Comparative phenotypic and genomic analysis of the methanogen Methanomethylovorans thermophila L2FAW and its phylogenomic placement within the Genome Taxonomy Database
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The genome of the methylotrophic methanogen Methanomethylovorans thermophila L2FAW is not included in the Genome Taxonomy Database (GTDB) so far, even though the strain was first described in 2005. To evaluate its genomic characteristics and placement in the GTDB, we sequenced the genome of M. thermophila L2FAW via Illumina shot-gun and Oxford Nanopore sequencing and subsequently did hybrid assembly. The assembled genome consists of 2.25 Mbp (contigs ≥ 500 bp) with a G+C content of 37.97%. The quality of the genome is good, which is already apparent from the low L50 (= 1) and L90 (= 2) metrics. Our assembled genome was highly similar to the metagenome assembled genome Methanomethylovorans sp014361205 (GCA_014361205.1_ASM1436120v1_genomic) with an average nucleotide identity (ANI) of 99.9%. Even though KEGG Mapper Reconstruction results revealed that M.thermophila L2FAW harbors all the enzymes necessary for acetoclastic and hydrogenotrophic methanogenesis, and gapseq predicted formate as potential substrate for M. thermophila L2FAW, no metabolic activity could be observed on acetate, H2-CO2 (80:20 vol/vol, 2000 mbar), and on a mixture of H2-CO2 and formate in lab tests thus the obligate methylotrophic lifestyle of the phenotype was confirmed.
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Thank you very much for submitting your revised manuscript to Access Microbiology addressing the reviewer concerns. While the reviewers appreciate the efforts made to improve the manuscript, they have requested further amendments. Please address them in a revised manuscript. Please provide a revised manuscript (including a tracked-changed document) along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
Firstly, I would like to thank and congratulate the authors on incorporating and modifying their manuscript. The minor changes made to the introduction and results sections make the work easier to follow. With regard to the genes used for phylogeny, I was not suggesting the inclusion of a list, but rather a brief comment on the type of genes. This is because, for this type of phylogeny involving multiple genes, it is necessary to select genes with a similar evolutionary rate. In other words, selecting ribosomal subunits (which are highly stable) is not the same as selecting genes that code for membrane proteins. In your case, the vast majority of genes are related to ribosomes, which is ideal and should be mentioned briefly, as this reflects a higher degree of reliability. Nevertheless, I still …
Comments to Author
Firstly, I would like to thank and congratulate the authors on incorporating and modifying their manuscript. The minor changes made to the introduction and results sections make the work easier to follow. With regard to the genes used for phylogeny, I was not suggesting the inclusion of a list, but rather a brief comment on the type of genes. This is because, for this type of phylogeny involving multiple genes, it is necessary to select genes with a similar evolutionary rate. In other words, selecting ribosomal subunits (which are highly stable) is not the same as selecting genes that code for membrane proteins. In your case, the vast majority of genes are related to ribosomes, which is ideal and should be mentioned briefly, as this reflects a higher degree of reliability. Nevertheless, I still believe that a molecular phylogeny should be performed using species from other genera that are closely related to Methanomethylovorans. Using Barrnap + MAFFT + IQTREE is quick and easy. The authors think that this is beyond the scope of their article, but they are proposing a new genome, and a genome-level comparison does not provide good coverage with described species of the genus (even if they are MAGs).
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Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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
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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments and detailed assessments are attached below. They both agree that this is a nice addition to the literature, however they have flagged some important concerns about the replicability of the experimental work, the dataset size and the methodological approch used for the evolutionary analysis (ANI). For these reasons, the manuscript would need to go through major revisions to be considered suitable for Access Microbiology, which may include additional experimental work. I encourage you to provide a revised manuscript addressing the reviewer comments. Please provide a revised manuscript (including a tracked-changes version) along with a point-by-point response to the reviewer comments within 3 …
Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments and detailed assessments are attached below. They both agree that this is a nice addition to the literature, however they have flagged some important concerns about the replicability of the experimental work, the dataset size and the methodological approch used for the evolutionary analysis (ANI). For these reasons, the manuscript would need to go through major revisions to be considered suitable for Access Microbiology, which may include additional experimental work. I encourage you to provide a revised manuscript addressing the reviewer comments. Please provide a revised manuscript (including a tracked-changes version) along with a point-by-point response to the reviewer comments within 3 months.
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Comments to Author
In their manuscript, Wunderer et al. presented their work titled "Phylogenomic placement of Methanomethylovorans thermophila L2FAW in the Genome Taxonomy Database". This article presents the assembly of the species Methanomethylovorans thermophila L2FAW. Subsequently, an evolutionary estimate was derived employing an ANI approach in conjunction with archaeal core genes. Consequently, a functional annotation centered on methane metabolism was conducted. From my point of view, certain methodologies employed by the authors exhibit a paucity of rigor and coherence. Please find attached the majors' comments for your review. 1. The introduction is lacking in clarity and contributes minimal new information. From my perspective, it would be beneficial to have access to the characteristics of the species …
Comments to Author
In their manuscript, Wunderer et al. presented their work titled "Phylogenomic placement of Methanomethylovorans thermophila L2FAW in the Genome Taxonomy Database". This article presents the assembly of the species Methanomethylovorans thermophila L2FAW. Subsequently, an evolutionary estimate was derived employing an ANI approach in conjunction with archaeal core genes. Consequently, a functional annotation centered on methane metabolism was conducted. From my point of view, certain methodologies employed by the authors exhibit a paucity of rigor and coherence. Please find attached the majors' comments for your review. 1. The introduction is lacking in clarity and contributes minimal new information. From my perspective, it would be beneficial to have access to the characteristics of the species analyzed in the article. The following are examples of comments that offer minimal contribution: The National Center for Biotechnology Information (NCBI) Assembly Database serves as a repository for genome-related data. 2. Perhaps it would be useful to compare the genome obtained with those of other species of the genus. For instance, does it have the expected size for a species of the genus? If not, what distinguishes it from the rest? 3. Could you please clarify why the genomes available in the RefSeq database were the only ones used? In the context of this study, the most logical option is to utilize all the genomes available in GenBank, which typically encompasses the RefSeq genomes. In your case, increasing the number of genomes (approximately 20 genomes) would greatly enrich the analysis. 4. Regarding phylogeny, it is rigorous, but the type of genes used for it could be indicated (this could be included in the database introduction). Additionally, I believe that incorporating metadata for select characteristics could enhance the visual presentation. Please indicate whether your species shares characteristics with MAG Methanomethylovorans sp014361205. 5. The incorporation of the ANI metric for genome evaluation is noteworthy. However, it is necessary to include a panel B with the percentage of coverage. It is important to note that presenting 100% identity of aligned sequences with 50% coverage is not equivalent to presenting 100% coverage. It would be advisable to change the colors to a color range suitable for colorblind individuals. 6. It is unclear why the authors chose to utilize Nanopore sequencing information exclusively for genome assembly, given the availability of Illumina data for addressing common errors observed in assemblies using this technology. 7. My primary concern pertains to the ANI supplementary table, which indicates that the coverage percentages are notably low for species within the same genus. A 16S phylogeny should be performed with groups outside the genus to confirm their membership in the genus. 8. There is no conclusions section. I am unaware of whether this is due to the format of this type of article.
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?
Not at all
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
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Comments to Author
Review notes The paper summarises finding from short and long read sequencing and genomic assembly of the methanogenic archaeon Methanomethylovorans therhmophila L2FAW. Methanomethylovorans represents the dominant dimethyl sulfide degrading methanogen in river sediments (not sure if generally or in Netherlands specific). The key findings surround the addition of a genome to and expansion of a potentially under represented genus. A close match (ANI 99.81%) exists within the GTDB and the authors provide an in silico analysis suggesting the strain has the genes responsible for acetoclastic and hydrogenotrophic methanogenesis (the later potentially utilising formate). Both illumina and ONT reads are publically available and assembly methods are detailed enough to validate the assemblies/annotations …
Comments to Author
Review notes The paper summarises finding from short and long read sequencing and genomic assembly of the methanogenic archaeon Methanomethylovorans therhmophila L2FAW. Methanomethylovorans represents the dominant dimethyl sulfide degrading methanogen in river sediments (not sure if generally or in Netherlands specific). The key findings surround the addition of a genome to and expansion of a potentially under represented genus. A close match (ANI 99.81%) exists within the GTDB and the authors provide an in silico analysis suggesting the strain has the genes responsible for acetoclastic and hydrogenotrophic methanogenesis (the later potentially utilising formate). Both illumina and ONT reads are publically available and assembly methods are detailed enough to validate the assemblies/annotations and while the metabolic modelling is not my field of expertise, the authors used published tools. My only concern is the replication of data found in Figure S1, this would need to be replicated at least 3 times to be accepted under ACMI remit. Any negative comments reflect this and not the genome assembly etc. Beyond this its a nice paper reporting and interesting organism. Title: Perhaps include some comment on relevance i.e. to expand reference database? Or perhaps because it is an important or present dimethyl sulfide-degrader? I encourage a snappier title to draw the reader. Abstract: Again, a line indicating importance/relevance would be valuable. While appreciate this is a short communication, you had a reason for sequencing the strain, this would be appropriate to communicate. Is it simply it grew from the sludge? Taxonomic evaluation: As indicated in the aims, I would suggest some/most of the value comes from a more complete genome assembly. I would suggest even including in table S1 showing the difference between/ improvement comparing the closest match. I.e. 53(3) contigs vs e.g. 100(15) etc. Table S2: included "s" e.g. 5s - 16s - 23s. Seems pedantic but might not be clear to non-expert eyes. Figure S1, is this replicated in anyway? - If it was I would include it as primary data rather than supplementary and if it is not, it does not fall within the ACMI robust science remit. Minor comment: Line 47: spelling? - Methanomethlovorans
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly 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
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