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by
  • Tae Gyun Kim †,
  • Chaeyun Hong † and
  • Seoung Rak Lee *
  • et al.

Reviewer 1: Anonymous Reviewer 2: Hidayat Ullah Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review highlights the genomic landscape, ecological functions, and specialized metabolites of the Roseobacter group. It was well prepared and structured. I only have minor comments.

  1. 2.1. Taxonomic Position and Representative Genera: I think this part is not up to date. I can't get a framework on the newest taxonomic diversity of Roseobacter clade/Roseobacteraceae. To my knowledge, a paper in 2023 (https://doi.org/10.1099/ijsem.0.006156) showed the diversity of Rosebacteraceae with the biggest dataset up todate. The authors can also refer LPSN (https://lpsn.dsmz.de/domain) and GTDB (https://gtdb.ecogenomic.org/) to improve this section. Roseobacteraceae and Paracoccusaceae are merged in GTDB, but the diversity of the genus Roseobacter can be introduced.
  2. Many bacteria of this group are red or orange because of producing carotenoids. Carotenoids are also important for survival and association with other organisms. Can the authors introduce in relevant sections?

Author Response

Thank you for your comments. 

Reviewer #1: This review highlights the genomic landscape, ecological functions, and specialized metabolites of the Roseobacter group. It was well prepared and structured. I only have minor comments.

Thank you for the positive assessment and for the helpful suggestions!

 

  1. Please update Section 2.1 using the recent phylotaxonomic framework, LPSN, and GTDB. Clarify the treatment of Roseobacteraceae and Paracoccaceae in GTDB and introduce the diversity of the genus Roseobacter.

We revised Section 2.1 to distinguish the Roseobacter-Group from the type genus Roseobacter and to update the taxonomic context. We added the 2023 phylotaxonomic assessment of Roseobacteraceae and Paracoccaceae (Ref. 15), retained LPSN as the nomenclatural framework (Ref. 16), and explicitly identified GTDB R11-RS232 (Refs. 17 and 18). The revised text states that the type genera Roseobacter and Paracoccus, representing Roseobacteraceae and Paracoccaceae in LPSN, respectively, are both classified within Rhodobacteraceae in GTDB R11-RS232. We clarify that this reflects a broader genome-based family circumscription rather than formal nomenclatural synonymy. We retain the term 'Roseobacter-Group' as an ecological and evolutionary descriptor.

 

  1. Many bacteria in this group are red or orange because they produce carotenoids. Please introduce carotenoids in the relevant sections, including their importance for survival and association with other organisms.

We added a Section 2.2 paragraph on carotenoid production by aerobic anoxygenic phototrophic Roseobacter-Group members, covering light harvesting and protection from photooxidative stress. It cites spheroidenone in Dinoroseobacter shibae and evidence that it reduces singlet-oxygen transfer under phytodetritus-associated conditions, while noting that this does not establish a direct host benefit (Refs. 19 and 25).

Reviewer 2 Report

Comments and Suggestions for Authors

Please check the attached draft for comments / suggestions.

Comments for author File: Comments.pdf

Author Response

Thank you for your comments. 

Reviewer #2: The annotated manuscript requested revisions to the title, abstract, scope, novelty statement, taxonomy, biosynthetic discussion, terminology, and activity tables.

Thank you very much for the detailed annotations and constructive recommendations. We have carefully revised the manuscript as follows.

 

  1. Please modify the title and focus the review on host-associated metabolites rather than natural products in general. Use 'Roseobacter group' consistently and state the host-association organizing framework.

Thank you for your suggestion. The title has been changed to 'Host-Associated Metabolites of the Marine Roseobacter-Group: Chemical Diversity, Biosynthesis, Ecological Functions and Bioactivities.' Throughout the title, Abstract, and main text, we consistently use 'Roseobacter-Group' for the broader ecological lineage.

 

  1. Please revise the abstract to name representative bioactivity classes with examples, add a host-association clause, make the biosynthetic dimension explicit, and focus on structurally characterized metabolites.

The abstract now specifies that the review concerns structurally characterized, host-associated metabolites and gives tropodithietic acid and roseobacticides as antibacterial and algaecidal examples. It also states the host-association framework and explicitly describes the phenylacetate-sulfur and tryptophan-sinapic-acid biosynthetic connections.

 

  1. Please explain why this review is needed now and how it differs from earlier reviews of the marine Roseobacter lineage.

We revised the final paragraph of the Introduction to define the review's scope and novelty. In contrast to earlier broad ecological and genomic reviews, this article focuses on structurally characterized metabolites recovered from host-associated settings or induced by host-derived cues, and it incorporates recent advances in host-cue-based elicitation, genetic pathway-to-product linkage, and structure elucidation from trace quantities.

 

  1. Please describe the relationships between the phenylacetate/TDA/roseobacticide pathway and the tryptophan/sinapic-acid hybrid pathways.

We added explicit discussion of the phenylacetate and sulfur-metabolism network linking TDA and roseobacticide production (Sections 4.1.1 and 4.1.2). We also describe the cooperative use of tryptophan and sinapic acid to elicit the sinacidin, sinatryptin, and sinamicin hybrid metabolites (Section 4.2.1). These connections are now also summarized in the abstract and Introduction.

 

  1. How many predicted NRPS/PKS clusters have a linked product?

Section 2.2 now reports that a PCR-based survey detected NRPS-like and PKS-like fragments in seven and five, respectively, of 22 strains. We now state explicitly that the targeted PCR survey did not reconstruct complete biosynthetic gene clusters or test pathway-to-product relationships and therefore cannot provide a numerical count of product-linked clusters. Thus, the seven and five values indicate biosynthetic potential, not direct metabolite assignments. The igi locus for indigoidine is presented separately as a genetically validated NRPS-like example, not as a product assignment generated by that screening dataset.

 

  1. Please recheck the placement of the Roseobacter group within Alphaproteobacteria, add the missing supporting reference, and standardize capitalization and italics for the genus Roseobacter and other microbial terms.

We updated the taxonomic discussion in Section 2.1, added a global metagenomic reference for uncultivated pelagic lineages, and completed a manuscript-wide taxonomic-formatting check. Roseobacter-Group is now consistently hyphenated, with Roseobacter italicized and '-Group' in Roman type; Roseobacteraceae, Rhodobacteraceae, Paracoccaceae, and affected genus/species names were likewise corrected. Gene symbols were also checked for consistent formatting.

 

  1. Please avoid describing Roseochelin B as potent without an IC50 value, revise the corresponding statement in Table 2, and correct the strain designation DSM 10.

We revised the Roseochelin B description to state that it showed algaecidal activity against Emiliania huxleyi, while noting that an IC50 value was not reported. Table 2 now uses the same wording. We also standardized the DSM 10 strain designation in Table 1. We also corrected the cell-line designation to MCF-10A and describe it as non-tumorigenic human mammary epithelial cells in Section 4.1.1 and Table 3.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This review provides a useful overview of natural products produced by marine Roseobacter bacteria. It covers their chemical diversity, biosynthesis, ecological roles, and biological activities. The manuscript is generally well organized and includes recent studies. However, several points need clarification or correction before publication.

  1. In lines 476–477, the authors refer to Table 4 when discussing the effects of cysteinolides A and B on 5-lipoxygenase (5-LOX). However, these compounds and their activities are not included in the table. Please add the relevant data to Table 4 or remove the table reference. The authors should also clarify that the reported activity was measured in a cell-free enzyme assay at 30 μM.
  2. In line 489, the authors describe DMSP mainly as an algal precursor. Although DMSP is often derived from algae in marine algal–bacterial interactions, some marine heterotrophic bacteria can also synthesize this compound. Curson et al. (2017) demonstrated bacterial DMSP biosynthesis and identified the dsyB gene involved in this process. Please revise this section to acknowledge bacterial DMSP production and clarify why DMSP is excluded from the list of numbered compounds. Reference: https://doi.org/10.1038/nmicrobiol.2017.9
  3. In lines 552–569, the names R. tolerans, R. mucosus, and R. nubinhibens are abbreviated. Since both Ruegeria and Roseovarius are discussed in the manuscript, these abbreviations may cause confusion. Please use the full genus name, Roseovarius, at the first mention of each species in this section. Also, please use different abbreviations for different genera.
  4. Reference 28 lists only Geng and Belas as authors. However, the original publication has five authors: Geng, H.; Bruhn, J.B.; Nielsen, K.F.; Gram, L.; Belas, R. Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters. Applied and Environmental Microbiology 2008, 74, 1535–1545. https://doi.org/10.1128/AEM.02339-07
  5. Compound 43 is marked with an asterisk (*) in Table 1, but the meaning of this symbol is not explained. The structure of this compound has been questioned in a later study. Please add footnote explaining this issue.

Author Response

Thank you for your comments.

Reviewer #3: This review provides a useful overview of natural products produced by marine Roseobacter bacteria. It covers their chemical diversity, biosynthesis, ecological roles, and biological activities. The manuscript is generally well organized and includes recent studies. However, several points need clarification or correction before publication.

Thank you for the positive assessment.

 

  1. Cysteinolides A and B and their 5-lipoxygenase (5-LOX) activities are discussed in the text but were not included in Table 4. Please add the relevant data or remove the table reference, and clarify that the activity was measured in a cell-free enzyme assay at 30 uM.

We added cysteinolides A and B (15 and 16) to Table 4, including their source, 5-LOX target, cell-free assay system, and activity at 30 uM. The text now states that cysteinolide B showed greater inhibition than cysteinolide A at this concentration and that no IC50 value was reported. We further clarify that these data do not establish anti-inflammatory efficacy in vivo.

 

  1. DMSP should not be described solely as an algal precursor because some marine heterotrophic bacteria can synthesize it. Please acknowledge bacterial dsyB-dependent DMSP biosynthesis and explain why DMSP is excluded from the numbered compounds.

We revised Section 4.1.5 to state that, although DMSP is commonly supplied by phytoplankton in algal-bacterial interactions, some marine heterotrophic bacteria can synthesize it through a dsyB-dependent pathway (Ref. 41). We also explain that DMSP is not assigned a compound number because it is treated as a widespread primary osmolyte and central sulfur metabolite rather than a structurally characterized specialized metabolite produced by the Roseobacter-Group.

 

  1. The abbreviations R. tolerans, R. mucosus, and R. nubinhibens may be confused with Ruegeria. Please use the full genus name Roseovarius at the first mention of each species and distinguish abbreviations for different genera.

We now use the full species names Roseovarius tolerans, Roseovarius mucosus, and Roseovarius nubinhibens at their first mention in this section and checked the surrounding text to distinguish Roseovarius from Ruegeria consistently.

 

  1. Reference 28 should list the complete five-author author group for the original publication on tropodithietic acid biosynthesis.

We revised the relevant reference entry (now Ref. 34 after the consolidated renumbering) to include the complete author group: Geng, H.; Bruhn, J.B.; Nielsen, K.F.; Gram, L.; and Belas, R.

 

  1. Compound 43 is marked with an asterisk in Table 1, but the meaning of this symbol is not explained. Please add a footnote addressing the later study that questioned the structural assignment.

We added a Table 1 footnote explaining that the original assignment of compound 43 as cyclo-(Gly-L-Pro-L-Glu) has been questioned and that a cyclic hexapeptide, cyclo-(Gly-Pro-Glu)2, was proposed in a later synthetic study. The text now also states that direct comparison with the natural isolate is still required for definitive structural resolution.