Novel Genomes of Sphingomonadales Strains Isolated from Diverse Environments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an interesting and useful topic, focusing on targeted isolation of Sphingomonadales using SPT-based PCR screening across diverse environments. The study is clearly based on extensive experimental work and the authors show strong technical skills. However, the manuscript is very heavy in methods and supplementary material, while the main paper lacks clear figures and visual summaries. A major concern is that almost all figures are placed in the Supplementary Information, including key phylogenetic and genome comparison results. This strongly affects readability and makes it difficult for the reader to evaluate the main conclusions directly from the paper.

The title is clear and informative, but it is quite long and very technical. It could be slightly simplified or made more outcome-oriented, for example by highlighting the recovery of novel strains or the improvement of isolation strategy. This would help attract a broader readership.

The abstract is well written and covers background, aims, methods, and outcomes, but it is quite dense and method-focused. It would benefit from including one or two clear quantitative results, such as the number of isolates obtained, the improvement in true-positive rate with streptomycin, or how many potentially novel species were identified. The phrase “novel genomic space” is interesting but somewhat vague and could be clarified briefly.

The introduction is strong, well referenced, and logically structured. The background on glycosphingolipids and Sphingomonadales is appropriate, and the rationale for targeting the SPT gene is convincing. However, the introduction is relatively long and could be slightly condensed. The specific knowledge gap addressed by this study could be stated more clearly toward the end, followed by a short paragraph explicitly summarizing what this work achieved.

The Materials and Methods section is extremely detailed and technically rigorous, but the level of detail is excessive for the main manuscript. Many subsections, particularly those describing minor protocol modifications, incubation times, and centrifugation steps, would be better placed entirely in the Supplementary Methods. The workflow is sometimes difficult to follow due to very long paragraphs. A simple schematic figure summarizing sampling, isolation, PCR screening, sequencing, and bioinformatic analysis would greatly improve clarity.

The Results section contains important findings but is very text-heavy. In the sections describing isolation from soil, soap scum biofilms, and cyanobacterial microbiomes, key outcomes such as number of isolates, success rates, and selection effects are only described in text. There are no main figures summarizing these results, which makes the section harder to follow. At least one main figure showing isolation efficiency across environments would significantly improve the presentation.

The subsection on streptomycin resistance and colony pigmentation presents one of the most practically useful findings of the study. The improvement in true-positive rate with streptomycin selection is convincing, but again this result is only described in text. Percentages such as 50% versus 72% would be much clearer if shown in a simple figure in the main manuscript rather than remaining purely descriptive.

The whole genome sequencing and comparative genomics section is central to the manuscript, yet almost all supporting figures are placed in the Supplementary Information. Key phylogenomic trees, OrthoANI comparisons, and evidence supporting novelty of species are not shown in the main text. As a reader, it is difficult to evaluate claims of novel genomic space without constantly referring to supplementary figures. At least one representative phylogenomic tree and one ANI comparison figure should be moved to the main manuscript.

The Discussion section is generally well written and balanced. The authors successfully relate their findings to previous literature and provide useful practical insights into isolation strategies. The discussion of PCR false positives is honest and appropriate. However, the discussion could benefit from a short limitations paragraph addressing issues such as primer specificity and culturable bias, and some repetition with the Results section could be reduced.

The Supplementary Information is extensive and well organized, but too much essential data is placed there. The Supplementary section should support the paper, not replace the core Results. A key question for the authors is why all figures are placed in the Supplementary Information, especially when many of them are essential for understanding and evaluating the main conclusions.

The data availability statement is clear and appropriate, but it would help to include a clear main-table mapping strains to genome accession numbers for transparency and ease of use.

Comments on the Quality of English Language

The manuscript is generally understandable and written in acceptable scientific English; however, several sections are overly dense and method-heavy, which affects clarity and flow. Some sentences are long and could be simplified to improve readability, particularly in the Introduction, Methods, and Results sections. Careful language editing focusing on conciseness and structure would help the authors express their findings more clearly and make the manuscript more accessible to a broader readership.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Dear Authors,

the manuscript entitled “Serine Palmitoyltransferase Primers Guide Isolation of Sphin-2 gomonadales Strains Across Diverse Environments” is of interest for the readers and fits to the scope of this journal. The manuscript deals with new designed PCR primers to target the SPT gene of Sphingomonadales for their isolation from different habitats, such as soil, soap scum biofilms, and xenic cyanobacterial cultures, to build a strain library for the study of novel glycosphingolipid chemistry.

 

The Abstract is a good and precise summary of the whole manuscript.

 

The Introduction part gives a comprehensive of the relevance and significance of the topic as well as the current state of the art. However, the statement in lines 35–37 should be more thoroughly explained regarding its relevance for human health.

 

The Material and Methods part is well structured, straight forward and can easily be followed. All methods are explained in detail. However, it is not mentioned how the new

Primers (Tab.1. S1 PB005, PB006, PB007, PB008 “this work”) were designed. Did the authors use ARB; Primer3, MEGA….?

Line 151-160: Detailed description of agarose gel preparation and loading is unnecessary.

Line 222-229: Abbreviations must be defined upon first use, please add strain names within in the appropriate section to the text. E.g. as cross reference to Tab. 3 S1.

Line 232: 1:2 is it meant in the way of µL or volume?

Line 309: Plasmids were nowhere mentioned before…. Please add in the appropriate section.

 

The Results part presents all results in a clear and comprehensive manner.

In section 3.2.1 : Table has no caption and should be presented in landscape format.

 

The Discussion part is much too short and fails to address all isolated Sphingomonadales strains from the different habitats. It must be substantially expanded into a proper discussion, structured as follows: (1) summary of key results (e.g., primer specificity, isolation success rates); (2) comparison with literature on SPT gene function and glycosphingolipid chemistry; (3) implications for environmental microbiology and strain library utility; (4) limitations (e.g., habitat biases, sequencing depth); and (5) outlook for future studies.

 

Please avoid placing two headings directly one after the other without text in between:

2. and 2.1; 2.2 and 2.2.1; 3.and 3.1; 3. and 3.1 and 3.1.1; ………

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript "Serine Palmitoyltransferase Primers Guide Isolation…" is devoted to designing PCR primers that allowed authors to isolate some Sphingomonadales strains by targeting the SPT gene. The search for producers of biologically active compounds remains relevant, and from this point of view, the reviewed work is of particular interest.

Although the authors found several new glycosphingolipid producers, the article looks like a protocol. The work does not solve any scientific problem; therefore, the results section looks like a list of laboratory manipulations and does not contain a discussion of the results of the scientific problem. For this reason, the conclusion section is also missing.

The advantages of the article are a good introduction and a detailed description of the methods. However, this is not enough for a scientific article. Therefore, the manuscript should be submitted to journals that publish protocols, not scientific results.

The authors also provided a supplementary file containing important information on the phylogeny of the discovered microorganisms. Section 4 of this file contains 12 figures obtained from the analysis of genome sequencing data for the discovered microorganisms. These results can be transferred into the main article. A detailed discussion of these results could enhance the article's value.

Therefore, the article cannot be accepted for publication in its current form and must be revised to meet scientific article standards. Alternatively, it must be submitted to a specialized journal/section that publishes protocols.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors took the comments into account and significantly improved the manuscript. The corrections were made, and the work boasts novelty and provides readers with new findings. The supplementary materials are very impressive. One inaccuracy remains to be corrected. Because the authors revised the main text, they cite 48 sources. However, the reference list retains the same number of references as the first version of the manuscript. The list of references needs to be revised.

Author Response

We apologize for not updating the bibliography with the revision, that mistake has been corrected.