Quantitative Metaproteomic Characterization of Acetic Acid Bacteria Reveals Functional Dynamics During Verdejo Wine Acetification

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors set out to identify the main microbes involved in vinegar production in Spain from Verdejo wine and to assess the metabolic, stress response and other pathways that might contribute towards microbial survival under high ethanol and acetic acid conditions. They carried out semi-continuous submerged culture and used LC-MS/MS to quantify protein abundances at 4 time points during the acetification process and applied differential expression and clustering analysis to group proteins with similar expression patterns. They presented the results as heatmaps, Upset charts and protein interaction networks, making it much easier to see the underlying patterns and relationships involved. 

Ethanol concentration was reduced and acetic acid concentration increased throughout the acetification process. Volcano plots, comparing protein abundances between two time points suggested that changes in protein abundance were more gradual during the earlier 3 stages but then more dramatic towards the end. The greatest differences were seen when comparing the first and last time points. Gene ontology analysis was carried out for the up-regulated proteins between the first and last time point (all were K. europaeus proteins). A network was constructed, showing the GO term connections among these upregulated proteins. These included proteins with roles in amino acid metabolism, membrane assembly and redox reactions. Redox balance and oxidative stress responses are highlighted as important. The membrane is important for two reasons: it protects the organism from stress, caused by high levels of acetic acid outside the cell and most of the enzymatic processes for acetate production are carried out in the periplasmic space. The authors relate their findings to reports in the literature that the insertion of proteins (transporters and porins) into the membrane is important. Downregulated proteins (in the last compared with the first time points) reveal that translation is downregulated, which is a normal response to stress. Principal component analysis showed that there is only a small degree of overlap among time points 1, 2 and 3, while timepoint 4 is completely separate from the other timepoints.

GO biological processes analysis indicated that stress responses increase throughout the acetification process in Acidiphilium species but that K. europaeus was more resistant to acetic acid stress. K. europaeus was also responsible for most of the metabolic related protein activity, indicating that this species was chiefly responsible for acetate production. Enriched GO cellular localisation terms among upregulated proteins in K. europaeus (in contrast to other species) included "periplasmic" space, which reflect the importance of this space for acetification.

STRING analysis using data from the KEGG and uniprot databases was used to construct networks, showing the relationships among different processes in the cells. Finally, a very detailed, clear diagram was produced, showing the likely flow of metabolites, electrons etc. in K. europaeus, based on the protein abundance data.

The authors clearly showed that one species Komagataeibacter europaeus dominated the culture at all time points during the process.  They demonstrated that this species was much more (acid) stress resistant than various other species in the culture but that this came with a high energy cost. 

The study has elucidated details of metabolism, stress responses etc. that help make K. europaeus effective in the acetification process. It also serves as an excellent example of how to carry out such analyses and obtain maximum data from an LC-MS/MS experiment. It is of interest to researchers in academia and industry and could be of use to biotechnology in the future. I recommend publication with minor changes (see below)

122/123: I think the technical and biological replicates are the wrong way round

276: should "five clusters" be "four"?

326: add space before "was"

491: "metabolites exchange" should be "metabolite exchange"

492/499/520/581/587: "between" should be "among"

544-546: sentence does not make sense. Please reformat

685: " an increase in" or " increased"

Generally (throughout manuscript) I suggest changing "AAB" to "AABs" when it refers to a plural

 

Author Response

The authors set out to identify the main microbes involved in vinegar production in Spain from Verdejo wine and to assess the metabolic, stress response and other pathways that might contribute towards microbial survival under high ethanol and acetic acid conditions. They carried out semi-continuous submerged culture and used LC-MS/MS to quantify protein abundances at 4 time points during the acetification process and applied differential expression and clustering analysis to group proteins with similar expression patterns. They presented the results as heatmaps, Upset charts and protein interaction networks, making it much easier to see the underlying patterns and relationships involved. 

Ethanol concentration was reduced and acetic acid concentration increased throughout the acetification process. Volcano plots, comparing protein abundances between two time points suggested that changes in protein abundance were more gradual during the earlier 3 stages but then more dramatic towards the end. The greatest differences were seen when comparing the first and last time points. Gene ontology analysis was carried out for the up-regulated proteins between the first and last time point (all were K. europaeus proteins). A network was constructed, showing the GO term connections among these upregulated proteins. These included proteins with roles in amino acid metabolism, membrane assembly and redox reactions. Redox balance and oxidative stress responses are highlighted as important. The membrane is important for two reasons: it protects the organism from stress, caused by high levels of acetic acid outside the cell and most of the enzymatic processes for acetate production are carried out in the periplasmic space. The authors relate their findings to reports in the literature that the insertion of proteins (transporters and porins) into the membrane is important. Downregulated proteins (in the last compared with the first time points) reveal that translation is downregulated, which is a normal response to stress. Principal component analysis showed that there is only a small degree of overlap among time points 1, 2 and 3, while timepoint 4 is completely separate from the other timepoints.

GO biological processes analysis indicated that stress responses increase throughout the acetification process in Acidiphilium species but that K. europaeus was more resistant to acetic acid stress. K. europaeus was also responsible for most of the metabolic related protein activity, indicating that this species was chiefly responsible for acetate production. Enriched GO cellular localisation terms among upregulated proteins in K. europaeus (in contrast to other species) included "periplasmic" space, which reflect the importance of this space for acetification.

STRING analysis using data from the KEGG and uniprot databases was used to construct networks, showing the relationships among different processes in the cells. Finally, a very detailed, clear diagram was produced, showing the likely flow of metabolites, electrons etc. in K. europaeus, based on the protein abundance data.

The authors clearly showed that one species Komagataeibacter europaeus dominated the culture at all time points during the process.  They demonstrated that this species was much more (acid) stress resistant than various other species in the culture but that this came with a high energy cost. 

The study has elucidated details of metabolism, stress responses etc. that help make K. europaeus effective in the acetification process. It also serves as an excellent example of how to carry out such analyses and obtain maximum data from an LC-MS/MS experiment. It is of interest to researchers in academia and industry and could be of use to biotechnology in the future. I recommend publication with minor changes (see below)

122/123: I think the technical and biological replicates are the wrong way round

Thank you for your comment. We agree that the original wording could be misleading. In our experimental design, S1-S4 do not represent technical replicates, but different sampling points of the acetification process defined according to ethanol concentration. For each sampling point, four independent samples were collected from different reactor cycles in order to obtain replicate measurements under the same process condition. Therefore, these correspond to biological replicates. The manuscript has been revised accordingly to clarify this distinction (lines 122-123).

276: should "five clusters" be "four"?

We thank the reviewer for this careful observation. The term “five clusters” was incorrect. Although the initial sentence stated that 1,409 proteins were grouped into five clusters, only four clusters (A-D) were actually identified, described in the text, and represented in the figure. This has been corrected to “four clusters” in the revised manuscript.

326: add space before "was"

We thank the reviewer for this careful observation. The missing space before “was” has been corrected in the revised manuscript.

491: "metabolites exchange" should be "metabolite exchange"

We thank the reviewer for this careful observation. The expression has been corrected from “metabolites exchange” to “metabolite exchange” in the revised manuscript.

492/499/520/581/587: "between" should be "among"

We thank the reviewer for this helpful comment. The term “between” has been revised to “among” in the indicated lines throughout the revised manuscript (lines 469,525, 534, 555, 62, and 629).

544-546: sentence does not make sense. Please reformat

We thank the reviewer for this observation. The sentence has been reformulated in the revised manuscript (lines 579-581) to improve its clarity and meaning.

685: " an increase in" or " increased"

We thank the reviewer for this careful observation. The expression has been corrected, and the sentence has been modified accordingly in the revised manuscript (line 729).

 

Generally (throughout manuscript) I suggest changing "AAB" to "AABs" when it refers to a plural

We thank the reviewer for this helpful suggestion. The use of “AAB” and “AABs” has been carefully revised throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript title was “Quantitative Metaproteomic Characterization of Acetic Acid Bacteria Reveals Functional Dynamics during Verdejo Wine Acetification” . In this study, the functional dynamics of acetic acid bacteria(AAB) during Verdejo vinegar acetification were analyzed using a quantitative metaproteomic approach. The research was meaningful and worth for the control of wine. The specific revision advice was as follow.

1. The introductionshould provide a detailed account of the scientific basis of this research.
2. The significance and novelty of this research are not sufficiently emphasized.
3. In the result analysis, the logic needs to be clarified.

Author Response

The manuscript title was “Quantitative Metaproteomic Characterization of Acetic Acid Bacteria Reveals Functional Dynamics during Verdejo Wine Acetification”. In this study, the functional dynamics of acetic acid bacteria(AAB) during Verdejo vinegar acetification were analyzed using a quantitative metaproteomic approach. The research was meaningful and worth for the control of wine. The specific revision advice was as follow.

1. The introduction should provide a detailed account of the scientific basis of this research.
2. The significance and novelty of this research are not sufficiently emphasized.

We thank the reviewer for these valuable comments. To respond to comments 1 and 2 of the reviewer, the Introduction has been expanded to strengthen the scientific basis of the study and to better emphasize its significance and novelty. Specifically, we have further clarified the current knowledge gap regarding functional microbial dynamics during industrial acetification, the limitations of taxonomic approaches alone, the relevance of metaproteomics to identify metabolically active pathways, and the originality and industrial relevance of applying this approach to characterize the acetification cycle of Verdejo wine under submerged conditions (lines 77-86).

 

3. In the result analysis, the logic needs to be clarified.

We thank the reviewer for this valuable comment. The analysis of the results has been carefully revised to improve clarity, consistency, and the logical interpretation of the findings throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Line 59: What is the complexity of the vinegar microbiota? The manuscript mainly focuses on acetic acid bacteria (AAB), so what does it intend to convey by describing the colonies as complex?

Line 248: Figure 3 shows that the relative abundance of Acetobacteraceae is only 6%, but the result is described as 94.5% in the text. The results should be presented separately at the family level and genus level; the current presentation in the manuscript is likely to cause misunderstanding.

Line 250: As only identification down to the genus level was performed in Figure 3, how was the species Komagataeibacter europaeus identified? Moreover, judging solely from the figure, Acidocella should be the most abundant genus.

Line 359: In Figure 6, which two groups were compared to calculate the fold change?

Line 436: Improve the clarity of the images (Figure 8 and 9).

Author Response

Line 59: What is the complexity of the vinegar microbiota? The manuscript mainly focuses on acetic acid bacteria (AAB), so what does it intend to convey by describing the colonies as complex?

We thank the reviewer for this valuable comment. The term “complex” refers to the fact that, although acetic acid bacteria (AAB) constitute the dominant microbial group during acetification, the vinegar microbiota is not restricted to this functional group. Previous studies have reported the presence of other taxonomic groups potentially involved in the process, including yeasts, lactic acid bacteria, and archaea, some of which are still not fully characterized. In addition, many acetic acid bacteria are fastidious microorganisms, and several species are difficult to isolate using conventional plate-culture methods, which may lead to an underestimation of the actual microbial diversity of the system. Therefore, the use of the term “complex” refers to both taxonomic diversity and the dynamic functional interactions occurring within the acetification ecosystem.

Line 248: Figure 3 shows that the relative abundance of Acetobacteraceae is only 6%, but the result is described as 94.5% in the text. The results should be presented separately at the family level and genus level; the current presentation in the manuscript is likely to cause misunderstanding.

We thank the reviewer for pointing this out. The value of 94.5% refers to the proportion of proteins assigned to the family Acetobacteraceae relative to the total common proteins identified (1,409/1,489). In contrast, Figure 3 shows the relative distribution of genera only within the Acetobacteraceae subset, which therefore sums to 100%. The apparent 6% corresponds to a magnified view of the low-abundance genera and not to the total abundance of Acetobacteraceae. We have revised the text (lines 247-256) and figure legend to avoid misunderstanding.

Line 250: As only identification down to the genus level was performed in Figure 3, how was the species Komagataeibacter europaeus identified? Moreover, judging solely from the figure, Acidocella should be the most abundant genus.

We thank the reviewer for this valuable comment. Protein identification was performed using the UniProt database, which allowed taxonomic assignment at the species level when supported by the corresponding protein annotations. Therefore, the identification of Komagataeibacter europaeus was based on species-level protein annotation and can be consulted in Table S1 of the Supplementary Material. Figure 3 was designed to display only genus-level information for visualization purposes and readability. Specifically, the figure represents the relative frequency (%) of each identified genus within the proteins assigned to the family Acetobacteraceae at each sampling point (S1-S4). Within this genus-level representation, Komagataeibacter is the predominant genus across the acetification process.

Line 359: In Figure 6, which two groups were compared to calculate the fold change?

We thank the reviewer for this valuable comment. The fold change values shown in Figure 6 were calculated from the comparison between sampling points S1 and S4, which represent the beginning and the end of the acetification cycle, respectively. This information has been clarified in the revised figure legend and in the corresponding section of the manuscript.

Line 436: Improve the clarity of the images (Figure 8 and 9).

The image clarity has been improved, as the reviewer noted.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors evaluated the functional dynamics during verdejo wine　acetification by quantitative metaproteomic characterization of acetic acid bacteria. Overall, the manuscript was planned well, and it also demonstrated good innovation. The current manuscript contains valuable information for publication, but some revisions were needed. Here are some specific comments.

• Some important parameters of results must be included in Abstract.
• L49-51, The authors cited a large number of literature and I suggest providing some research examples.
• L104-109，The microorganisms used in this research was not found, and it should be emphasized.
• Figure 6 is beautiful, but the interpretation is not deep enough. For example, the author points out that upregulated proteins are enriched in "arginine biosynthesis" and "membrane protein insertion", but does not provide a detailed explanation of why these pathways are involved.
• Figure 12: This diagram is highly valuable, but its presentation is not good. Please clearly indicate in the caption or text whether all proteins shown in the figure were detected in this study? For example, certain enzymes in the TCA cycle may not be detected (marked in blue or gray).

Author Response

The authors evaluated the functional dynamics during verdejo wine acetification by quantitative metaproteomic characterization of acetic acid bacteria. Overall, the manuscript was planned well, and it also demonstrated good innovation. The current manuscript contains valuable information for publication, but some revisions were needed. Here are some specific comments.

• Some important parameters of results must be included in Abstract.

We thank the reviewer for this valuable comment. The Abstract has been revised to improve the presentation of key quantitative results and relevant parameters.

• L49-51, The authors cited a large number of literature and I suggest providing some research examples.

We thank the reviewer for this valuable suggestion. The sentence has been revised to include representative examples of acetic acid resistance mechanisms described in the cited literature.

• L104-109，The microorganisms used in this research was not found, and it should be emphasized.

We thank the reviewer for this valuable comment. The description of the microorganisms used in the study has been clarified and emphasized in Section 2.2 (Microorganisms). Specifically, we now indicate that the inoculum consisted of a resident mixed microbial consortium obtained from an industrial active fermentation tank.

• Figure 6 is beautiful, but the interpretation is not deep enough. For example, the author points out that upregulated proteins are enriched in "arginine biosynthesis" and "membrane protein insertion", but does not provide a detailed explanation of why these pathways are involved.

We thank the reviewer for this valuable comment. The interpretation of Figure 6 has been further expanded in the revised manuscript.

 

• Figure 12: This diagram is highly valuable, but its presentation is not good. Please clearly indicate in the caption or text whether all proteins shown in the figure were detected in this study? For example, certain enzymes in the TCA cycle may not be detected (marked in blue or gray).

We thank the reviewer for this valuable comment. The information distinguishing detected and non-detected proteins was already included in the graphical legend and figure caption. However, to improve clarity, the caption has been revised while maintaining the original structure. Specifically, it now more explicitly indicates that red symbols correspond to proteins experimentally detected in this study, whereas blue symbols represent pathway components that were not detected in the present dataset. The meaning of the abundance triangles has also been clarified.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

None.

Reviewer 3 Report

Comments and Suggestions for Authors

accept