There found mixed expression of the number for subunit compositions as subscriptions throughout the manuscript. Please correct them properly.
RE: Corrected as suggested.
The names of authors are not properly cited in “Reference#9”
RE: The authors names have been corrected, thank you.
