Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis
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Evaluation Summary:
This manuscript is of interest to microbiologists and protein biochemists who study biofilm formation, haem proteins, and cellular responses to extracellular oxidative stress. It proposes a mechanism for biofilm protection from reactive oxygen species (ROIs) through the examination of the Gram positive, Bacillus subtilis. The data support many of the conclusions of the paper and highlight the importance of cell surface-localized protein peroxidase activity for proper biofilm assembly in a model species. Further evidence is needed to fully support the proposed mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis , prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr 63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI -deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.
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Author Response
Reviewer #1 (Public Review):
In their paper, titled ‘Group II truncated haemoglobin YjbI prevents reactive oxygene species-induced protein aggregation in Bacillus subtilis’, Imai et al., suggest that the protein YjbI acts as a hydroperoxide peroxidase and therefore it may protect cell-surface cells from oxidation. Using AFM and contact angle measurements they show that yjbI mutants lead to changes in cell surface properties as well as to the formation of more hydrophilic biofilms, relative to the wild-type (WT) strain. Since both tasA and yjbI mutants experienced a similar phenotypic behaviour, the authors linked between the two proteins, TasA and YjbI, and in a series of biophysical and biochemical tests they tried to establish this link. This study touches upon an important question, how do biofilms protect …
Author Response
Reviewer #1 (Public Review):
In their paper, titled ‘Group II truncated haemoglobin YjbI prevents reactive oxygene species-induced protein aggregation in Bacillus subtilis’, Imai et al., suggest that the protein YjbI acts as a hydroperoxide peroxidase and therefore it may protect cell-surface cells from oxidation. Using AFM and contact angle measurements they show that yjbI mutants lead to changes in cell surface properties as well as to the formation of more hydrophilic biofilms, relative to the wild-type (WT) strain. Since both tasA and yjbI mutants experienced a similar phenotypic behaviour, the authors linked between the two proteins, TasA and YjbI, and in a series of biophysical and biochemical tests they tried to establish this link. This study touches upon an important question, how do biofilms protect themselves from reactive oxygene species (ROIs), that is nicely described in the introduction; The link between the above proteins in very interesting and relevant to the main question proposed in the study. However, the experiments presented does not always directly support the conclusions made.
The points that I find necessary to clarify/extend:
- A major claim in the paper is that biofilms that do not harbour the tasA gene (tasA-) are flat, and therefore their contact angle is low, indicating that they are less hydrophobic than WT strains. However, the phenotype of biofilms of tasA mutants are normally not that flat (see for example Romero et al., PNAS 2010; Vlamakis et al., Genes and Development, 2008; Erskine et al., Molecular Microbiology 2018). As a matter of fact, even the WT biofilms that are used as a control in this study are much more flat than the biofilms that serve as standards in the papers referenced above.
We appreciate the reviewer’s comment. As we explained above (answer to Essential Revisions, point 4), there are differences in the morphology of colonies between the 168 and NCBI3610 strains of B. subtilis, as previously pointed out in the literature (Arnaouteli et al. Nat. Rev. Microbiol., 19:600-614, 2021; Mielich-Süss and Lopez, Environ. Microbiol., 17:555-565, 2014). We employed B. subtilis strain 168 because this strain is a close representative of B. subtilis, as described by Zeigler et al. (J. Bacteriol., 190:6983-6995, 2008), and serves as a model organism for wider aspects of basic research, including oxidative damage responses.
To clarify this point, we have added the following text to the revised manuscript in lines 269–277: “Most studies on biofilm formation in B. subtilis use the B. subtilis NCBI3610 strain as a model bacterium because of its ability to form well-structured three-dimensional biofilms (Arnaouteli et al., 2021, Mielich-Süss et al., 2014). The biofilms of the wild-type and tasA mutant strains of the B. subtilis 168 strain are known to be morphologically different from those of the B. subtilis NCBI3610 strain (Romero et al., 2010, Vlamakis et al., 2008, Erskine et al., 2018). In this study, the B. subtilis 168 strain was used because it is the most representative of B. subtilis and serves as a model organism for a wider range of research aspects (Zeigler et al., 2008) as we were not only interested in evaluating biofilm formation but also in more general aspects of oxidative damage responses in bacteria.”
- Figure 1. The authors use AFM phase imaging to probe differences in cellular stiffness. This AFM mode is not quantitative and the differences presented could also result from differences in adhesion between the tip and the sample. A more quantitative means to evaluate stiffness is a direct measurement of moduli in Force mode, a standard AFM module.
Thank you for your comment. As mentioned above (answer to Essential Revisions, point 3), the AFM data have been removed.
- Line 147. The authors link between the lack of monomeric TasA in YjbI mutants and the formation of covalent cross linking in TasA aggregates. This is a strong statement that unfortunately is not supported by any of the experiments described in the manuscript.
As mentioned above (answer to Essential Revisions, point 5), we have removed the statement regarding the lack of monomeric TasA in the mutant. The following has been included to highlight the potential involvement of covalent bonds in the TasA aggregate formation in lines 126–129 in the revised manuscript: “No monomeric TasA was detected in the insoluble fraction of the yjbI-deficient mutant strain. An aggregate of TasA was observed under strong reducing and heat-denaturing conditions in SDS sample buffer, suggesting that covalent bonds may be involved in aggregate formation.”
- The authors seek to make a connection between YjbI and TasA. However, this link is either not well established or only hinted indirectly in this manuscript, through precipitation assays, contact angle measurements and growth curves. To establish such a link, a more molecular approach is advised. Experiments that would provide a direct link between the two proteins and mark specific molecular changes of the proteins include for example titration NMR studies of labelled proteins (at least one of the proteins). In cases where the authors need to show protein localization to the cell surface, it would be of help to use TEM or high-end fluorescence microscopy.
We thank the reviewer for this valuable advice. In response to this comment, we carried out additional experiments, as described above (answer to Essential Revisions, point 1). We will consider the suggested studies, mainly with a molecular approach including titration NMR and TEM, for future studies, as facilities for these specific studies are currently not available.
- This paper suggests that the protein YjbI acts as an electron donor. Given that there are other proteins with a similar role (in other organisms), it would be nice to show whether there is any homology (by sequence and/or structure) to these proteins.
Thank you for your comments. We have added a description of animal peroxiredoxins and selenomethionine (with GSH or a thioredoxin system) that have been shown to scavenge protein hydroperoxides to the revised manuscript. We also added a description of how YjbI differs from peroxiredoxin and selenomethionine.
The corresponding sentences have been added to the revised manuscript in lines 254–268: “Peroxiredoxins have been reported to repair intracellular protein peroxidation in mammals (Peskin et al., 2010). However, YjbI is distinct from peroxiredoxins in that it is a haem protein with no significant sequence homology (<15%). The second-order rate constants (M-1·s -1) for the reactions of mammalian peroxiredoxins 2 and 3 with BSA-OOH are 160 and 360, respectively, and have been shown to reduce protein peroxides more efficiently than GSH under physiological conditions (Peskin et al., 2010). Although direct comparison is difficult due to different experimental conditions, YjbI and peroxiredoxins are likely to have a similar catalytic rate, as both proteins can reduce BSA-OOH in the order of several mM in roughly 5 min at similar protein concentrations (Fig. 3e) (Peskin et al., 2010). Interestingly, selenomethionine can catalyze the removal of hydroperoxides from proteins in the presence of GSH or a thioredoxin system (Rahmanto & Davies, 2011). However, this system, as well as peroxiredoxins, localises in the cytoplasm of cells, which is a significant difference between YjbI and these proteins. Moreover, whether bacteria utilize peroxiredoxins and the selenomethionine system to remove hydroperoxides from proteins remains unclear.”
- (Minor point). The use of Pymol to demonstrate that the YjbI's pocket could serve as a binding site for haem molecule is nice, but using Molecular Dynamics (or any other calculation) would be more quantitative and convincing of the specificity of the interaction.
We appreciate your comment regarding this point. However, we believe that analysis using molecular dynamics (or other calculations) is largely difficult because the structure of the hydroperoxidised protein substrates is not available. Further, the degree of similarity between the structure of TasA or BSA and the hydroperoxidised form is unclear. A calculation analysis with a small model substrate can be adopted in future work. Therefore, we only showed the surface opening of the YjbI structure, which is potentially relevant for binding to a hydroperoxidised protein substrate.
Reviewer #2 (Public Review):
In this study, Imai et al. uncover a role for the truncated haemoglobin protein YjbI in biofilm formation by the model bacterium B. subtilis. They show that yjbI gene disruption results in altered biofilms, with increased wettability and different matrix stiffness relative to cells. The absence of YjbI activity results in aggregation of the amyloid-like TasA matrix protein, and the biofilm wettability defect of the yjbI mutant can be recapitulated by anti-YjbI immune serum, suggesting that YjbI is located on the cell surface. Absence of YjbI also modestly increases the sensitivity of cells growing on agar plates to the oxidant AAPH. Using the model protein substrate BSA, purified YjbI can at least partially reverse oxidant-induced BSA aggregation in vitro, convincingly showing the YjbI has protein hydroperoxide peroxidase activity, which is evidently an unusual enzymatic activity. Finally, the authors examine lipid peroxidation and conclude that YjbI is not involved. The results are interesting in that they connect YjbI to a biofilm phenotype and convincingly show protein hydroperoxide peroxidase activity by a truncated haemoglobin protein, an activity not previously attributed to this class of proteins.
The experiments are largely well done, but some of the corresponding conclusions are overinterpreted, connecting ideas without experimental support. Moreover, the yjbI mutant has a narrow and relatively mild phenotype.
- The paper identifies two separate properties of YjbI: its mutant phenotype with respect to biofilm formation, and its peroxidase activity against oxidant-induced aggregation of TasA and BSA. The authors conclude that these properties are connected, but this is not formally tested. While purified YjbI can reverse hydrogen peroxide-induced aggregation of purified TasA in vitro, and the yjbI mutant shows more TasA in the insoluble fraction of B. subtilis pellicle lysates, these experiments do not show that the TasA aggregates in pellicle lysates are caused by peroxidation, nor do they show that TasA aggregation is normally kept at bay by YjbI peroxidase activity (it is possible that YjbI has a separate role in biofilm integrity). Some experiments that might lend support to this connection include examining the biofilm phenotype of a catalytically dead point mutant of YjbI (perhaps Y25 or Y63, l. 298, or other residues informed by the crystal structure of Giangiacomo et al.) to establish whether peroxidase activity is important for biofilm formation. Such a mutant would be particularly valuable, as it could also be used to test whether inactivation of enzyme activity affects other phenotypes (cell stiffness, for example). Another approach would be to use a soluble antioxidant molecule, purified YjbI, or another peroxidase to see if the yjbI biofilm can be rescued.
We greatly appreciate this comment, which is critical for improving our manuscript. To address this issue, we performed additional experiments using the Y25F, Y63F, and W69F variants of YjbI. The introduction of the Y63F variant gene into the yjbI-deficient strain failed to complement the defective phenotype of the yjbI-deficient strain in biofilm repellency (revised Fig. 1b). We found that the purified Y63F lost its hydroperoxide peroxidase activity (revised Fig. 3g). These results show a connection between the protein hydroperoxide peroxidase activity of YjbI and the abnormal biofilm phenotype of the yjbI-deficient strain. Accordingly, Figs. 1b and 3g have been added to the revised manuscript and figure descriptions have been included in lines 220–226, 322–324, and 327–328 (as explained above in the answer to Essential Revision, Point 2).
- The authors conclude on the basis of the AFM data in Fig. 1 that yjbI mutant cells are less stiff than WT cells, but the data only show relative stiffness. It is also unclear why a change in cell envelope stiffness would relate to biofilm wettability (ll. 130-131). If there truly is a change in cell envelope stiffness, a high-resolution, head-to-head AFM comparison of planktonically grown cells would be informative.
We appreciate the reviewer’s comment on this point. As mentioned above (answer to Essential Revision, points 3 and 6), we realized that our interpretations of the AFM data were not appropriate and not relevant to biofilm repellency. Accordingly, the AFM data were removed.
- The data in Fig. 2F showing hypersensitivity of yjbI mutant cells to AAPH were generated in an unusual way: stationary-phase liquid culture was spotted on an LB plate, and the colonies were "fractionated" at the noted intervals and resuspended in saline for OD measurement. Measuring sensitivity to AAPH just in shaking liquid planktonic culture would make this phenotype more convincing. Under non-biofilm forming conditions, is a surface-associated peroxidase important for cell growth or survival under oxidant challenge?
We appreciate your comment regarding this point and apologize for the error in the description “'Planktonically grown B. subtilis strains under AAPH-induced oxidative stress'” in the Methods section. No solid medium was used in the experiments. The description in the Figure legend of Fig. 2f is correct. The sentence in the text has been rewritten in the revised manuscript in lines 813.
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Evaluation Summary:
This manuscript is of interest to microbiologists and protein biochemists who study biofilm formation, haem proteins, and cellular responses to extracellular oxidative stress. It proposes a mechanism for biofilm protection from reactive oxygen species (ROIs) through the examination of the Gram positive, Bacillus subtilis. The data support many of the conclusions of the paper and highlight the importance of cell surface-localized protein peroxidase activity for proper biofilm assembly in a model species. Further evidence is needed to fully support the proposed mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
In their paper, titled 'Group II truncated haemoglobin YjbI prevents reactive oxygene species-induced protein aggregation in Bacillus subtilis', Imai et al., suggest that the protein YjbI acts as a hydroperoxide peroxidase and therefore it may protect cell-surface cells from oxidation. Using AFM and contact angle measurements they show that yjbI mutants lead to changes in cell surface properties as well as to the formation of more hydrophilic biofilms, relative to the wild-type (WT) strain. Since both tasA and yjbI mutants experienced a similar phenotypic behaviour, the authors linked between the two proteins, TasA and YjbI, and in a series of biophysical and biochemical tests they tried to establish this link. This study touches upon an important question, how do biofilms protect themselves from reactive …
Reviewer #1 (Public Review):
In their paper, titled 'Group II truncated haemoglobin YjbI prevents reactive oxygene species-induced protein aggregation in Bacillus subtilis', Imai et al., suggest that the protein YjbI acts as a hydroperoxide peroxidase and therefore it may protect cell-surface cells from oxidation. Using AFM and contact angle measurements they show that yjbI mutants lead to changes in cell surface properties as well as to the formation of more hydrophilic biofilms, relative to the wild-type (WT) strain. Since both tasA and yjbI mutants experienced a similar phenotypic behaviour, the authors linked between the two proteins, TasA and YjbI, and in a series of biophysical and biochemical tests they tried to establish this link. This study touches upon an important question, how do biofilms protect themselves from reactive oxygene species (ROIs), that is nicely described in the introduction; The link between the above proteins in very interesting and relevant to the main question proposed in the study. However, the experiments presented does not always directly support the conclusions made.
The points that I find necessary to clarify/extend:
A major claim in the paper is that biofilms that do not harbour the tasA gene (tasA-) are flat, and therefore their contact angle is low, indicating that they are less hydrophobic than WT strains. However, the phenotype of biofilms of tasA mutants are normally not that flat (see for example Romero et al., PNAS 2010; Vlamakis et al., Genes and Development, 2008; Erskine et al., Molecular Microbiology 2018). As a matter of fact, even the WT biofilms that are used as a control in this study are much more flat than the biofilms that serve as standards in the papers referenced above.
Figure 1. The authors use AFM phase imaging to probe differences in cellular stiffness. This AFM mode is not quantitative and the differences presented could also result from differences in adhesion between the tip and the sample. A more quantitative means to evaluate stiffness is a direct measurement of moduli in Force mode, a standard AFM module.
Line 147. The authors link between the lack of monomeric TasA in YjbI mutants and the formation of covalent cross linking in TasA aggregates. This is a strong statement that unfortunately is not supported by any of the experiments described in the manuscript.
The authors seek to make a connection between YjbI and TasA. However, this link is either not well established or only hinted indirectly in this manuscript, through precipitation assays, contact angle measurements and growth curves. To establish such a link, a more molecular approach is advised. Experiments that would provide a direct link between the two proteins and mark specific molecular changes of the proteins include for example titration NMR studies of labelled proteins (at least one of the proteins). In cases where the authors need to show protein localization to the cell surface, it would be of help to use TEM or high-end fluorescence microscopy.
This paper suggests that the protein YjbI acts as an electron donor. Given that there are other proteins with a similar role (in other organisms), it would be nice to show whether there is any homology (by sequence and/or structure) to these proteins.
(Minor point). The use of Pymol to demonstrate that the YjbI's pocket could serve as a binding site for haem molecule is nice, but using Molecular Dynamics (or any other calculation) would be more quantitative and convincing of the specificity of the interaction.
-
Reviewer #2 (Public Review):
In this study, Imai et al. uncover a role for the truncated haemoglobin protein YjbI in biofilm formation by the model bacterium B. subtilis. They show that yjbI gene disruption results in altered biofilms, with increased wettability and different matrix stiffness relative to cells. The absence of YjbI activity results in aggregation of the amyloid-like TasA matrix protein, and the biofilm wettability defect of the yjbI mutant can be recapitulated by anti-YjbI immune serum, suggesting that YjbI is located on the cell surface. Absence of YjbI also modestly increases the sensitivity of cells growing on agar plates to the oxidant AAPH. Using the model protein substrate BSA, purified YjbI can at least partially reverse oxidant-induced BSA aggregation in vitro, convincingly showing the YjbI has protein …
Reviewer #2 (Public Review):
In this study, Imai et al. uncover a role for the truncated haemoglobin protein YjbI in biofilm formation by the model bacterium B. subtilis. They show that yjbI gene disruption results in altered biofilms, with increased wettability and different matrix stiffness relative to cells. The absence of YjbI activity results in aggregation of the amyloid-like TasA matrix protein, and the biofilm wettability defect of the yjbI mutant can be recapitulated by anti-YjbI immune serum, suggesting that YjbI is located on the cell surface. Absence of YjbI also modestly increases the sensitivity of cells growing on agar plates to the oxidant AAPH. Using the model protein substrate BSA, purified YjbI can at least partially reverse oxidant-induced BSA aggregation in vitro, convincingly showing the YjbI has protein hydroperoxide peroxidase activity, which is evidently an unusual enzymatic activity. Finally, the authors examine lipid peroxidation and conclude that YjbI is not involved. The results are interesting in that they connect YjbI to a biofilm phenotype and convincingly show protein hydroperoxide peroxidase activity by a truncated haemoglobin protein, an activity not previously attributed to this class of proteins.
The experiments are largely well done, but some of the corresponding conclusions are overinterpreted, connecting ideas without experimental support. Moreover, the yjbI mutant has a narrow and relatively mild phenotype.
The paper identifies two separate properties of YjbI: its mutant phenotype with respect to biofilm formation, and its peroxidase activity against oxidant-induced aggregation of TasA and BSA. The authors conclude that these properties are connected, but this is not formally tested. While purified YjbI can reverse hydrogen peroxide-induced aggregation of purified TasA in vitro, and the yjbI mutant shows more TasA in the insoluble fraction of B. subtilis pellicle lysates, these experiments do not show that the TasA aggregates in pellicle lysates are caused by peroxidation, nor do they show that TasA aggregation is normally kept at bay by YjbI peroxidase activity (it is possible that YjbI has a separate role in biofilm integrity). Some experiments that might lend support to this connection include examining the biofilm phenotype of a catalytically dead point mutant of YjbI (perhaps Y25 or Y63, l. 298, or other residues informed by the crystal structure of Giangiacomo et al.) to establish whether peroxidase activity is important for biofilm formation. Such a mutant would be particularly valuable, as it could also be used to test whether inactivation of enzyme activity affects other phenotypes (cell stiffness, for example). Another approach would be to use a soluble antioxidant molecule, purified YjbI, or another peroxidase to see if the yjbI biofilm can be rescued.
The authors conclude on the basis of the AFM data in Fig. 1 that yjbI mutant cells are less stiff than WT cells, but the data only show relative stiffness. It is also unclear why a change in cell envelope stiffness would relate to biofilm wettability (ll. 130-131). If there truly is a change in cell envelope stiffness, a high-resolution, head-to-head AFM comparison of planktonically grown cells would be informative.
The data in Fig. 2F showing hypersensitivity of yjbI mutant cells to AAPH were generated in an unusual way: stationary-phase liquid culture was spotted on an LB plate, and the colonies were "fractionated" at the noted intervals and resuspended in saline for OD measurement. Measuring sensitivity to AAPH just in shaking liquid planktonic culture would make this phenotype more convincing. Under non-biofilm forming conditions, is a surface-associated peroxidase important for cell growth or survival under oxidant challenge?
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