Genomic Insights into Marinovum sedimenti sp. nov., Isolated from Okhotsk Sea Bottom Sediments, Suggest Plasmid-Mediated Strain-Specific Motility
, Viacheslav Eremeev 1, Evgeniya Bystritskaya 1, Peter Velansky 2, Valeriya Kurilenko 1 and Marina Isaeva 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLine 45–47:Please verify the taxonomic wording and ensure it is technically accurate (e.g., “the family … was proposed” rather than “was created”). Also confirm that reference [5] explicitly supports the specific taxonomic statement you are making.
Line 51–53:The phrase “deep bottom sediments” is redundant/awkward. Consider revising to either “deep-sea sediments” or “bottom sediments from the Okhotsk Sea” (choose one for clarity and consistency).
Line 63–65: After stating that M. algicola KCTC 22095^T is the closest relative, you then refer to M. algicola DG898. Please clarify what DG898 refers to (e.g., a different strain, a genome-sequenced isolate, or another reference genome).
Line 84: For “as described by [11],” please confirm that [11] is a methods reference that clearly covers all assays listed (Gram staining, oxidase, catalase, motility). If it does not, either add the appropriate additional citations or briefly note any methodological deviations.
Line 117–119: GGDC is best known for genome-based relatedness metrics (e.g., dDDH), and the DSMZ phylogenomics pipeline is typically genome-oriented. If you adapted these tools for a single-gene (16S rRNA) phylogeny, briefly describe the workflow (alignment method, trimming, and model/algorithm used). Otherwise, consider replacing this with a standard 16S workflow description (e.g., MAFFT/MUSCLE alignment followed by an ML tree using IQ-TREE/RAxML, with the substitution model specified).
Line 164: Please provide the accession IDs for the genome sequences you retrieved from NCBI.
Line 188–190: If bootstrap support is low, please report the actual ML/MP bootstrap values (in the tree, the text, or the figure caption). Describing support as “low” without values is too vague.
Line 192–193: You report 96.4% 16S rRNA gene similarity to the type strain M. algicola DSM 10251^T, which is lower than the 97.0–97.4% similarities stated earlier. Please reconcile these values and, if M. algicola remains the closest phylogenomic relative, briefly explain that 16S similarity may not mirror genome-scale relatedness in this case. Also ensure genus/species names are italicized and type-strain formatting is consistent throughout.
Line 358: “939 unique enzymes” is ambiguous. Please define “unique” (e.g., unique EC numbers, unique annotated functions, non-redundant proteins after clustering at X% identity, etc.).
Line 360–361: Revise to: “supporting the biochemical test results.”
Author Response
Response: Thank you very much for taking the time to review our manuscript and for your critical feedback on our work.
Comment 1: Line 45–47: Please verify the taxonomic wording and ensure it is technically accurate (e.g., “the family … was proposed” rather than “was created”). Also confirm that reference [5] explicitly supports the specific taxonomic statement you are making.
Response 1: Thank you for your remark. It has been changed to what you suggested.
Comment 2: Line 51–53: The phrase “deep bottom sediments” is redundant/ awkward. Consider revising to either “deep-sea sediments” or “bottom sediments from the Okhotsk Sea” (choose one for clarity and consistency).
Response 2: We agree with your remark. The word "deep" has been removed. Lines: 66, 85, 515 and 546.
Comment 3: Line 63–65: After stating that M. algicola KCTC 22095^T is the closest relative, you then refer to M. algicola DG898. Please clarify what DG898 refers to (e.g., a different strain, a genome-sequenced isolate, or another reference genome.
Response 3. Thank you for your question. This is one of the strains of M. algicola that we refer to and compare with further, since DG898 has a genome with the status of a chromosomal assembly and contains a flagellar chromid for motility. We have added this clarification to the text. Please, check lines 60-63.
Comment 4: Line 84: For “as described by [11],” please confirm that [11] is a methods reference that clearly covers all assays listed (Gram staining, oxidase, catalase, motility). If it does not, either add the appropriate additional citations or briefly note any methodological deviations.
Response 4: We confirm that all assays (gram-staining, oxidase, catalase activities, and motility (the hanging drop method)) were examined according to the methods described by Gerhardt et al. (1994). In addition, we would like to clarify that an oxidase reagent kit was used for testing oxidase activity. Please, look at lines 86-88.
Comment 5: Line 117–119: GGDC is best known for genome-based relatedness metrics (e.g., dDDH), and the DSMZ phylogenomics pipeline is typically genome-oriented. If you adapted these tools for a single-gene (16S rRNA) phylogeny, briefly describe the workflow (alignment method, trimming, and model/algorithm used). Otherwise, consider replacing this with a standard 16S workflow description (e.g., MAFFT/MUSCLE alignment followed by an ML tree using IQ-TREE/RAxML, with the substitution model specified).
Response 5: In addition to the aforementioned servers GGDC and TYGS, GGDC also provides gene phylogenies and similarities from DSMZ single-gene phylogeny server. For convenience, we have added a more precise link: https://ggdc.dsmz.de/phylogeny-service.php in the Methods (Line 138).
Comment 6: Line 164: Please provide the accession IDs for the genome sequences you retrieved from NCBI.
Response 6: The lack of the accession IDs was due to a delay in receiving them from NCBI due to their temporary shutdown; by now they have been received and included in the manuscript.
Comment 7: Line 188–190: If bootstrap support is low, please report the actual ML/MP bootstrap values (in the tree, the text, or the figure caption). Describing support as “low” without values is too vague.
Response 7: The DSMZ Single Gene Phylogeny Server provides results with bootstrap support values above 60%. We agree with the policy. Furthermore, we believe that even the default bootstrap support value of 60% on the server indicates moderate to weak reliability. A more or less reliable confidence level starts at 70%. Therefore, the actual bootstrap value is irrelevant if it is below 70%.
Comment 8: Line 192–193: You report 96.4% 16S rRNA gene similarity to the type strain M. algicola DSM 10251^T, which is lower than the 97.0–97.4% similarities stated earlier. Please reconcile these values and, if M. algicola remains the closest phylogenomic relative, briefly explain that 16S similarity may not mirror genome-scale relatedness in this case. Also ensure genus/species names are italicized and type-strain formatting is consistent throughout.
Response 8: Thank you for your very special question. A detail explanation of this observed fact is provided in the text. Please, check lines 219-226.
Comment 9: Line 358: “939 unique enzymes” is ambiguous. Please define “unique” (e.g., unique EC numbers, unique annotated functions, non-redundant proteins after clustering at X% identity, etc.).
Response 9: The term "unique genes" is used on the Protologger server for genes that are specific or highly characteristic of a particular taxonomic group (clade), which allows one to determine the evolutionary position of new microorganisms.
Comment 10: Line 360–361: Revise to: “supporting the biochemical test results.”
Response 10: It has been revised. Please, look at line 398-399.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors submitted a manuscript that explores the genomic, morphological, and biochemical characteristics of Marinovum sedimenti sp. nov. The description of the new species is appropriate; however, to support plasmid-mediated strain-specific motility, experimental validation is required rather than relying solely on bioinformatic evidence. I therefore suggest that the following comments be addressed:
Introduction
At the end of the Introduction, an excessive summary of the manuscript’s results is presented, which is redundant with the Abstract. It is recommended to reduce the description of the findings in this section and instead expand the biochemical, morphological, and ecological background information on the genus Marinovum.
Materials and Methods
Describe the strategy used for strain isolation and the main characteristics of the study area.
Include references for the analysis of cell morphology, and specify the conditions and growth phase at which the cells were observed.
Indicate the pH at which the biochemical and substrate assimilation tests were performed.
Explicitly state which tests are included in the API 20E, API ID32 GN, and API ZYM kits.
Indicate how many bootstrap replicates were used in the phylogenetic analyses.
Specify whether long and short reads were preprocessed in a similar manner prior to assembly. If not, clearly distinguish the software and pipelines used for each data type.
In this section, state the criteria used for selecting genomes for phylogenomic analyses and provide a supplementary table including at least the species name, genome ID, and completeness.
Describe the strategy used to identify horizontal gene transfer (HGT).
Results
When reporting 16S rRNA gene sequence identity percentages, also indicate the sequence coverage.
It is recommended that the results be discussed in parallel in order to contrast the findings with supporting evidence for the proposed new species.
Provide experimental evidence for the presence of plasmids in the strains, as well as their estimated sizes.
Is there experimental evidence demonstrating that the chromid is responsible for motility? For example, does curing the strain of the plasmid/chromid result in loss of motility, and does transfer of this element to a non-motile strain confer motility? In the absence of such evidence, the interpretation of the results should be more cautious.
Where appropriate, describe the distinction between plasmids and chromids.
Please clarify whether the pangenome analysis included P. aestuarivivens. If so, justify the inclusion of a genus different from Marinovum. It is recommended to focus the results on the analyzed the novel strains and to avoid broader statements about Paraglaciecola unless it is also examined in depth.
Data Availability Statement
Include the accession IDs and links providing access to the genomic data
Author Response
The authors submitted a manuscript that explores the genomic, morphological, and biochemical characteristics of Marinovum sedimenti sp. nov. The description of the new species is appropriate; however, to support plasmid-mediated strain-specific motility, experimental validation is required rather than relying solely on bioinformatic evidence. I therefore suggest that the following comments be addressed:
Response: Thank you very much for taking the time to review our manuscript and for your critical remarks on our work.
Comment 1: Introduction. At the end of the Introduction, an excessive summary of the manuscript’s results is presented, which is redundant with the Abstract. It is recommended to reduce the description of the findings in this section and instead expand the biochemical, morphological, and ecological background information on the genus Marinovum.
Response 1: Thank you for your recommendation. We have added the requested information about the genus Marinovum. Additionally, we have transferred a part of text that illuminates the role of the Marinovum algae strains in the study of multipartite genome phenomena, as well as classification of fla-type clusters, higher to supplement this background information (Lines 50-63). We consider that the brief summary of findings is a vital part of taxonomic papers and we would like to keep it.
Comment 2: Materials and Methods: Describe the strategy used for strain isolation and the main characteristics of the study area. Include references for the analysis of cell morphology, and specify the conditions and growth phase at which the cells were observed. Indicate the pH at which the biochemical and substrate assimilation tests were performed. Explicitly state which tests are included in the API 20E, API ID32 GN, and API ZYM kits.
Response 2: Some more details have been added to the text. Please, look at lines 86-88, 102-104, 116-117, and 119.
Comment 3: Materials and Methods: Indicate how many bootstrap replicates were used in the phylogenetic analyses.
Response 3. It has been fixed.
Comment 4: Specify whether long and short reads were preprocessed in a similar manner prior to assembly. If not, clearly distinguish the software and pipelines used for each data type. In this section, state the criteria used for selecting genomes for phylogenomic analyses and provide a supplementary table including at least the species name, genome ID, and completeness.
Response 4. We specified long and short reads processing tools. Please, look at lines 154-156.
We have expanded this Section with the criteria used for genome selection for phylogenomic analysis and provided a supplementary table. Please look at lines 175-177. The list of selected bacterial genomes is given in Table S1.
Comment 5: Describe the strategy used to identify horizontal gene transfer (HGT).
Response 5: From https://www.sanger.ac.uk/tool/alien_hunter/. Alien_hunter is an application for the prediction of putative Horizontal Gene Transfer (HGT) events with the implementation of Interpolated Variable Order Motifs (IVOMs). An IVOM approach exploits compositional biases using variable order motif distributions and captures more reliably the local composition of a sequence compared to fixed-order methods. Optionally the predictions can be parsed into a 2-state 2nd order Hidden Markov Model (HMM), in a change-point detection framework, to optimize the localization of the boundaries of the predicted regions.
Comment 6: Results: When reporting 16S rRNA gene sequence identity percentages, also indicate the sequence coverage.
Response 6: We have added this information. Please, look at lines 221-222.
Comment 7: It is recommended that the results be discussed in parallel in order to contrast the findings with supporting evidence for the proposed new species.
Response 7: We did not quite understand this sentence. Could you, please, rephrase your comment?
Comment 8: Provide experimental evidence for the presence of plasmids in the strains, as well as their estimated sizes. Is there experimental evidence demonstrating that the chromid is responsible for motility? For example, does curing the strain of the plasmid/chromid result in loss of motility, and does transfer of this element to a non-motile strain confer motility? In the absence of such evidence, the interpretation of the results should be more cautious.).
Response 8: The research goal set for this study was description of a new species. The experimental estimation of plasmid sizes goes beyond the scope of this goal and would require a specialized set of skills and equipment (for example, PFGE systems). However, the complete circular assembly obtained with two sequencing technologies validates both the presence and sizes of these plasmids. The experimental evidence of the pMsK2 providing the motility of strain KMM 9879 in this case can be derived from genome analysis and the electron micrographs (More pictures of KMM 9879 showing its flagella were provided in the Supplementary Materials). The only flagellar cluster found in the genomes of KMM 9879 and KMM 9989T belongs to the fla2 family and is located on the pMsK2 of KMM 9879. It was estimated that ANIs values between KMM 9879 and KMM 9989T chromosome and pMkK1 are 99.9996 % and 99.9977 %, respectively (see picture). This means that they are nearly identical; the only significant difference in their genomes being the pMsK2. As the KMM 9879 strain is motile, and the KMM 9989T is not, it can be safely concluded that this phenotypic difference originates from the composition of the pMsK2.
Comment 9: Where appropriate, describe the distinction between plasmids and chromids.
Response 9: Unfortunately, this question is debatable and goes beyond the scope of a taxonomic paper. To address this, we have sited the work of Harrison, P.W. et al [Harrison, P.W.; Lower, R.P.; Kim, N.K.; Young, J.P. Introducing the bacterial ‘chromid’: not a chromosome, not a plasmid. Trends Microbiol. 2010, 18, 141–148]. To ascertain if extrachromosomal genetic elements found in KMM 9989T and KMM 9879 belong to chromids or plasmids, we have estimated their codon usage. Chromids have the same codon usage as the chromosome, which evidences their coevolution. We have additionally supplemented the genetic characteristic with the indication of core genes that are spread out through the chromids (Lines 302-304).
Comment 10: Please clarify whether the pangenome analysis included P. aestuarivivens. If so, justify the inclusion of a genus different from Marinovum. It is recommended to focus the results on the analyzed the novel strains and to avoid broader statements about Paraglaciecola unless it is also examined in depth
Response 10: We have included P. aestuarivivens in the pangenome to figure out if it contains enough unique gene clusters to distinguish it from the representatives of the Marinovum genus. However, the results were inconclusive as indicated in lines 383–390.Sadly, we could not obtain the culture of Prymorskyibacter aestuariivivens OITF-36T and therefore, we could not verify its taxonomic position.
Comment 11: Data Availability Statement. Include the accession IDs and links providing access to the genomic data
Response 11: The lack of the accession IDs was due to a delay in receiving them from NCBI due to their temporary shutdown; by now they have been received and included in the manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsNo further comments.
Author Response
Comment 1: No further comments.
Response 1: Thank you again for taking the time to review our manuscript and for your critical comments on our work.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis new version addresses the comments and suggestions. However, the authors do not provide substantial evidence of plasmid involvement in mobility. I suggest taking this information with caution and describing the experimental limitations and future directions for characterizing this finding in both the abstract and the discussion. In the title, you may change “reveals” to “suggests,” given the absence of evidence, or something more appropriate.
Author Response
Comment 1: This new version addresses the comments and suggestions. However, the authors do not provide substantial evidence of plasmid involvement in mobility. I suggest taking this information with caution and describing the experimental limitations and future directions for characterizing this finding in both the abstract and the discussion. In the title, you may change “reveals” to “suggests,” given the absence of evidence, or something more appropriate.
Response 1: Thank you for your suggestion. We have added a probabilistic element to the manuscript title and to several sentences in the abstract (lines 29-30) and discussion section (lines 314-316).
