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zytbill

铜虫 (小有名气)

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在线: 34.1小时
虫号: 34009
注册: 2004-01-05
性别: GG
专业: 环境微生物学

[交流] 文章被据了。二个reviewer，一个可以，一个据。已有3人参与

最后一个说英文不好，英文是老板定稿的，我老板是几个杂志的编委，每个月都转些文章给我审稿。改下再投高一点的。

Reviewers' comments:

Reviewer #1: see attached PDF

Title: ” XXXXXX“ Describes differences in protein abundance that may be used to further understand 2-KLG production in G. oxydans and improve facile synthesis of L-AA from glucose. This paper is of interest to the research community, and the results should be published.
The reviewer has three important scientific concerns that require the author’s attention prior to acceptance in this journal:
1. Why wasn’t the exoproteome (i.e. proteins present and increasing in abundance in the culture media) studied? This may more directly lead to interacting products stimulating 2-KLG production in G. oxydans.
2. Why were only 5 of the 11 differentially abundant (and common to both bacteria) analyzed by MALDI-ToF-ToF? Please disclose whether attempts were made (and unsuccessful) or why attempts were not made to identify these (potentially) interesting proteins.
3. Why is spot 1116 (SpoIVB) demonstrating reduced abundance at 36 h post-culture when it is theoretically a protein contributing to sporulation events? Please disclose the specific function and rationale for why this protein is decreasing during peak induction of sporulation.
Lastly, the reviewer suggests some minor corrections in the technical writing to increase readability by the audience:
1. Abstract: Change 12th and 36th to 12 and 36, respectively
2. Remove significance section from body of manuscript
3. Line 67: please change “combined” to “combining”.
4. Lines 255-256: Do the authors mean 50% acetonitrile and 50 mM NH4HCO3?
5. Line 274: What is the “matrix”? There are many MALDI-ToF matrices, please specify which one was used for this study.

Reviewer #2: 2-keto-L-gulonic acid (2-KLG), the key intermediate in vitamin C production, is produced by Gluconobacter oxydans. Co-cultivation with Bacillus strains like B. licheniformis and B. cereus greatly enhance growth of G. oxydans and production of 2-KLG. The aim of the work presented by Jiang and co-workers is to identify possible factors that stimulate growth and 2-KLG production by means of comparative genomics and proteomics approaches.
Main comments:
1. According to the abstract (line 24) and the introduction (line 121) authors performed comparative genomics. Where are the resulting data? Table 1 only depicts number of genes and proteins of different Bacillus species, which is hardly a comparative genomics approach. I would expect a more detailed analysis of genome structure and function. What are the similarities and differences especially between the genomes of B. cereus and B. licheniformis, since these are the strains of interest in this manuscript?
2. The authors show that co-cultivation leads to enhanced production of 2-KLG during the first 18 hours (line 306); but according to Figure 1 the 2-KLG amount reaches its maximum amount after 36 to 42 hours.
3. The authors show further that the supernatants from B. licheniformis and B. cereus cultures alone are sufficient for 2-KLG synthesis (line 315 ff.). Supernatants of cultures after 36 h fermentation of the Bacillus strains yielded highest induction rates of the metabolite. The authors state that at this time point the Bacillus cells are in the stationary growth phase and spore formation was observed. The authors conclude that sporulation might be the reason for the release of the bioactive product by the Bacillus strains (line 425). There are no data presented, however, showing number of spores. How was the formation of spores demonstrated?  Also, a growth curve of the bacterial cultures would help interpretation. Without these data it is hardly possible for the reader to appraise the results. After time point 36 h, when the Bacillus cells enter stationary phase no more 2-KLG is produced. In my opinion, this rather suggests that processes before entering the stationary phase and sporulation stimulate 2-KLG synthesis. In this respect, the title of the manuscript should be reconsidered, because sporulation is never shown.
4. The cytoplasmic proteome of B. cereus and B. licheniformis at logarithmic growth and stationary phase was analyzed to identify possible bioactive products. For this reason 2D gels were prepared. According to the methods section only 2 biological replicates were analyzed (line 219). This is a really low number of replicates and in my opinion it is not sufficient for a statistical analysis. I would recommend at least a third biological replicate to account for biological variances and to use a second method to validate the results from the 2D gel analysis regarding the expression profiles of the five proteins that were analyzed further, for example q-PCR.
5. The proteomic approach revealed that both Bacillus strains show altered protein pattern during log phase and stationary phase. Comparison of the gel pictures of both strains showed that 942 spots appear on the same positions on the respective gel and therefore the protein spots were comparable (line 347). Even with closely related bacterial strains I would be careful to make such a statement. Without identification of the spots one cannot tell if the proteins are the same even if they appear on the same position. Differences in the amino acid composition of the same protein can lead to an altered position of the respective protein spot on the gel when comparing two strains. For further analysis, only spots that are on the same position on the gel were considered. Surprisingly, only 11 proteins showed identical regulation (3-fold threshold) between both strains. Five of these spots were identified by MALDI-TOF MS. I would expect more similarities; an overlap of 11 proteins seems quite small. Since the authors only compare protein spots which appear on the same position on the gels a high number of proteins with identical function in both strains but slightly different amino acid composition (and therefore different molecular weight and/or pI) escape analysis. I would suggest that identification of all proteins with altered amount under the tested conditions would lead to more comprehensive data sets. This would yield an overall-view on the physiological state of the Bacillus cells switching from exponential growth to stationary phase. Knowing the physiology of B. licheniformis and B. cereus would help to identify pathways or enzymes that might be involved in production of bioactive factors
6. In line 398 ff. the authors describe a B. licheniformis  urophorphyrin-III C-methyltransferase with an unusual length. The respective methyltransferase in the exact same position on the B. cereus gel is much shorter with only half the size of the B. licheniformis enzyme. The authors declare possible post-translational modifications as a reason. Can they exclude annotation errors in the database?
7. In the discussion, the authors do not describe how the five cytosolic proteins identified as possible bioactive factors might affect the synthesis of 2-KLG by G. oxydans. In the same section the authors state that they provided a comprehensive proteomic profiling which, according to the presented limited data, is not the case (line 489).
8. Since the supernatant of Bacillus cultures alone can stimulate synthesis of 2-KLG, I would also recommend the analysis of the extracellular proteins to identify possible bioactive factors. Also extracellular metabolites produced by both Bacillus species should be considered as possible factors and therefore should be subjected to analysis. A chinese publication of Feng and coworkers, 2000 (S. Feng, Z. Zhang, C. Zhang, Z. Zhang Effect of Bacillus megaterium on Gluconobacter oxydans in mixed culture Ying Yong Sheng Tai Xue Bao, 11 (2000), pp. 119-122) described a similar co-cultivation experiment (G. oxydans cultivated with B. megaterium) where bioactive compounds for the stimulation of 2-KLG synthesis were found in the supernatant of B. megaterium cultures. With this publication in mind - why didn't the authors focus on the extracellular proteins as well? In my opinion this would be the more promising approach.
Minor comments
Fig 1 and  2 should be presented with similar layout (color schemes for both strains are confusing, because they are swapped between both diagrams), labeling of axes showed be in identical fonts.
Fig 5 very good quality gels,  but for a better visualization of differences and interesting spots I would prefer false color overlay images which should be possible with the Image software,
In line 211 the authors describe analytic and preparative gels and that the analytic gels were stained with silver nitrate. What staining method was used for preparative gels?
Line 253:  "Detected spots in non-stained gel areas were excised…" I don't understand what the authors mean. How were spots in non-stained gels detected?
Line 289: The authors used the non-redundant NCBI database for protein identification. Why did they not use a Bacillus specific database?
The English language should be checked by a native speaker.
Knowledge on stimulation factors of the 2-KLG synthesis will help to optimize its production process. Therefore this is an important research field. However, for a successful publication I advise the authors to conduct additional experiments as mentioned above.

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