A bacterial membrane sculpting protein with BAR domain-like activity
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Abstract
Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli . Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.
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###Reviewer #3
This manuscript reports the first description of a eukaryotic-like Bin/Amphiphysin/RVS (BAR) domain protein in a bacterium (Shewanella oneidensis MR-1), BdpA, with conserved roles in membrane curvature control during outer membrane vesicle (OMV) formation. Consistent with this, a BdpA-defective mutant had defects in the size and shape of redox-active membrane vesicles and formed outer membrane extensions (OMEs) lacking the characteristic tubular structure. Heterologous expression of the BdpA proteins in model (Escherichia coli) and non-model (Marinobacter atlanticus) bacteria hosts promoted OME formation. The authors propose BdpA as a new subclass of prokaryotic BAR proteins with eukaryotic-like roles in membrane curvature modulation. This is an interesting finding that could be strengthened with topological studies of …
###Reviewer #3
This manuscript reports the first description of a eukaryotic-like Bin/Amphiphysin/RVS (BAR) domain protein in a bacterium (Shewanella oneidensis MR-1), BdpA, with conserved roles in membrane curvature control during outer membrane vesicle (OMV) formation. Consistent with this, a BdpA-defective mutant had defects in the size and shape of redox-active membrane vesicles and formed outer membrane extensions (OMEs) lacking the characteristic tubular structure. Heterologous expression of the BdpA proteins in model (Escherichia coli) and non-model (Marinobacter atlanticus) bacteria hosts promoted OME formation. The authors propose BdpA as a new subclass of prokaryotic BAR proteins with eukaryotic-like roles in membrane curvature modulation. This is an interesting finding that could be strengthened with topological studies of BdpA in OMV/OME and quantitative analyses to validate the many qualitative microscopic observations.
Numbered summary:
To my knowledge this is the first description of a BAR-domain protein in a prokaryotic organism. But the role of prokaryotic proteins with amphipathic α-helical domains in membrane binding/curvature is not new. A review by Dowrkin1 describes some of these structural homologs and their role in membrane binding and curvature control via their amphipathic domains (e.g., Bacillus subtilis SpoVM, which controls forespore membrane curvature during sporulation using its helical domain). This information is important in the introduction and could help with the phylogenetic analyses (comment 4 below).
I am having a hard time reconciling the presence of a galactose-binding domain in BdpA and LPS sugar binding. This would suggest that the proteins coat the OMV rather than interacting with the periplasmic side of the outer membrane to promote OMV formation and release (which I somehow assume based on the role of some eukaryotic BARs). The lack of topological studies makes these models highly speculative and weakens some of the conclusions. The paper would be strengthened with the addition of topological studies in OMVs and OMEs.
Many experiments rely on microscopic observations of cells, OMVs and OMEs to support conclusions based on (at most) semiquantitative data. These experiments require validation with methods that quantitatively determine critical variables such as OMV size and size distribution. Also note the microscopic methods are poorly described or not described at all in the methods section. Thus, it is not clear how many cells they examined microscopically and how many biological replicates (cultures) they used. The variability associated with this type of microscopic assessments makes sample size (number of cells, typically in the hundreds) and replication in independent cultures critical.
Many of the branch points in the phylogenetic tree (Fig. 5) have very low confidence values. The authors did not provide the alignments so I could not evaluate the accuracy of the approach to offer suggestions for improvement. The predictive value of the tree may improve by including prokaryotic amphipathic helical domains such as those from SpoVM, MinD and FtsA. These issues are not as concerning in the tree presented in Fig. S6 although I note that this tree is supposed to show the distribution of "BdpA orthologs in other prokaryotes" but most of the branches are for eukaryotic proteins. I also note that the Methods section describes important results about the homology (or lack of homology) between BdpA and other prokaryotic and eukaryotic proteins. This information is more appropriate in the Results section.
References:
Dworkin, J. Cellular polarity in prokaryotic organisms. Cold Spring Harbor perspectives in biology 1, a003368-a003368, doi:10.1101/cshperspect.a003368 (2009).
Gorby, Y. et al. Redox-reactive membrane vesicles produced by Shewanella. Geobiology 6, 232-241, doi:10.1111/j.1472-4669.2008.00158.x (2008).
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###Reviewer #2 Some Gram-negative bacteria, such as Shewanella oneidensis, produce outer membrane extensions (OME) that mediate electron transfer to extracellular substrates. Many of the players involved in the transfer of electrons via these nanowires have been discovered but the mechanisms of outer membrane remodeling have remained mysterious. Here, Phillips, Zacharoff, and colleagues, identify BdpA as a protein that stabilizes OMEs in Shewanella oneidensis and perhaps displays outer membrane remodeling activity in other bacterial species. Given its homology to eukaryotic BAR-domain proteins, the authors suggest that BdpA and its homologs define the first prokaryotic family of BAR proteins or pBARs.
This works tackles a number of significant questions that span broad areas of microbiology and cell biology. First, it explores a …
###Reviewer #2 Some Gram-negative bacteria, such as Shewanella oneidensis, produce outer membrane extensions (OME) that mediate electron transfer to extracellular substrates. Many of the players involved in the transfer of electrons via these nanowires have been discovered but the mechanisms of outer membrane remodeling have remained mysterious. Here, Phillips, Zacharoff, and colleagues, identify BdpA as a protein that stabilizes OMEs in Shewanella oneidensis and perhaps displays outer membrane remodeling activity in other bacterial species. Given its homology to eukaryotic BAR-domain proteins, the authors suggest that BdpA and its homologs define the first prokaryotic family of BAR proteins or pBARs.
This works tackles a number of significant questions that span broad areas of microbiology and cell biology. First, it explores a critical area of bacterial cell biology: how do gram negatives remodel their outer membranes? Second, it focuses on an underappreciated aspect of extracellular electron transfer, an activity widespread amongst bacteria with clear relevance to basic and applied fields. Finally, it provides a possible glimpse into the evolution of BAR-domain proteins which play diverse cellular roles in eukaryotes. Despite the substantial advances presented here, I have some concerns which, if addressed, can lead to more certain conclusions about the cellular role of BdpA.
I liked the comparative proteomics approach as a tool to identify unique OME components. I was surprised that the two fractions differed so much in their protein composition. Based on the materials and methods the OM and OME fractions were isolated from cells grown under very different conditions. Could this account for the large differences between these two fractions? Looking at the list of proteins enriched in either fraction is there any indication of significant contamination from other cellular fractions? What controls were used to ensure that the purification procedure was working effectively?
The authors conclude that the OM vesicles are conductive. However, some controls are needed since other cellular components (such as OM fractions containing Mtr proteins) may have contaminated the OME fraction. Is the OME fraction "enriched" for this activity compared to just the OM fraction?
Is BdpA really a BAR-domain protein? The authors use computational tools (such as BLAST and homology modelling) to posit that BdpA is a BAR-domain protein. This hypothesis is strengthened by the phenotype of mutants missing bdpA. While OMEs are not absent, their architecture is visibly altered which may point to some instability in the membrane extensions. Significantly, BdpA is sufficient to induce OME-like structures when expressed in planktonic Shewanella cells, a condition during which OMEs are not normally produced. However, as authors state, BdpA barely meets the cutoff (as set by the program used) for a BAR-domain protein. Furthermore, some of its homologs that share high levels of sequence identity don't pass the bar set by these computational methods. However, we cannot say that BdpA is actually a BAR-domain protein. Its effects on membrane stability could be indirect or the result of binding to outer membrane features in a manner distinct from other BAR proteins. Therefore, some biochemical corroboration of its activity on membranes or structural data are needed to confirm its relationship to eukaryotic Bar domain proteins. On a minor note I would prefer "bacterial" rather than "prokaryotic" since BdpA Bar-like domain is not found in archaea. Also, other groups have proposed that bacterial proteins contain BAR domains (for instance, Tanaka et al in reference 28). How similar is BdpA to these proteins?
Heterologous expression of BdpA in other bacteria provides one of the most compelling arguments for its central role in producing OMEs. However, the imaging data provided here (at least in my pdf) do not provide the clearest evidence for induction of OMEs in M. atlanticus and E. coli. This is especially the case with the E. coli images. The extended web of staining in 4c does not resemble the tubules seen in S. oneidensis. It would be great to have some electron microscopy data and/or higher resolution fluorescence images of these bacteria as corroborating evidence. Additionally, only a few cells are shown so some quantification of the proportion of cells with OMEs is needed.
Other than the predicted signal peptide, does BdpA have any predicted features that indicate it is an outer membrane protein? The authors hypothesize that the putative Galactose-binding domain of BdpA mediates binding to LPS. However, it is also possible that it binds to peptidoglycan components. Therefore, independent data on localization of BdpA via microscopy or higher resolution biochemical fractionation would provide greater confidence that the protein is acting in the appropriate cellular location.
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###Reviewer #1 In the manuscript "A Prokaryotic Membrane Sculpting BAR Domain Protein" the authors describe the identification of the first bacterial membrane sculpting BAR domain protein, and the characterization of its function. In eukaryotes this protein is important for shaping membrane curvature. Here they identify a protein containing a BAR domain in the bacterium Shewanella oneidensis, which they name BdpA (BAR domain-like protein A). The authors show that BdpA is enriched in outer membrane vesicles (OMVs) and outer membrane extension (OMEs), regulates the size of OMVs and the shape of OMEs. They show this by characterizing and quantifying membrane vesicles and extension comparing WT with a BdpA mutant and the BdpA mutant with heterologous BdpA expression. They further show that heterologous expression of BdpA promotes OME in E. …
###Reviewer #1 In the manuscript "A Prokaryotic Membrane Sculpting BAR Domain Protein" the authors describe the identification of the first bacterial membrane sculpting BAR domain protein, and the characterization of its function. In eukaryotes this protein is important for shaping membrane curvature. Here they identify a protein containing a BAR domain in the bacterium Shewanella oneidensis, which they name BdpA (BAR domain-like protein A). The authors show that BdpA is enriched in outer membrane vesicles (OMVs) and outer membrane extension (OMEs), regulates the size of OMVs and the shape of OMEs. They show this by characterizing and quantifying membrane vesicles and extension comparing WT with a BdpA mutant and the BdpA mutant with heterologous BdpA expression. They further show that heterologous expression of BdpA promotes OME in E. coli.
In my opinion this paper provides solid support for the presence of these proteins in bacteria with an important function in membrane vesicles and membrane extensions.
Minor Comments:
In the introduction the authors summarize what is known about BAR eukaryotic protein in terms of membrane localization and their role in membrane curvature and tubulation events. I think it is important to also provide a summary of what is known about the functional biological implication of these proteins in eukaryotes. Namely, if the main function of BAR proteins in eukaryotes is always related to tubulation formation or if there are other functions attributed to these proteins.
Contrast and resolution in Figure 3, panel a, is weak making it difficult to see tubules described by the authors.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.
###Summary
In this manuscript the authors propose the identification of a novel protein involved in outer membrane remodelling, named BdpA (BAR domain-like protein A). According to the proposed model BdpA has a conserved role in membrane curvature control during formation of outer membrane vesicle (OMV) and of outer membrane extension (OMEs) in Shewanella oneidensis. The authors also provide evidence that heterologous expression of BdpA promotes formation of OMEs in other bacteria (namely in E. coli), and that BdpA is …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.
###Summary
In this manuscript the authors propose the identification of a novel protein involved in outer membrane remodelling, named BdpA (BAR domain-like protein A). According to the proposed model BdpA has a conserved role in membrane curvature control during formation of outer membrane vesicle (OMV) and of outer membrane extension (OMEs) in Shewanella oneidensis. The authors also provide evidence that heterologous expression of BdpA promotes formation of OMEs in other bacteria (namely in E. coli), and that BdpA is sufficient to induce OME-like structures when expressed in conditions where OMEs are normally not formed. In eukaryotes proteins containing BAR domains are important for shaping membrane curvature. Given the homology of BdpA to eukaryotic BAR-domain proteins, the authors suggest that BdpA and its homologs define the first prokaryotic family of BAR proteins or pBARs, with eukaryotic-like roles in membrane curvature modulation.
Overall, the reviewers think that this is a very interesting study, and provided that further support is obtained to substantiate the proposed model the reviewers agree that the findings described here tackle a number of significant questions of broad interest. However, the reviewers also think that the evidence provided in this manuscript still does not fully support the conclusion that BdpA protein is involved in membrane curvature control as the eukaryotic proteins containing the BAR domains.
We have compiled a list of comments that we hope will help the authors address the concerns of the reviewers to obtain stronger support for the function of BdpA.
The reviewers are concerned that some of the conclusions are based on qualitative observations of microscopy analysis of OMVs and OMEs, and quantitative analyses are lacking to validate qualitative observations. As specified in with examples in the list of minor points below the reviewers propose that the data should be re-analyzed to obtain quantitative results. Specifically, a size distribution analysis could be applied to some microscopy data. Also note that the microscopy methods are poorly described, and as the calculation methods used are not fully available it is difficult to understand if the appropriate methods were used. Please specify how many cells were examined microscopically and how many biological replicates (cultures) were used in each experiment.
Statistical analyses were not always the most accurate. In figure 2 unpaired t-test was used for samples that have high variance, this approach may inflate the statistical difference between the strains. For figure 2 a histogram of size distribution analyses could be shown for each strain.
The reviewers are concerned that the proteomic data is not clear enough to conclude that the BdpA protein is localized to or enriched in OMV/OME. Could the results be complemented with some other method to confirm BdpA localization? The reviewers are particularly concerned by the fact that a large number of proteins were identified in the OMV fraction. Could it be that some of the OMV/OME fractions were contaminated? What controls were used to ensure that the purification procedure was working effectively? Could the data be strengthened by some quality control analyses to determine how many of those proteins are actually predicted to localize to the outer membrane and periplasm? From the methods it seems that the culture conditions used to prepare the OM versus OMV were different, is this so? If yes, why were the culture conditions different? This could affect protein expression? Please include the detailed growth conditions in the method section.
The conclusion that BdpA is a BAR-domain protein is largely based on homology. The supplementary information file includes homology models that show striking similarity with eukaryotic BAR proteins. However, as the authors state, BdpA barely meets the cutoff for a BAR-domain protein. The results with the phenotype of the BdpA mutant, complementations and sufficiency data provide good support to the functional role of BdpA in membrane remodelling. However, the effect of BdpA on membrane stability could be indirect or the result of binding to outer membrane features in a manner distinct from other BAR proteins. Could these results be strengthened with some biochemical corroboration of its activity on membranes or structural data to confirm its relationship to eukaryotic Bar domain proteins? Or structural data to confirm its relationship to eukaryotic BAR domain proteins?
The reviewers propose that the paper would be strengthened with the addition of topological studies in OMVs and OMEs. The reviewers had problems in reconciling the presence of a galactose-binding domain in BdpA and LPS sugar binding. The authors hypothesize that the putative Galactose-binding domain of BdpA mediates binding to LPS. However, it is also possible that it binds to peptidoglycan components. This would suggest that the proteins interact with the periplasmic side of the outer membrane rather than coat the OMV to promote OMV formation and release (which one could assume based on the role of some eukaryotic BARs). The addition of topological studies (or some biochemical approach) could make these models less speculative, strengthening the conclusions.
Heterologous expression of BdpA in other bacteria provides important compelling arguments for its central role in producing OMEs. However, the imaging data provided do not provide the clearest evidence for induction of OMEs in M. atlanticus and E. coli. This is especially the case with the E. coli images. The extended web of staining in 4c does not resemble the tubules seen in S. oneidensis. It would be great to have some electron microscopy data and/or higher resolution fluorescence images of these bacteria as corroborating evidence. Additionally, only a few cells are shown and quantification of the proportion of cells with OMEs is needed. Thus, as already discussed in point 1, quantitative analyses could improve this important point.
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