The EMBO Journal Peer Review Process File - EMBO-2010-74824
Manuscript EMBO-2010-74824
Identification of FtsW as a transporter of lipid-linked cell
wall precursors across the membrane
Tamimount Mohammadi, Vincent van Dam, Robert Sijbrandi, Thierry Vernet, Andre Zapun,
Ahmed Bouhss, Marlies Diepeveen-de Bruin, Martine Nguyen-Disteche, Ben de Kruijff and Eefjan
Breukink
Corresponding author: Eefjan Breukink, Utrecht University
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Editorial Decision:
Additional correspondence:
Accepted:
18 May 2010
30 June 2010
07 December 2010
04 January 2011
14 January 2011
31 January 2011
04 February 2011
09 February 2011
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
30 June 2010
Thank you for submitting your manuscript for consideration by The EMBO Journal. Let me first of
all apologise for the delay in getting back to you with a decision. Unfortunately, two of the referees
were not able to return their reports as quickly as initially expected.
Your manuscript has now finally been seen by three referees whose comments to the authors are
shown below. You will see that the referees are generally positive about your work and that they
would support its ultimate publication in The EMBO Journal after appropriate revision. I would thus
like to invite you to prepare a revised manuscript in which you need to address the referees'
criticisms in an adequate manner. In particular, it would be important to sort out the role MurJ/MviN
as put forward by all three referees.
I should remind you that it is EMBO Journal policy to allow a single round of revision only and that,
therefore, acceptance of the manuscript will depend on the completeness of your responses included
in the next, final version of the manuscript.
When preparing your letter of response to the referees' comments, please bear in mind that this will
form part of the Peer Review Process File, and will therefore be available online to the community.
For more details on our Transparent Editorial Process initiative, please visit our website:
http://www.nature.com/emboj/about/process.html
© European Molecular Biology Organization
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Thank you for the opportunity to consider your work for publication. I look forward to your
revision.
Yours sincerely,
Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
This paper describes the identification of FtsW as the membrane transporter responsible for
translocating the peptidoglycan precursor lipid II across the bacterial cytoplasmic membrane. This is
an important discovery. Although mechanistic aspects remain for the future, the identification of a
'ATP-independent' lipid transporter is major step forward. This class of transporters was proposed to
exist almost 30 years ago and - until now - no member of this class had been identified.
The data are qualitatively convincing but quantitatively less so. I have a number of specific points
that the authors should consider
Page 3: The rationale for choosing FtsW is a bit weak. In a sense it does not matter since the data
bear out the authors' choice, but it would be interesting to have a firm rationale.
Line 103: The term FRET signal should be clearly defined. Do the authors mean the sensitized
emission from TMR-labeled vancomycin or the reduction in NBD fluorescence?
Lines 105-109: The experiment with RSO vesicles should be better explained. At a minimum, the
reader should be told of the freeze-thaw steps to introduce Lipid II precursors to enable the synthesis
of fluorescent Lipid II on the inner face. The structure of Lipid II should be shown in the main
paper, with the NBD modification highlighted.
Lines 109 & 112: The time course of the 'FRET signal' change should be graphed. The raw data are
good to see, but it would be useful to have the FRET change presented. The change appears to have
a burst phase for the RSO vesicles derived from normal cells, followed by a linear increase. For the
FtsW over-expressors, the time course appears more like an exponential increase. Some discussion
of these data should be offered.
Line 126: The purified protein should be shown in some form - perhaps a Coomassie-stained SDSPAGE analysis should be included in Figure 4.
Line 129: How large are the LUVs? If they are ~150 nm in diameter, reconstitutions done at a
protein: phospholipid molar ratio of 1:20,000 will generate vesicles with ~10 proteins per vesicle.
This is information that should be presented.
Line 138-139: The ability of KcsA to flip phospholipids (as reported in Kol et al., 2003) is restricted
to NBD-PG. In these studies, the protein:phospholipid ratio was 1:2000, compared to 1:20,000 in
the present paper, and the time course of flipping was over several hours. The statement on lines
138-139 should be modified so that it is more accurate.
Lines 155-158: Figure S6 should be moved to the main paper since the protein-dependence of
flipping is an important component of the argument that FtsW is the Lipid II flippase. However,
analysis of Fig. S6 reveals a problem that the authors need to address. The data suggest that >10
FtsW molecules need to be reconstituted per vesicle in order to have 1 functional transporter per
vesicle. Are most of the purified FtsW molecules inactive? Is there another component to this
system? Do the authors have an independent way of assessing whether their FtsW preparations
contain properly folded protein?
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Line 160: The reference to Shukla 2007 should be replaced with Sanyal et al. Biochemistry 2008.
Why not explicitly test the role of MviN since the assay is available?
Figures: Please clarify A.U. on the fluorescence spectra. Perhaps normalization of the data or simply
a direct reference to 'arbitrary units' would be better
General comment: Single experiments, mainly raw data, are shown. This makes it hard to assess the
quality/reproducibility of the data.
Referee #2 (Remarks to the Author):
Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane
This paper represents a significant advance in our understanding of bacterial cell wall synthesis and
provides a new assay method for the biochemical analysis of this key activity in cell wall
biosynthesis. The paper was well written and presented in the data in clear and rigorous manner.
Although in this respect the authors have perhaps been a little too scientific in their representation of
the data and have reduced the significance that this paper has for a general reader. Here I refer
primarily to the figures that provide the key data. From my point of view it would be good to modify
Fig. 1 to incorporate a hypothetical trace for what might be expected to occur in the assays shown in
later figures and to provide a clear explanation of the trends seen. This would then allow readers to
understand the later plots more easily. While incorporating this change it would also be very helpful
to re-think the format of the later figures: these figures are very simple and appear to be straight out
of a lab book. In this respect it is nice to see "raw" data. But, the trends shown are relatively small
(by the scales used) and so the reader is likely to question the significance. This in some way could
be countered by the introduction of the "hypothetical plot" in Fig. 1, but also might be significantly
helped by processing the data in a different way - one suggestion that occurs to me would be to
display the data as changes 530/580 nm ratio over time rather than showing the spectrum at each
time point - perhaps put these in supplementary data? The key point being that the figures need to be
simple and communicate the key points in a way that can be understood at a glance rather than
careful reference to the text. It should also be considered that the figures will be much smaller that
the submitted files - will the data be visible when reduced to the size of the journals normal format.
At the scientific level several questions/points were raised in my mind that i think need to be
attended to. I have listed them below in no specific order.
On reading the introduction and discussion sections I feel that the biological significance of the data
is not well communicated. Here there is a need to provide information about RodA and its role in
cell wall biosynthesis, as for those in the field, this protein should have the same role and be equally
active. There should also be an explanation as to why this activity is not evident in some of the
assays - I presume that it, or the complex containing it is in, is not stable enough?
The specificity of the transporter is not well explored. The data clearly shows that lipid II is
transported. Can another labelled lipid be put into the system and is this transported at the same
rate?
MurJ - is this a transporter, the conflicting data that exists puts this to question - could this be solved
by this methodology. It would strengthen the paper significantly to have this data.
Finally there are a few minor typos that should be attended to:
Lines 84-87 and 122-123 - more about RodA is required here, particularly in the later section as it
should be active here - could a rodA pbp mutant be used?
Line 93 ",thereby connecting cell wall synthesis and the division machinery." Reads better, but may
not have the same meaning as the authors had in mind?
Line 400 - blank line?
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Referee #3 (Remarks to the Author):
Manuscript EMBOJ-2010-74824 is the product of four European laboratories of matching expertise,
each well established in their studies of molecular aspects of bacterial peptidoglycan synthesis. The
authors report experimental evidence which they interpret as the proof that FtsW has the specific
biochemical function to catalyze the trans-membrane movement of lipid II: the bactoprenyl linked
precursor of peptidoglycan biosynthesis also know as lipid II.
The localization of FtsW on cwd islands in the proximity of the murG gene which encodes for the
protein that completes the biosynthesis of lipid II and the essentiality of FtsW make it indeed
conceivable that this protein is the much looked for catalyst of lipid II transport, i.e., the much
looked for "flippase".
The authors developed an elegant fluorescence resonance energy transfer (FRET) based assay to
produce direct experimental evidence for this complex transport process which they succeeded to
reproduce in bacterial membrane vesicles in vitro.
As an additional line of evidence, the authors were able to show that purifying E. coli FtsW can
catalyze redistribution of lipid II in model membranes using unilamellar vesicles. As controls, the
experiment is repeated with several conceivable alternative candidates and/or membrane proteins
such as Mray and SecYEG with negative results.
Complex control experiments are also done to exclude the possibility that the observed effects
simply represent leakiness of the model membranes caused by the FtsW protein rather than an actual
translocation.
I am convinced by the experimental data together with the well established competence of the
collaborating laboratories that their claim is justified: this is a major finding indeed which provides a
specific role for the mysterious FtsW protein and also provides a much looked for catalyst for the
flippase reaction.
The only thing which remains "up in the air" is the counter-claim by the groups of Inoue and the
group of Ruiz. Both of these groups reported as recently as 2008 that the protein catalyzing the
flippase reaction is not FtsW but rather the gene product of murJ.
The authors discuss the pro and contra arguments, eventually suggesting that perhaps the murJ
protein is also involved in peptidoglycan synthesis in a more indirect way rather than as an
alternative flippase.
This is an impressive piece of work which may have solved one of the major outstanding questions
of the peptidoglycan synthesis field.
On the other hand the manuscript, particularly the Abstract and the Introduction are written in a
sloppy way which would require corrections. For instance, right at the beginning the authors should
reveal that the work is done in E. coli. In the introduction, lines 68 and 69, what the authors
probably want to say is that "the cytoplasmic phase of peptidoglycan precursor synthesis culminates
in the production of the UDP-MurNAcpentapeptide and (rather than from) UDP-GlcNAc.
Technical abbreviations like "NBD" (see line 78) or TMR (see line 103) should be defined when
they first show up in the text. "FRET" should also be spelled out. In line 140 Mray is not "involved
in the first step of peptidoglycan biosynthesis" but rather in the first "membrane linked step". In the
polemics in line 200, I suggest to change the sentence to say "in spite of the reasoning of Ruiz et
al......". In line 209, I would suggest to add - for fairness sake the word "also" (i.e, "this protein
might also be involved".....).
Throughout the paper the expression "cell walled bacteria" sounds awkward, I suggest to change it
to eubacteria producing cell wall peptidoglycan.
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With these caveats, I suggest publication.
1st Revision - authors' response
07 December 2010
We thank the referees for their careful review of our manuscript, their positive comments and useful
suggestions, which have strengthened the manuscript significantly. We have addressed their valid
concerns with textual and additional experimentation. In particular, we have performed experiments
with MviN (the recently proposed Lipid II flippase) to sort out the role of this protein as raised by all
three referees and requested by you. The results are included in the revised manuscript. Using the
assays described in our paper MviN did not show any flippase activity indicating that this protein is
not directly involved in the transport of Lipid II across the bacterial membrane. Thus the results are
supportive of the findings reported in the original submission. In revising our manuscript to address
the Reviewers comments concerning the representation of the data we also have included additional
figures showing the time course of the FRET signal.
A point-by-point response is described below. Changes in the text are highlighted. We trust that our
revisions address all the issues raised by the Reviewers fully and adequately, rendering the
manuscript now suitable for publication.
Referee #1
The referee commented that our manuscript ‘is an important discovery and that although
mechanistic aspects remain for the future, the identification of an ATP-independent lipid transporter
is a major step forward. This class of transporters was proposed to exist almost 30 years ago and
until-until now- no member of this class had been identified’.
We thank the Reviewer for this positive comment.
The reviewer added that ‘the data are qualitatively convincing but quantitatively less so’ and had a
number of specific points, which we address below (and with revisions):
1.
Page 3: The rationale for choosing FtsW is a bit weak. In a sense it does not matter since
the data bear out the authors' choice, but it would be interesting to have a firm rationale.
Response: In addition to the reasons stated on page 3 and 4 (lines 83-99); FtsW has been proposed
to be the missing Lipid II transporter in different studies. However no (experimental) evidence has
been provided yet. To confirm flippase activity of this protein, or to rule it out, we adopted a
photocrosslink approach using photoactivatable Lipid II (a description of this method can be found
in Van Dam et al, ChemBioChem 2009). Photo-cross-linking analyses of E. coli RSO vesicles using
this approach revealed the presence of various cross-linked proteins. When this approach was
applied to membrane vesicles isolated from a strain overexpressing FtsW a dramatic change in the
pattern of crosslinked proteins was obtained pointing to a role of this protein in the translocation
process. This result prompted us to further assess this hypothesis using the assays described in the
present paper.
2.
Line 103: The term FRET signal should be clearly defined. Do the authors mean the
sensitized emission from TMR-labelled vancomycin or the reduction in NBD fluorescence?
Response: We thank the reviewer for pointing this out. A FRET signal is a fluorescence signal
yielded by the energy transfer between NBD and TMR fluorophores when they are in close
proximity of each other. In the assays the FRET signal is reflected by an increase in the emission of
the TMR fluorescence accompanied by a decrease in the emission of the NBD fluorescence. The
definition of the FRET signal has now been added in the main text on page 4 lines 109-110 and was
given in the legend of Figure 2. See also the additional explanation on page 4 lines 115-120.
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3.
Lines 105-109: The experiment with RSO vesicles should be better explained. At a
minimum, the reader should be told of the freeze-thaw steps to introduce Lipid II
precursors to enable the synthesis of fluorescent Lipid II on the inner face. The structure of
Lipid II should be shown in the main paper, with the NBD modification highlighted.
Response: We appreciate that the Reviewer points this out. Following this suggestion we have
included more details about the experiment with RSO in the main paper (page 4 lines 115-122). We
have also included an additional figure panel in Fig. 1 (Fig 2B in the revised manuscript) illustrating
what might be expected to occur when RSO vesicles are used in the FRET assays (as also suggested
by Referee #2).
Supplementary Fig. 1, with the structure of Lipid II and NBD-Lipid II, has been incorporated into
the main paper (Fig. 1 in the revised manuscript).
4.
Lines 109 & 112: The time course of the 'FRET signal' change should be graphed. The raw
data are good to see, but it would be useful to have the FRET change presented. The
change appears to have a burst phase for the RSO vesicles derived from normal cells,
followed by a linear increase. For the FtsW over-expressors, the time course appears more
like an exponential increase. Some discussion of these data should be offered.
Response: We agree with the Reviewer that it is useful to present the FRET change over time. The
time course of the FRET signal has now been displayed as a 578/534 ration where 578 nm is the
emission maximum wavelength of vancomycin-TMR and 534 nm the emission maximum
wavelength of NBD-Lipid II under each figure (see figure 3C, 4C, S3C and S4C).The time course of
both the normal cells and the cells overexpressing FtsW follows a linear increase that achieved a
plateau after ~10 min. However upon overexpression of FtsW the increase is much more
pronounced.
5.
Line 126: The purified protein should be shown in some form - perhaps a Coomassiestained SDS-PAGE analysis should be included in Figure 4.
Response: following the Reviewer’s suggestion we have included a Coomassie-stained SDS-PAGE
analysis of the purified FtsW in Figure 5A (see also main text on page 5 line 145).
6.
Line 129: How large are the LUVs? If they are ~150 nm in diameter, reconstitutions done
at a protein: phospholipids molar ratio of 1:20,000 will generate vesicles with ~10 proteins
per vesicle. This is information that should be presented.
Response: the Reviewer is correct. The LUVs prepared following the reconstitution method
described in the paper are expected to generate unilamellar vesicles with a diameter of ~150 nm
(Lèvy et al, 1990; Sanyal et al, 2008; Sahu et al, 2008)(and also confirmed by size distribution
analysis). The information has now been included in the manuscript in page 5 line 148 (see also
below our response to point 8).
7.
Line 138-139: The ability of KcsA to flip phospholipids (as reported in Kol et al., 2003) is
restricted to NBD-PG. In these studies, the protein: phospholipid ratio was 1:2000,
compared to 1:20,000 in the present paper, and the time course of flipping was over
several hours. The statement on lines 138-139 should be modified so that it is more
accurate.
Response: To exclude any confusion and to draw the attention of the readers to the conditions used
in the assay reported in Kol et al, 2003, we have modified the statement (page 6 lines 157-159).
8.
Lines 155-158: Figure S6 should be moved to the main paper since the protein-dependence
of flipping is an important component of the argument that FtsW is the Lipid II flippase.
However, analysis of Fig. S6 reveals a problem that the authors need to address. The data
suggest that >10 FtsW molecules need to be reconstituted per vesicle in order to have 1
functional transporter per vesicle. Are most of the purified FtsW molecules inactive? Is
there another component to this system? Do the authors have an independent way of
assessing whether their FtsW preparations contain properly folded protein?
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Response: i) Figure S6 has been moved to the main paper (Figure 6) as suggested by the Reviewer.
See also page 6 lines 179-181.
ii) We will first address the issue raised by the Reviewer about the number of FtsW molecules that
need to be reconstituted per vesicle in order to have 1 functional transporter per vesicle.
We do not agree with the statement of the Reviewer that more than 10 FtsW molecules need to be
reconstituted per vesicles in order to have 1 functional transporter per vesicle. First of all the data in
Figure 6 (formerly S6) show that when reconstituted at a protein:phospholipid ratio of 1:20,000 our
system will generate about 10 proteins per vesicle. The analysis demonstrates that vesicles
reconstituted at a protein:phospholipid molar ratio of 1:40,000 (~5 FtsW molecules per vesicle) still
display flippase activity. Accordingly, about 5 FtsW (and not more than 10) molecules need to be
reconstituted per vesicle in order to have one functional transporter per vesicles. Moreover, it is
expected that 50% of the transporter proteins are reconstituted in the appropriate orientation in the
vesicles. Hence, most of the purified FtsW molecules are active (see also page 6 and 7 lines 175186).
iii) the Referee wonders whether most of the purified FtsW molecules are inactive.
It is difficult to assess the number of active FtsW molecules in the purified fraction. Considering the
arguments mentioned above, we estimate the number of inactive molecules to be low.
iv) The Referee asks whether there is another component to this system.
This is an interesting point that will be addressed in further studies. It is indeed possible that other
components are involved in this system, i.e. the regulation at the molecular and cellular level where
for example interactions/complex forming with MurG and PBPs are expected to play a role.
However, it would be too speculative to discuss this here.
v) Unfortunately, an independent assay to assess whether our FtsW preparations contain properly
folded protein is not (yet) available.
9.
Line 160: The reference to Shukla 2007 should be replaced with Sanyal et al. Biochemistry
2008.
Response: We have corrected the reference (line 186).
10. Why not explicitly test the role of MviN since the assay is available?
Response: We have assessed the effect of MviN in the transport of Lipid II using both the FRETbased assay and the reconstituted system. As shown in Fig. S7 the overproduction of MviN did not
result in an increased transport of Lipid II across the inner membrane of the RSO vesicles. When
purified MviN was tested using the biochemical assay (Fig. S8) no difference was observed
regarding the level of reduction of fluorescence by dithionite indicating that MviN does not facilitate
the transbilayer movement of Lipid II in model membranes.
11. Figures: Please clarify A.U. on the fluorescence spectra. Perhaps normalization of the data
or simply a direct reference to 'arbitrary units' would be better
Response: A direct reference to arbitrary units has been added in the figure legends as suggested by
the Reviewer.
12. General comment: Single experiments, mainly raw data, are shown. This makes it hard to
assess the quality/reproducibility of the data.
Response: As described above we have included additional figures to improve the clarity of the data.
All results shown in this paper are representative of at least two independent experiments, which is
now also stated in the manuscript where appropriate.
Referee #2
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This Reviewer commented that our paper ’represents a significant advance in our understanding of
bacterial cell wall synthesis and provides a new assay method for the biochemical analysis of this
key activity in cell wall biosynthesis. The paper was well written and presented in the data in clear
and rigorous manner’.
We are grateful to the Reviewer for his/her very positive view about the importance of our work.
1.
A major concern of the Reviewer is the representation of the data. The Reviewer proposed
to modify Fig. 1 and to incorporate a hypothetical trace in Fig. 1 for what might be
expected to occur in the assays shown in later figures and to provide a clear explanation of
the trends seen. And also to re-think the format of the figures.
Response: We thank the Reviewer for these constructive comments. Following his/her suggestions
we have incorporated a hypothetical trace in Fig. 2 (in the revised manuscript) illustrating what
might be expected to occur during the FRET assays using RSO vesicles (see also page 4 lines 115120). We have also modified the size of the figures.
2.
Another helpful suggestion of the Reviewer was to improve understanding is as follows: to
display the data as changes 530/580 nm ration over time rather than showing the spectrum
at each time point, perhaps put these in supplementary data?
Response: As mentioned in our response to Reviewer 1 (point 4) we have included additional
figures where the FRET change is presented as change in the 578/530 ratio over time.
3.
At the scientific level several questions/points were raised in my mind that I think need to
be attended to. I have listed them below in no specific order. On reading the introduction
and discussion sections I feel that the biological significance of the data is not well
communicated. Here there is a need to provide information about RodA and its role in cell
wall biosynthesis, as for those in the field, this protein should have the same role and be
equally active. There should also be an explanation as to why this activity is not evident in
some of the assays - I presume that it, or the complex containing it is in, is not stable
enough?
Response: We have now provided information about RodA and SpoVE in the Introduction (page 34, lines 85-99) and the Results section (page 5 lines 140-141).
We agree that RodA (as a homolog of FtsW) should have the same role as FtsW in cell wall
biosynthesis. We have performed experiments with RodA purified from Streptococcus pneumonia
using the reconstituted system and found that indeed this protein is able to facilitate the transbilayer
movement of Lipid II in model membranes (data not shown). However, we do not agree with the
Reviewer that the role of RodA was not evident in our assays. As shown in Fig. 4 depletion of FtsW
resulted in reduced (but not complete absence) Lipid II transport. The remaining transport is mostly
likely due to the presence of RodA. This has now been stated in the manuscript (page 5 lines 140141).
4.
The specificity of the transporter is not well explored. The data clearly shows that lipid II is
transported. Can another labelled lipid be put into the system and is this transported at the
same
rate?
MurJ - is this a transporter, the conflicting data that exists puts this to question - could this
be solved by this methodology. It would strengthen the paper significantly to have this data.
Response: While we agree with the Reviewer that the substrate specificity of the transporter is an
interesting idea that should be explored (and would certainly be of interest in elucidating the
mechanism of action of the transporter), we think that this is beyond the scope of the current
manuscript and are of the opinion that this is more suitable for a future study.
We have not (yet) studied another labelled lipid in the reconstituted system.
MviN/MurJ: As there is no experimental proof of a role of MviN in Lipid II transport, we are not
aware of any existing conflicting data. Yet, we have tested the effect of MviN using the assays
described in the paper and demonstrated that this protein has no direct role in the transport of Lipid
II (see also our response to Referee #1 point 10).
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The Reviewer had a few minor typos, which we address below:
5.
Lines 84-87 and 122-123 - more about RodA is required here, particularly in the later
section as it should be active here - could a rodA pbp mutant be used?
Response: We have included information about RodA (see Introduction, page 3-4 lines 85-99) and
from Fig. 4 (page 5 lines 138-141) it is clear that RodA is active.
A rodA pbp mutant is not available.
6.
Line 93 "thereby connecting cell wall synthesis and the division machinery." Reads better,
but may not have the same meaning as the authors had in mind?
Response: Machineries in this sentence include the cell wall synthesis machinery and the division
machinery. Yet, to exclude any confusion the sentence has now been modified as suggested by the
Reviewer (page 4 line 96).
7.
Line 400 - blank line?
Response: We have deleted the blank line.
Referee #3
The Reviewer commented that ‘this is an impressive piece of work which may have solved one of the
major outstanding questions of the peptidoglycan synthesis field’. We thank the reviewer for the
very positive comments.
1.
The reviewer also commented that ‘On the other hand the manuscript, particularly the
Abstract and the Introduction are written in a sloppy way which would require
corrections. For instance, right at the beginning the authors should reveal that the work is
done in E. coli. In the introduction, lines 68 and 69, what the authors probably want to say
is that "the cytoplasmic phase of peptidoglycan precursor synthesis culminates in the
production of the UDP-MurNAcpentapeptide and (rather than from) UDP-GlcNAc’.
Response: We apologize for the inconsistencies in the manuscript and we appreciate the Reviewer
pointing this out. We have revised the text in the Abstract and the Introduction and included the
changes (the changes in the text are highlighted) as suggested by the Reviewer.
2.
Technical abbreviations like "NBD" (see line 78) or TMR (see line 103) should be defined
when they first show up in the text. "FRET" should also be spelled out. In line 140 MraY
is not "involved in the first step of peptidoglycan biosynthesis" but rather in the first
"membrane linked step". In the polemics in line 200, I suggest to change the sentence to
say "in spite of the reasoning of Ruiz et al......". In line 209, I would suggest to add - for
fairness sake the word "also" (i.e, "this protein might also be involved".....).
Response: We have defined the abbreviations in the main text (page 3 line 79, page 4 lines 104-105
and 108). We have modified the sentence in line 140 (line 159-161 in the revised manuscript). We
have modified the sentence in line 200 (line 227-229in the revised manuscript). We have added
‘Also’ (line 236) to the sentence as suggested by the Reviewer.
3.
Throughout the paper the expression "cell walled bacteria" sounds awkward, I suggest to
change it to eubacteria producing cell wall peptidoglycan.
Response: We have modified the expression in the revised manuscript (line 83 and line 88).
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2nd Editorial Decision
04 January 2011
Thank you for sending us your revised manuscript. Our original referees have now seen it again. In
general, the referees are now positive about publication of your paper. However, referee 1 feels that
there are a few issues that still need to be addressed (see below) before we can ultimately accept
your manuscript. Also referee 2 has minor suggestions. I would therefore like to ask you to deal with
the issues raised in an amended manuscript. I would like to clarify that the "hypothetical blot" (see
referee 1 point 4) needs to be more clearly identifiable as hypothetical. I think that the best way to
deal with this point would be to keep the blot, but move it to the supplementary material with a
clearer explanation. Regarding the figures (see referee 2) I need to ask you to include all panels of
every figure into one page/one file per figure. Also, it would be good to check the labels for the
different traces again: different grey scales are not the best way to discriminate between the different
conditions in our view.
Please let us have a suitably amended manuscript as soon as possible.
Yours sincerely,
Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
I have a number of major points concerning quantitative aspects.
1. Line 70; the sentence does not make sense - perhaps the phrase 'and UDP-N-acetylglucosamine
(UDP-GlcNAc)' should be removed
2. Line 73; should be Fig 1A
3. Line 80; cite Fig. 1B
4. Line 120 and Fig. 2B; the 'hypothetical' plot is quite dangerous to show. I thought that these were
the actual data and wondered about the significance/relevance of Fig. 3A before realizing that Fig.
2B was 'hypothetical'. A reviewer asked for this in the original round of review, but I am
uncomfortable with such a graph. Perhaps the editors can intervene here.
5. Line 123; why 14ºC? Also, the temperature at which the dithionite reduction assays should be
stated in the methods and/or figure legends.
6. Line 128 and Fig. 3C; If the RSO vesicles contain more FtsW-flippase (overexpression of FtsW
vs normal expression) then the rate of flipping should increase, not the final amplitude of the
578/534 ratio. It would seem from the raw data that the same amounts of NBD-Lipid II are
synthesized in the FtsW overexpressors as in the normal RSOs, and presumably the same amount of
fluorescent vancomycin was added, so why are the amplitudes of the two lines in Fig. 3C different?
What is the maximum 578/534 ratio that can be obtained in this system in a particular experiment?
Is NBD-Lipid II synthesis ongoing during the experiment?
7. Fig. 3C, also line 252; The rate of FRET change seems very slow; it is not possible from the data
to obtain a half-time, but this would certainly exceed 10 minutes. This contrasts with the data
obtained from the dithionite assay where the read-out has a half-time of less than 10 seconds. This
issue need to be fully analyzed and discussed.
8. Fig. 4C: the authors suggest that the FRET increase in FtsW-depleted cells is due to residual
RodA. As in an earlier comment, this should enable the same final amplitude to be reached as in
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FtsW-replete cells - only the kinetics would be decreased. From the figure it is hard to judge
whether this would be the case. If the same amplitude were to be reached, it would take hours.
9. Fig. 6; There are still issues with the experiment showing that more translocation can be detected
when more FtsW is reconstituted. The authors present a series of traces showing that as FtsW
content per vesicle increases from 0 to 5, 10, 20 and 40 FtsW molecules per vesicle on average, the
amount of NBD-Lipid II that is reduced increases exponentially. Thus there is no doubt that there is
an FtsW-dependent increase in the amount of NBD-Lipid II that is reduced. However, there is a
calibration problem. Because the assay measures the fraction of vesicles that contains a flippase, and
flippases are distributed statistically among the vesicles, then it can be expected that with an average
of 5 flippases per vesicle (1:40,000 sample), most vesicles would have at least 1 flippase and the
fluorescence reduction should be maximal.
A possible explanation is that the majority (90%?) of the reconstituted FtsW molecules is inactive,
so that at the highest amount that the authors reconstitute, they really only have 4 active FtsW
flippases per vesicle on average. Perhaps co-expression with PBP3 (Fraipont et al. 2010) would
improve the FtsW preparation?
Alternatively, as the authors suggest, the FtsW may be aggregated and thus not incorporated into the
vesicles, i.e., the real average number of FtsW flipapses per vesicle is much lower than expected
based on the amount used for reconstitution. In this case, the authors should isolate the vesicles by a
simple flotation protocol and determine the amount of vesicle associated FtsW. This is important to
establish and will strengthen the paper.
10. Line 189; the authors should allow for a third possibility that the reconstitution of FtsW results
in the asymmetric reconstitution of NBD-Lipid II. The control experiments that they provide would
not address this issue.
11. Line 228; what is the FtsW-equivalent protein in V. okutanii?
12. Specificity of transport. One of the reviewers indicated that specificity of the transporter was not
well explored. While I agree that a detailed study is beyond the scope of the paper, it is surely
informative to test whether FtsW flips NBD-modified phospholipids (commercially available). The
authors provide the converse control by showing that KcsA does not flip NBD-Lipid II but (in
published work) transports NBD-PG on a time scale of hours. It would be relatively simple to
provide this comparison for FtsW in the present paper.
13. Graphs such as those shown in Fig. 3C and 4C should really contain error bars.
Referee #2 (Remarks to the Author):
Identification of FtsW as a transporter of lipid linked cell wall precursors across the membrane.
Mohammadi et al.
The revised version of the paper is a significant improvement upon the original in terms of the text
and as such I no-longer have any problems here (although there are a few typos that should be
addressed - the author list has lower case for one persons name and this also occurs in the main text.
A careful proof read is needed here).
However, the figures are still in need of modification. Although they do present the data they are
very basic and will not reproduce well in the journal format (unless A4 size figures are permitted!).
This point I leave to the editor as the risk here is that the final versions will not present the data due
to the loss of resolution upon reduction in size etc.
Referee #3 (Remarks to the Author):
I read the authors response to Editorial criticism. I find the revised manuscript an excellent addition
to the literature and it should be accepted for publication without further changes.
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2nd Revision - authors' response
14 January 2011
We thank the referees for their careful evaluation of the revised manuscript. We are glad to learn
that they are now positive about publication of our paper.
We appreciate Referees 2 minor suggestions concerning the typos and the size of the figures. We
have included these in the revised paper according to your recommendations. We are also grateful
for your useful suggestions about how to improve the quality of the figures.
Regarding the few issues raised by Referee 1, we have included a point-by-point response (see
below).
Referee #1
1.
Line 70; the sentence does not make sense-perhaps the phrase ‘and UDP-Nacetylglucosamine (UDP-GlcNAC)’should be removed
The sentence has been changed to ‘the production of UDP-N-acetylmuramyl-pentapeptide (UDPMurNAc-pentapeptide) from UDP-N-acetyl-glucosamine (UDP-GlcNAc).’
2.
Line 73; should be Fig 1A
We have corrected the citation.
3.
Line 80; cite Fig. 1B
We have cited Figure 1B.
4.
Line 120 and Fig. 2B; the 'hypothetical' plot is quite dangerous to show. I thought that
these were the actual data and wondered about the significance/relevance of Fig. 3A before
realizing that Fig. 2B was 'hypothetical'. A reviewer asked for this in the original round of
review, but I am uncomfortable with such a graph. Perhaps the editors can intervene here.
We have moved Figure 2B to the Supplementary Material as suggested by the Editor. To circumvent
any confusion we have labelled the figure with the term ‘hypothetical plot’ and included a more
detailed explanation in the figure legend (see Supplementary Figure S2).
5.
Line 123; why 14ºC? Also, the temperature at which the dithionite reduction assays should
be stated in the methods and/or figure legends.
We would like to refer the Referee to the results published by van Dam et al, (2007). Here the
authors described that at elevated temperatures the fluorescence signal of NBD-labelled Lipid II
decreases in time, which was corroborated by a decrease in the amount of Lipid II that could be
isolated from the vesicles. This was attributed to transglycosylase activity, most likely of PBPs. This
phenomenon causes the total fluorescence signal of our FRET measurements to decrease in time if
these are performed at temperatures around 25oC. We have now included information on this effect
in the main text (line 125) as a footnote on page 5 (assuming that a footnote is allowed). If a
footnote is not allowed, we can move the specified text to the legend of Figure 3.
The temperature at which the dithionite reduction assays were performed has now been stated in the
Methods section (line 375) and the corresponding figure legends as suggested by the Reviewer.
6.
Line 128 and Fig. 3C; If the RSO vesicles contain more FtsW-flippase (overexpression of
FtsW vs normal expression) then the rate of flipping should increase, not the final
amplitude of the 578/534 ratio. It would seem from the raw data that the same amounts of
NBD-Lipid II are synthesized in the FtsW overexpressors as in the normal RSOs, and
presumably the same amount of fluorescent vancomycin was added, so why are the
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amplitudes of the two lines in Fig. 3C different? What is the maximum 578/534 ratio that
can be obtained in this system in a particular experiment? Is NBD-Lipid II synthesis
ongoing during the experiment?
Although this is an important point raised by the Referee, there seems to be a misunderstanding
from his/her point of view. In order to clarify this point, we would like to draw the attention of the
Referee to Figure 2 and Figure 3A and B of the manuscript. The maximum 578/534 ratio can be
deduced from Figure 2 and is equal to 3.879 since under these conditions all NBD-labelled Lipid II
molecules are bound by TMR-labelled vancomycin. This number is far from the highest ratio of
~2.0 obtained for vesicles containing overexpressed FtsW (Figure 3C). Thus, from these spectra (see
gradual increase in the FRET signal) it is evident that not all synthesized NBD-Lipid II is
transported. The transport seems somehow to be constrained by an as yet unclear factor(s). This
latter may be a component(s) present in the biological membranes that coordinates/regulates the
transport process at the molecular and cellular level. We suggest that the maximal FRET-signal
obtained in these experiments reflects the actual amount of (active) transporters. Hence the Lipid II
transported to the outer leaflet of the bacterial membrane and bound by vancomycin remains trapped
in the transporter (FtsW) blocking further transport of Lipid II molecules present in the inner leaflet.
Taking this into account, the use of membranes containing more FtsW-flippase in our FRET assay is
not expected to result in an increase in the rate of flipping, but rather expected to result in an
increased ratio as is seen in Figure 3. We have now clarified this point in the Discussion section
(lines 257-269).
i) The Reviewer asks why the amplitude of the two lines in Figure 3C is different. We agree with the
Reviewer that RSOs containing more FtsW-flippase would result in an increase in the rate of
flipping (and not the final amplitude of the 578/534 ratio) when all NBD-Lipid II would be
transported. However this is not the case (as mentioned earlier). Therefore the difference in the two
lines can be attributed to the aspect explained above.
ii) The Reviewer asks for the maximum 578/534 ratio that can be obtained in this system in a
particular experiment.
As mentioned above, the maximum 578/534 ratio can be deduced from Figure 2 and is equal to
3.897.
iii) The Reviewer wonders whether NBD-Lipid II synthesis is ongoing during the experiment.
This is difficult to determine, but given the results described in van Dam et al. (2007), where a
decrease in NBD-labelled Lipid II was shown during the transport assay, we consider it likely that
no Lipid II synthesis occurs (which would cause the levels of NBD-labelled Lipid II to remain
constant). Moreover, the blocking of the transport process could cause the available pool of
bactoprenol-phosphate at the inner leaflet of the RSOs to become depleted.
7.
Fig. 3C, also line 252; The rate of FRET change seems very slow; it is not possible from
the data to obtain a half-time, but this would certainly exceed 10 minutes. This contrasts
with the data obtained from the dithionite assay where the read-out has a half-time of less
than 10 seconds. This issue needs to be fully analyzed and discussed.
This difference can be explained by the regulation of the transport process in biological membranes
(see our response to point 6) that is absent in the biochemical assay.
8.
Fig. 4C: the authors suggest that the FRET increase in FtsW-depleted cells is due to
residual RodA. As in an earlier comment, this should enable the same final amplitude to be
reached as in FtsW-replete cells - only the kinetics would be decreased. From the figure it
is hard to judge whether this would be the case. If the same amplitude were to be reached,
it would take hours.
See our response to point 6. This is a further example that in our FRET assay the amplitude that is
reached is determined by the amount of (active) transporters.
9.
Fig. 6; There are still issues with the experiment showing that more translocation can be
detected when more FtsW is reconstituted. The authors present a series of traces showing
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that as FtsW content per vesicle increases from 0 to 5, 10, 20 and 40 FtsW molecules per
vesicle on average, the amount of NBD-Lipid II that is reduced increases exponentially.
Thus there is no doubt that there is an FtsW-dependent increase in the amount of NBDLipid II that is reduced. However, there is a calibration problem. Because the assay
measures the fraction of vesicles that contains a flippase, and flippases are distributed
statistically among the vesicles, then it can be expected that with an average of 5 flippases
per vesicle (1:40,000 sample), most vesicles would have at least 1 flippase and the
fluorescence reduction should be maximal. A possible explanation is that the majority
(90%?) of the reconstituted FtsW molecules is inactive, so that at the highest amount that
the authors reconstitute, they really only have 4 active FtsW flippases per vesicle on
average. Perhaps co-expression with PBP3 (Fraipont et al. 2010) would improve the FtsW
preparation?
Alternatively, as the authors suggest, the FtsW may be aggregated and thus not
incorporated into the vesicles, i.e., the real average number of FtsW flipapses per vesicle is
much lower than expected based on the amount used for reconstitution. In this case, the
authors should isolate the vesicles by a simple flotation protocol and determine the amount
of vesicle associated FtsW. This is important to establish and will strengthen the paper.
We like to stress here that the Reviewer acknowledges the most important point of Figure 6 that the
effect observed is directly related to the presence of FtsW in the vesicles. The cause of the
“calibration problem” pinpointed by the Referee could be due to the presence of inactive FtsW
molecules or by an aggregation effect during the reconstitution process (as suggested by us) or even
a combination of these phenomena. We cannot exclude that inactive FtsW molecules are present in
our preparation to some extent, as mentioned in our first rebuttal, but we do not agree that this
would represent the majority of the molecules. As also mentioned in the manuscript, this
“calibration problem” is reported in other articles dealing with reconstitution of transporters of lipidlinked substrates, including phospholipids. Up to now all studies reported on the translocation
process by other flippases using similar reduction assays demonstrated that a maximum quenching
of 100% was never achieved. This was obtained when translocation studies were performed in
membranes of eukaryotic cells (Chang et al, 2004; Vehring et al, 2007 and Sanyal et al, 2008),
membranes of plant cells (Sahu et al, 2008) and membranes of bacterial cells (Hrafnsdo’ttir et al,
2000). Our results are consistent with the findings of these studies. An efficiency of quenching of
less than 100% is likely to be related to the inherent properties of these types of assays, as stressed
in the abovementioned publications. Elaborating the details of this particular aspect of transport
assays would certainly be of interest and it is likely that our system is well suited to fully elucidate
this, but this is far beyond the aim of the current study.
10. Line 189; the authors should allow for a third possibility that the reconstitution of FtsW
results in the asymmetric reconstitution of NBD-Lipid II. The control experiments that they
provide would not address this issue.
Since the amount of NBD-Lipid II in our reconstituted system is always in excess compared to the
amount of FtsW (the reconstitution is performed with an FtsW:NBD-LipidII ratio of 1:20) we think
that the third possibility suggested by the Reviewer is unlikely. This is also supported by the fact
that FtsW does not contain any large periplasmic loops that would cause the protein to be biased for
a particular direction during the reconstitution process, which could possibly generate such an
asymmetric reconstitution.
11. Line 228; what is the FtsW-equivalent protein in V. okutanii?
Although Vesicomyosocious okutanni lacks FtsW, it has RodA (the close homologue of FtsW).
Thus, it is possible that in this bacterium RodA substitutes for the role of FtsW.
In addition, this bacterium forms symbioses with chemosynthetic gamma-proteobacteria. By
performing a comparative genomics analysis it was recently reported that the genome of this
symbiont lacks the region encoding proteins involved in the biosynthesis of peptidoglycan (Newton
et al, 2008). This may suggest that the synthesis of peptidoglycan is not essential in V. okutanii and
consequently it may not need an FtsW-equivalent.
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12. Specificity of transport. One of the reviewers indicated that specificity of the transporter
was not well explored. While I agree that a detailed study is beyond the scope of the paper,
it is surely informative to test whether FtsW flips NBD-modified phospholipids
(commercially available). The authors provide the converse control by showing that KcsA
does not flip NBD-Lipid II but (in published work) transports NBD-PG on a time scale of
hours. It would be relatively simple to provide this comparison for FtsW in the present
paper.
As stated in our response to Referee 2, who brought this up originally in the first round of review,
the specificity of transport is beyond the scope of the current study. This Referee apparently agreed
with us. Thus, we would like to explore this interesting aspect in a future study and trust that
Referee 1 (who already stated that he/she agrees that a detailed study is beyond the scope of the
paper) also agrees.
13. Graphs such as those shown in Fig. 3C and 4C should really contain error bars.
We have included the error bars in these figures.
Referee #2
The Referee commented that the revised version of the manuscript is a significant improvement
upon the original in terms of the text and as such no longer has any problems. The Referee
mentioned a few typos in the author list and in the main text.
We have carefully read the text and addressed the typos.
The Reviewer also commented that the figures are still in need of modification. We have now
amended the (size of) figures according to the Editor’s suggestions.
Referee #3
The Referee found our revised manuscript ‘an excellent addition to the literature and that it should
be accepted for publication without further changes’.
We thank the Reviewer for this very positive comment.
3rd Editorial Decision
31 January 2011
Thank you for sending us your re-revised manuscript. Our original referee 1 has now seen it again
and overall he/she is now supportive of publication of your manuscript. Still, he/she puts forward
two points that in his/her view have still not been addressed adequately (see below). The first issue
(point 9) appears to result from a mutual misunderstanding and I would like to ask you to address or
respond to this point. I accept your reply to second issue (point 12) in your point-by-point response
and would like to confirm that no further action is needed.
Please let us have a suitably amended manuscript as soon as possible. I will then formally accept the
manuscript.
Yours sincerely,
Editor
The EMBO Journal
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-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
The manuscript is further improved by clarifications to the text. A couple of issues remain that are
best handled by the journal.
Point 9. The authors have misunderstood my comment about the precise interpretation of the FtsWdependence of flipping - I am less concerned with the amplitude of the quenching (i.e., the problem
of why a 100% reduction is not achieved) than with the counting problem of how many FtsW
molecules there are per vesicle. Perhaps the editors will arbitrate this discussion.
Point 12. I am surprised that the authors resist the simple specificity experiment to test whether a
commercially available NBD-phospholipid is flipped by FtsW. Here again I leave the matter with
the editors.
Additional correspondence
04 February 2011
Thank you for the positive decision. We are glad to learn that Referee 1 is now supportive of
publication of our manuscript.
Our response to the issue raised by Referee 1 in point 9 about how many FtsW molecules are there
per vesicle follows below.
We would like to thank again Referee 1 for his/her comments and appreciate his/her interest and
efforts to clarify the effects seen in Figure 6. As mentioned in our previous rebuttal the Reviewer is
convinced that there is an FtsW dose-dependent effect on the transport of Lipid II. This is the most
important point that this figure displays. Understanding the details of this effect (in terms of issues
like efficiency of quenching, amount of active FtsW molecules per vesicle, orientation of the
transporter, the nature of transport i.e. unidirectional or bidirectional…etc) remains, however,
difficult. In an attempt to interpret the FtsW-dependence of transport, the Reviewer suggested
another aspect; ‘a calibration problem’ (which would mainly be due to the inactivity of our FtsW
preparation) that should be taken into account. He/she reasoned that at a reconstitution ratio of
1:40,000 most vesicles would have at least 1 flippase and the reduction should be maximal. It should
be noted here that the reviewer assumes that FtsW can work bidirectional, which is at this moment
unknown and thus speculative. Therefore, our reasoning to estimate the active fraction of the FtsW
preparations is as follows: reconstitution performed at a protein:phospholipid molar ratio of
1:40,000 indeed will generate about 5 FtsW molecules per vesicle. This is under the assumption that
the FtsW is evenly distributed over all vesicles (as was also supposed by the Referee). From this it
follows that if FtsW functions bidirectional 20% of the FtsW molecules is active. However, if FtsW
functions unidirectional, this would mean that on average at least two active FtsW molecules should
be present (to ensure that at least one is pointing in the right direction, i.e. outward) which would
increase the amount of functional FtsW molecules to 40%. However, this estimation which is
already prone to error due to all the assumptions, can be additionally influenced by several unknown
variables such as the amount of flippase-less vesicles, aggregation of the protein (as also mentioned
previously) and the inactivity of the FtsW protein (also pointed out by the Reviewer).
We hope that the reasoning as alluded to above has convinced you that currently all efforts to
precisely calculate the amount of (active) FtsW in our system are very complex and you may agree
with us that any attempt to do this in the current manuscript would be too speculative.
We trust that we now have addressed this point adequately.
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