The Draft genome sequence of Bacillus subtilis strain S2 Isolated from fermented bamboo drink.

  1. Priyanshi Mahendrakumar Jain
  2. Athira Cheruvari
  3. Helga Maria Tamilselvan Jenifer
  4. Rajagopal Kammara

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Abstract

This work reports the draft genome sequence of the fermented food probiotic Bacillus subtilis strain S2, which has antibacterial activity against Micrococcus luteus, Vibrio harveyi, Staphylococcus aureus, Listeria monocytogenes, and Streptococcus mutans. From the de novo assembly, a genome with 4,438 open reading frames and an approximate size of 4,208,679 bp was obtained. Phylogenetically, B. subtilis S2 and B. subtilis SRCM103571 are closely related. The organism is known to produce two bacteriocins (uniqueness), Sublancin and Subtilosin A, as per genome analyses by antiSMASH and BAGEL-4.

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  1. Access Microbiology

    Thank you very much for submitting your revised manuscript to Access Microbiology and responding the reviewer comments. We appreciate your time to improve the manuscript. After two rounds of revision, there are still methods and results that are mentioned in the text but not provided in full, such as Gram stain and endospore stain (L96-97). There are claims about uniqueness of the isolated strain because it produces two bacteriocins instead of one, despite this has been previously reported (10.1016/j.lwt.2021.112854). Furthermore, there is no evidence provided showing this Bacillus strain produces these bacteriocins (they are encoded in the genome, but they might not be synthesised). Additionally, there are claims on the antimicrobial activity of this strain against pathogens, and although explicit evidence of this was requested in the previous round of review, it is still not shown (neither methods or results of antimicrobial sensitivity testing). Reviewer requests on providing genome functional annotation data (GO, KEGG) were either dismissed (despite providing this type of information is standard practice) or accepted in the response to the reviewers, but not provided in the revised version of the manuscript (Table S3). Altogether, these considerations compromise the soundness of this work. For this, we have decided to no longer consider your manuscript for publication. I hope this does not discourage your from publishing with Access Microbiology in the future.

  2. Version published to 10.1099/acmi.0.000810.v3 on Access Microbiology
  3. Access Microbiology

    Thank you very much for submitting a revised version of your manuscript to Access Microbiology. We appreciate your efforts for summarising your work to make a genome announcement (however I would suggest that the work could be trimmed even more, removing conclusions that go beyond the description of the genome, its features and the source strain). The same reviewers that assessed your initial submission have provided comments on this new version and raised important concerns about it, which means the manuscript will need major revisions. Please pay special attention to all the concerns reported by the reviewers, they specially stress that the interpretation of the number of complete/partial rRNAs in this strain requires further explanation and re-assessment. Additionally, as per the Open Research nature of Access Microbiology, we do not allow authors to provide results as "data not shown". Please provide evidence of the antimicrobial properties of the strain presented in this work against pathogens or remove references to this conclusion from a future revised manuscript. Please provide a revised version of the manuscript (including a tracked-changes version) and a point-by-point response to the reviewer comments within 2 months

  4. Access Microbiology

    Comments to Author

    The author has shifted the focus of the manuscript from comparative genomics to announcing the draft genome of a Bacillus subtillis strain, which was one of the main requirements for changing from version 1. However, there are still some important modifications needed for publication: Major issues: * Lines 147-149: You state that Sublancin and Subtilosin A give your strain "unique characteristics," but in the introduction (Lines 45-46) you mentioned that "Apparently, B. subtilis is a producer of several bacteriocins like Subtilin, Subtilosin A, Sublancin, Betacin, and Mersacidin." Why then are these considered unique characteristics of your strain? Have you found these bacteriocins in other strains from your comparative analysis? * Lines 155-164: All the data described in these lines, such as GO annotation, KEGG pathway analysis, and CARD results, should be included as supplementary materials. * Lines 179-181: The authors claim that this B. subtilis strain possesses "one complete rRNA and eight copies of partial rRNAs (3 copies of 16s and five copies of 23s)," but how can this be guaranteed in a draft genome? Are the partial rRNAs at the ends of contigs? If so, the other genes could be in the gaps of the draft genome which remain unassembled. * Figure 1C: This figure should be independent, and the legend should explain what each ring corresponds to. I see three rings with GC skew and two with GC content. Why? Minor issues: * Line 20: "Some Bacillus spp. is used as a primary source…" should be changed to "Some Bacillus spp. are used as a primary source…". * Lines 58-59: The findings mentioned in this sentence are unclear, as is how they helped in B. subtilis isolation: "As a result of these findings, we were able to isolate B. subtilis from fermented bamboo beverages." * Lines 83-86: It is overly ambitious to state that comparative genomics with ten strains will allow for an understanding of genetic diversity in B. subtilis. Please revise this sentence. * Lines 88-91: Include data to support your statement. Also, clarify whether B. subtilis is the target of the other species or vice versa. * Line 107: The oligonucleotides used for amplifying and sequencing 16S should be indicated. * Line 152: Explain what is meant by CARD characteristics. CARD is a database of antibiotic resistance genes.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  5. Access Microbiology

    Comments to Author

    Thank you, authors, for this revision. Most major concerns have been aptly addressed. However, I have these two major concerns that fall in the literature analysis or discussion sections Line 180-185 "…is that it has only one complete rRNA and eight copies of partial rRNAs (3 copies of 16s and five copies of 23s)" Once again, I would not make this conclusion as how can you demonstrate that these numbers are not due to genome fragmentation. In fact, in your rebuttal you failed to do so. The PGAP pipeline cannot tell if that an rRNA gene is partial, not because it is actually partial in the bacterial chromosome, but because it is partial because it has been split over contigs during assembly. So you cannot use these annotation statistics as your basis for the partial rRNA fragments. Typically, rRNA genes are not partial and of an equal number (i.e., no of genes encoding 5s=16s=23s): when they are partial it is because of the quality of genome assembly (high vs low fragmentation). Please read up on bacterial rRNA operons and provide evidence of rigorous studies that show a partial rRNA gene in nature. Otherwise, I cannot approve this manuscript with its conclusions on the number of rRNA genes since the authors have failed to demonstrate that these numbers as well as the concomitant partiality rRNA genes, are not an artefact of genome assembly. Line 44 Bacteriocins are narrow spectrum, they are not broad spectrum. Please change this. Minor concerns I have the following concerns which include many that you may state as too detailed, yet such nuances are what distinguish the calibre of a paper. So, I would encourage you to attend to them all. Abstract Line 15 Unless you assembled a circularised genome, perhaps replace the word "chromosome" with "genome" as the former suggests that the assembly is not composed of contigs. Line 18 Replace "according to our investigations" with "as per genome analyses by antiSMASH and BAGEL-4". Introduction Line 30 Replace "catalase-positive bacteria" with "catalase-positive bacterium" as you are referring to the singular and not the plural form in this sentence. Line 36 Replace "is used as primary" to "are used as primary" as you have referred to many species by using the spp. abbreviation. Do the same change for line 41. Line 45 Remove "apparently" unless you do not trust the study you are citing in this sentence. Line 48 For accuracy, replace the current sentence with "High quality whole genome assemblies provide the raw data for accurate taxonomic classification and analyses of possible…" as sequencing alone does not guarantee any of what you stated; it merely provides the raw data for apt analyses that then can guarantee these benefits, and only when the resultant assembly is of high quality. Line 49 & 50, 51-54 Improve the grammar. Line 54-56 These sentences are ill-placed and distort the flow of the paragraph without adding vital information. I would delete them and continue from "Our interest…" Line 57 For accuracy and clarity, I would replace the cognate phrase with "… from our desire to isolate bacteriocins that are active against drug resistant bacteria, such as Staphylococcus aureus" Line 58 You need to first state the full name of S. aureus in the main text before you can abbreviate the genus epithet. Line 59 For clarity, I would replace the current last sentence of the paragraph with "Therefore, we isolated B. subtilis strain S2 from fermented bamboo beverages" Announcement section Line 79 For conciseness, I would remove "offered significant" Line 83-86 Delete lines 83-86 as that is not the main finding/focus of this study, as previously mentioned in the initial review of this study. Line 88 For clarity and accuracy, rephrase the start of the sentence with "The strain we isolated, B. subtilis S2, produced compounds that were inhibitory to both…" Lines 91-95 I would recommend deleting these lines to remove/deemphasize the aspects of comparative genomics as this was a very minor aspect, as previously discussed in the first review of this study. Line 98 Perhaps rephrase to "a sterile container under aseptic conditions". Line 100 Improve grammar by using the correct singular/plural form. Throughout the text, please note, bacteria are the plural form while bacterium is the single form. Make the appropriate changes in lines 100-108. Line 105 Poor grammar. Please rephrase from "Hence…" Line 106 For clarity, restate to "their 16s rRNA genes were characterised" or "using 16s rRNA molecular identification techniques" or something similar. Line 115-117 This is poorly written, and the terminologies used are not sufficiently descriptive. An example of better terminology would be " the obtained 16 rDNA sequences were queried against the NCBI Genbank database via nucleotide BLAST program". Please rephrase. Line 120 Please add the reference in regards to the phrase "as previously described". Line 125 Delete the reductant phrase "the genome was assembled using Unicycler v. 0.4.8 after sequencing on the Illumina HiSeq platform," Line 130 is missing some data Line 132 I would delete the following as it is ill-placed (methods section) "since the genome contains no plasmid, the threat of transferring resistance genes to other bacteria is substantially reduced" Line 146 I would remove aspects of examining nine genomes as you have only given results for the examination of one genome, that is S2. Line 152-154 Use better terminology and rephrase these lines for clarity. Line 155 I would restate as " ...tree showed that strain S2" Line 156 Poor word choices. I would say "Overall genome-relatedness indices were estimated using ANI and GGDC analyses" Line 157-160 Make them less redundant. Line 168 Clarify or delete the following phrase "thus persisting against various antibiotic-resistant bacteria and combating bacterial infections" Line 169 Poor word choice in "ensured". A better choice is revealed/showed/demonstrated Line 173 "be persistent to antibiotic resistance" is a poor choice of words. Perhaps use "persistently/continually resistant antibiotics". Also, please explicate in this section why the capacity to continually evolve antibiotic resistance is beneficial to probiotic candidature. Line 177 Rephrase "which is normal as most of the genomes analysed range" to which is within the normal range for B. subtilis strains. Lines 185-186 Delete these lines as you have already stated the close phylogenetic relationship between these two in lines 155. Perhaps add the reference to figure 1 here/delete lines 155 and retain these lines. Line 189 For clarity and accuracy, perhaps rephrase to "furthermore, we showed that strain S2 encodes resistance to numerous antibiotics". Presentation of results Lines 350 Poor terminology. Perhaps rephrase to "Genomic loci that encodes the production of Sublancin…" Line 351 Poor terminology; rephrase to "various genes required for their production" Figure 1A Remove the .faa and number at the beginning from each of the taxa names. Also, write the taxon names in the correct format for binomial nomenclature. For example, change "Bacillus subtilisKH2" to "Bacillus subtilis KH2" Make the lines of the phylogenetic tree thicker. This is easily done in MEGA. Alternatively, export the newick tree from MEGA and reedit the tree in easy-to-use online sites such iTOL so as to improve the appearance of the figure. Also, show use a consensus tree; present a tree with actual branch lengths and add a scale bar. Figure 1B is much better than that given in the first draft For a better look perhaps place the title "Sublancin" to the in the same X axis as the title for Subtilosin A Figure 1C. The font of the names of the genes is too small. Perhaps increase the font.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Version published to 10.1099/acmi.0.000810.v2 on Access Microbiology
  7. Access Microbiology

    Comments to Author

    In this study, the authors isolated a new Bacillus subtilis strain from a fermented bamboo drink. They sequenced, assembled and annotated the whole genome and compared it with other 9 B. subtilis genomes from the NCBI database. The objective of the work is of interest but it is not completely well conducted. I detected several major issues that should be addressed by the authors: 1. The title of the manuscript does not accurately represent its content. The comparative genomic analysis appears to be a secondary aspect rather than the primary focus. Consider revising the title to better reflect the study's objectives or expand the comparative genomics analysis by including a larger number of B. subtilis strains and performing a more comprehensive analysis of the pangenome, including core genome and accessory genes. 2. Line 55: When say "The strain was recognized as Bacillus sp. after the BLAST result." Specify the sequence used for the BLAST analysis that identified the strain as Bacillus sp., as well as the database it was compared against. If the 16S sequence was used, clarify this in the text. 3. Line 65: When say "One of the most recent and very common Bacillus spp. are B. subtilis…". Please, specify whether it refers to Bacillus subtilis being recently utilized in fermentation. Is Bacillus subtilis added to the fermentation or it just grows in the fermentation process without inoculation? Please, clarify. 4. Line 67: When say "Bacillus spp. can even cause diseases in human as well as animal, especially in immune compromised…". Specify which species of Bacillus can cause diseases, as earlier in the text it was mentioned that B. subtilis is generally recognized as safe (GRAS) microorganism. 5. Provide details on why the fermented bamboo drink was chosen for the study, including information on its preparation and whether a commercial brand was used or if it was prepared in-house. You should include this information in the introduction and state why you decided to search new Bacillus spp. in this drink. 6. Lines 104-109: You must indicate the purpose of using each bioinformatic tool. For example, you used CGView (Circular Genome Viewer) but there is not any circular diagram in the manuscript. 7. Specify the sequences and methodology used for the phylogenetic analysis, including whether the whole genome or only specific genes (e.g., 16S) were utilized. 8. Remove the table from Figure 1 (Panel A) and include it as an independent table (Table 1) in the manuscript. 9. Ensure uniformity in the text format within Figure 1 (Panel B) and consider revising it for consistency. 10. In Figure 1 (Panel C). Could you please replace the figure for a new one in which the text in the phylogenetic tree is not stretched out? Please, do not alter the proportions of the image. 11. In Figure 1 (Panel D). The indicated products are protein/enzymes but not secondary metabolites as say in figure caption. Also, they are generic/housekeeping functions (ATP synthases, ABC transporter…) that, despite can be related to the "stress response", are not a special characteristic to highlight from the strain. Are they found also in the other 9 strains? 12. In Figure 1 (Panel D). As you have the annotated genome, you must indicate the ID of the genes that were predicted for each "stress resistance". You could include this information in supplementary material. 13. Thoroughly review the English language usage throughout the manuscript for accuracy and clarity. Minor issues: 1. Replace "circumstances" with a more appropriate word in line 28-29. 2. Clarify the meaning of "in-vitro amplified" in line 83-84, possibly by rephrasing the sentence. 3. Line 122: It is not clear the meaning of the sentence "Our comparative genome analysis began with probiotic attributes,". Rewrite the sentence to make it more comprehensible. 4. Remove the word "shows" from lines 261-266 when describing the contents of Figure 1.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Access Microbiology

    Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments are attached below. They agree that, although the work has the potential to be a valuable contribution to the literature, the manuscript needs major amendments. They show particular concerns on the vague methodological descriptions (specially in the strain isolation protocol) and the excessive attention to minor details, which can be distracting from the main point of the work. They also agree that the title is misleading and should clearly reflect what the study is about, so I recommend that you consider other more appropriate title options. As a major issue, they also recommend that the study is re-framed as a genome announcement rather that a comparative genomics study. Please provide a revised version of the manuscript (including a tracked changes document) along with a point-by-point response to the reviewer comments within 2 months.

  9. Access Microbiology

    Comments to Author

    The authors isolated a putative Bacillus bacterium from a traditional bamboo drink. They then sequenced and assembled its high-quality draft genome, that was of low contamination and high completion. This genome opens up the use of advanced biotechnological tools in the traditional cuisine industry, and hence is of importance. However, the authors fail to present this finding clearly, mainly because of focusing on minor findings for which their data lacks sufficient well-presented support. Moreover, the specific cultivation step used to identify Bacillus from the bacterial community that I presume was present in bamboo drink is missing; without this description, the findings are not reproducible. Other crucial methods are also missing, the details of which are given herein. I can thus recommended the acceptance of this manuscript for publication, only upon major revision of the manuscript to focus on the sequencing, assembly, and species delineation via overall genome relatedness indices of strain S2, as well as rewriting major parts of this paper in a higher level of English and clarifying multiple parts of methodology. General comments The use of better word choices is recommended. For example, "is phylogenetically closely related" instead of line 17; a genome only encodes, it does not produce (line 18); "fewest rRNA operons among genomes analysed" instead of line 20. In line 28, I would use "niches" instead of "circumstances" "industrial sectors" instead of "factors" in line 33 and in line 61, I would use "most common" instead of "very common". In line 75, I would use "we conducted comparative genomics analyses" instead of "followed comparative genomics", In line 101, I would use "determined" instead of "understood". These are just a few examples. Please rewrite the entire manuscript avoiding poor word choices. Moreover, several sentences are poorly written, consider rewriting them to make them clearer, less redundant and more cohesive with the rest of succeeding/preceding sentences: line 20, lines 44-45, line 49, line 52, line 55, line 61, lines 73-75, 75-77, lines 80-83, lines 110-112, lines 115-116. Title: It is completely misleading as this paper primarily describes the isolation, genome sequencing and assembly of single putative Bacillus bacterium. The aspects of comparative genomics in this study are minimal. I would recommend changing the title to something similar to what the authors used for their NCBI Genbank master record page "Isolation of a Bacillus subtilis bacterium from fermented bamboo shoots and sequencing and assembly of its genome" Abstract I would recommend replacing lines 11-15, with more background information on strain S2. Explicitly state types of analyses (for example, illumine short read sequencing, short read assembly by Spades, genome annotation by Bakta, phylogenomic and secondary metabolite production analyses) as this is a genome analysis study. Line 18. Avoid undefined abbreviations in the abstract e.g., "WGS". Moreover, genome assembly is a better term that whole genome sequence, as the authors have repeatedly used the former term in the rest of the paper. Announcement section. Lines 65-67 are inaccurate statements. Bacillus subtilis subp. subtilis was last validly described in 1999 (https://lpsn.dsmz.de/subspecies/bacillus-subtilis-subtilis). It is not a recently described species. Lines 69-71 would have been better used to summarise research studies that highlight specific biotech applications of Bacillus subtilis that indeed benefited from its genome data, instead of general industrial uses of Bacillus. For example, https://doi.org/10.3389/fmicb.2021.714449, https://doi.org/10.3390%2Fijms23094853, https://doi.org/10.3390%2Fijms23094853, Line 41: the definition of a bacteriocin is partly inaccurate. A bacteriocin is defined by a narrow spectrum of activity and not a broad spectrum of activity. Line 53. Genus names should be in italics as they are Latin, i.e., Bacillus sp. instead of Bacillus sp. Methodology Bacillus isolation I suspect that Bacillus subtilis is not the only bacterium that is found in bamboo drink. If so, what steps were taken to specifically single out Bacillus bacteria from the other bacteria that would also be cultivated from the bamboo drink? Was there a differential or selective media used for the identification of Bacillus subtilis? Was this done by morphological characterisation? Without this description, the study cannot be reproduced. Lines 89-92: What was the purity 260/280 of DNA used for sequencing? What was the insert size used? Was it paired end sequencing? How many megabases of the genome was sequenced? In the NCBI Genbank master record you have stated the 1214x depth and the genome is 4.2MB and hence the total number of bases sequenced should be 5.1GB. However, the SRA archive states that the total numbers of bases sequenced was 2GB. Please can you clarify this? Lines 91: It is inaccurate to state that sequences were annotated, prior to assembly. Line 112: The hypothesis that S2 is unique because it has 9 rRNAs is highly refutable. The three rRNA genes occur in operons. Only when one has a genome in which all rRNA operons are intact, can one accurately state the number or rRNA genes in that genome. In your case, only one rRNA gene is complete and three are partial genes. This makes your enumeration of rRNA genes prone to overestimations. Line 115: It more accurate to state, 'in regards to its potential to produce antimicrobials' as no in vitro data on antimicrobial production is given in this study. Line 117: How was the phylogenetic analysis conducted? Lines 118. This conclusion is not supported by the data provided. How does the potential to produce antimicrobials make a bacterium a good candidate as probiotic? Lines 120-121 are unnecessary as there is already a data statement. Results Line 268: Figure 1 is a table. Why is it listed as a figure? The phylogenetic tree is poorly drawn and displays little effort to create a presentable diagram. The antismash representation shows little effort to be creative in making genes diagrams as the authors simply took a snapshot of the antismash output screen. Moreover, the presence of a homologous biosynthetic gene cluster (BGC) does not automatically indicate that the BGC is present in that genome. BLASTp sequence similarities (and concomitant e values) between all orthologs of BGCs and similar gene syntenies are more important and ANTIsmash does provide all this information. These data would have been better support for the hypothesis that strain S2 encodes the production of the aforementioned natural products. Figure 1 D is a table and not a figure. Lastly, the data does not support the hypothesis that this bacterium is Bacillus subtilis as no data from overall genome relatedness indices such as dDDH, has been given.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  10. Version published to 10.1099/acmi.0.000810.v1 on Access Microbiology