Taxonomic and Genomic Characterization of Niveibacterium terrae sp. nov., a New Species from Riparian Soil in the Seomjin River, Republic of Korea

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work presents a well-structured and detailed study of the taxonomic and genomic characterization of a novel bacterial species, Niveibacterium terrae sp. nov., isolated from riparian soil in Korea. The study effectively integrates phylogenetic, genomic, chemotaxonomic, and physiological analyses, providing strong evidence for proposing a new species. It is relevant to microbial taxonomy, environmental microbiology, and biotechnology, particularly in the context of bioremediation and ecological roles of freshwater bacteria.The ms is generally well-written, and the methodology is sound. However, there are some areas that require clarification, minor revisions, and additional discussion.

Could the authors elaborate on why they chose not to use additional phylogenomic methods such as MLSA or core-genome phylogeny?

How confident are the authors in the species delineation based on ANI and dDDH values alone? Would a concatenated marker gene approach strengthen the argument?

Have the authors considered functional assays to validate the predicted biosynthetic gene clusters (BGCs)?

Do the detected BGCs share homology with known antimicrobial or antioxidant gene clusters in related species?

What specific genes or metabolic pathways suggest that Niveibacterium terrae plays a role in bioremediation?

Could the authors provide experimental evidence or literature support linking Niveibacterium terrae to pollutant degradation or environmental remediation?

How do the fatty acid and polar lipid profiles compare to closely related genera within Zoogloeaceae?

Could variations in chemotaxonomic markers be due to different culture conditions rather than true species-level differences?

Were the sequence alignments checked for quality before tree construction?

Would the authors consider adding outgroups in the phylogenetic trees to improve taxonomic resolution?

Author Response

Dear Reviewer,

We sincerely thank you for your thorough and insightful review of our manuscript. Your constructive comments and suggestions have been invaluable in improving both the quality and clarity of our work. We have carefully addressed each of your points and implemented the requested revisions in the manuscript.

Your feedback has strengthened the scientific rigor and presentation of our findings on the taxonomic and genomic characterization of Niveibacterium terrae sp. nov. Your suggestions have significantly enhanced the overall quality of the manuscript, and we are sincerely grateful for the opportunity to improve our study through your expertise.

We hope that the revisions meet your expectations, and we are grateful for your insightful recommendations and continued support. Thank you once again for your valuable input, which has greatly contributed to strengthening our work.

 

1. Could the authors elaborate on why they chose not to use additional phylogenomic methods such as MLSA or core-genome phylogeny?

→ (Response) Thank you for your critical question. While we did not conduct a multilocus sequence analysis (MLSA) in this study, we performed a comprehensive phylogenomic analysis based on whole-genome sequences. The phylogenetic tree constructed using the Genome BLAST Distance Phylogeny (GBDP) approach (Figure 2) effectively supports the taxonomic position of strain 24SJ04S-2ᵀ within the genus Niveibacterium. This method provided high resolution in distinguishing the strain from closely related species.
Additionally, we ensured that our results were consistent with ANI and dDDH values, which are widely used for species delineation. Given this consistency, we believe that this approach sufficiently supports our findings. Nonetheless, we agree that MLSA or core-genome phylogeny could offer further insights, and we plan to incorporate these methods in future studies.

2. How confident are the authors in the species delineation based on ANI and dDDH values alone? Would a concatenated marker gene approach strengthen the argument?

→ (Response) Thank you for your insightful question. We have high confidence in the species delineation based on ANI and dDDH values, as these metrics are well-established and widely applied in prokaryotic taxonomy. The ANI values between strain 24SJ04S-2ᵀ and its closest relatives (Niveibacterium umoris and Niveibacterium microcysteis) ranged from 74.6% to 74.9%, while dDDH values were between 17.2% and 17.5%. These values are significantly below the respective thresholds of 95–96% and 70%, which strongly supports genomic distinctiveness.
Additionally, the phylogenomic analysis based on genome sequence data confirmed that strain 24SJ04S-2ᵀ occupies a distinct position within the genus Niveibacterium. While we believe these methods sufficiently validate the strain's status as a novel species, we acknowledge that additional approaches, such as concatenated marker gene analysis, could provide supplementary evidence. We plan to explore these methods in future studies to further enhance the resolution of phylogenetic relationships.

3. Have the authors considered functional assays to validate the predicted biosynthetic gene clusters (BGCs)?

→ (Response) Thank you for your valuable comment. We identified ten biosynthetic gene clusters (BGCs) in strain 24SJ04S-2ᵀ through antiSMASH analysis. Among these, the arylpolyene-resorcinol cluster showed 45% similarity to a known antioxidant cluster, and an NRPS-like cluster suggests potential antimicrobial activity. These findings indicate that the strain may produce secondary metabolites with ecological or biotechnological relevance.
However, since the primary focus of this study is the taxonomic and genomic characterization of the novel species Niveibacterium terrae, we were unable to perform functional assays to validate these BGC predictions. We fully acknowledge the importance of such experiments and plan to conduct follow-up studies, including metabolite profiling, antioxidant assays, and antimicrobial activity tests, to further explore the potential applications of these biosynthetic pathways.

4. Do the detected BGCs share homology with known antimicrobial or antioxidant gene clusters in related species?

→ (Response) Thank you for your insightful question. Among the ten biosynthetic gene clusters (BGCs) identified in strain 24SJ04S-2ᵀ, only a few showed notable homology with known clusters. For example, Region 6 (arylpolyene-resorcinol cluster) exhibited 45% similarity to the APE Vf cluster associated with antioxidant activity. Similarly, Region 7 (acyl_amino_acides cluster) displayed minimal homology (6%) with N-tetradecanoyl tyrosine. However, several other clusters, including Regions 1 (NAPAA), 2 (lassopeptide), and 3 (RiPP-like), did not match any known pathways, suggesting that strain 24SJ04S-2ᵀ may harbor novel biosynthetic mechanisms.
Further details regarding the homology and characteristics of these BGCs are provided in Table S2 of the Supplementary Materials. These findings indicate the potential for Niveibacterium terrae to produce unique natural products, which warrants further investigation through functional characterization.

5. What specific genes or metabolic pathways suggest that Niveibacterium terrae plays a role in bioremediation?

→ (Response) Thank you for your thoughtful question. Functional annotation of the genome of strain 24SJ04S-2ᵀ identified several genes and metabolic pathways associated with carbon and nitrogen cycling, which are critical for environmental processes. These include glycoside hydrolases involved in the degradation of complex organic matter and enzymes linked to nitrogen transformations, such as ammonification and denitrification.
Additionally, genes encoding antioxidant proteins were detected, suggesting that the strain has the ability to withstand oxidative stress and adapt to polluted environments. Based on these genomic features, Niveibacterium terrae is likely capable of contributing to bioremediation by facilitating both nutrient cycling and pollutant degradation in freshwater ecosystems. We have incorporated these findings into Sections 3.3, 3.4, and 4 of the manuscript for further clarification.

6. Could the authors provide experimental evidence or literature support linking Niveibacterium terrae to pollutant degradation or environmental remediation?

→ (Response) Thank you for your critical and insightful question. Currently, we do not have direct experimental evidence demonstrating pollutant degradation by strain 24SJ04S-2ᵀ. However, functional annotation of the genome identified genes involved in key processes such as carbon and nitrogen cycling, which are indicative of potential roles in environmental remediation.
Additionally, related bacteria within the family Zoogloeaceae have been reported to contribute to the degradation of organic pollutants in freshwater ecosystems. These studies provide indirect support for the potential environmental functions of Niveibacterium terrae. We plan to conduct experimental assays, including pollutant degradation tests and stress-response analyses, to verify these functions and further explore the ecological role of this novel species in future research.

7. How do the fatty acid and polar lipid profiles compare to closely related genera within Zoogloeaceae?

→ (Response) Thank you for your thoughtful question. We conducted a direct fatty acid analysis of strain 24SJ04S-2ᵀ and its closely related species within the genus Niveibacterium. The predominant fatty acids in strain 24SJ04S-2ᵀ were summed feature 3 (C16:1 ω6c and/or C16:1 ω7c) and C16:0, which are consistent with profiles observed in other Niveibacterium species. However, notable differences were observed in the concentration of C14:0, which was significantly higher in strain 24SJ04S-2ᵀ compared to the reference strains.
For polar lipids, the strain shared key components such as diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) with other members of Zoogloeaceae. However, strain 24SJ04S-2ᵀ was distinguished by the absence of certain unidentified lipids present in the reference species.
Additionally, a review of the literature on fatty acid profiles of genera within the family Zoogloeaceae revealed that common fatty acids, including C16:0 and C16:1, are frequently observed across various members of this family. These findings suggest both conserved and strain-specific chemotaxonomic features in Niveibacterium terrae.

8. Could variations in chemotaxonomic markers be due to different culture conditions rather than true species-level differences?

→ (Response) Thank you for your critical question. We understand that culture conditions can influence chemotaxonomic markers. To minimize such variations, all strains were grown under standardized conditions on R2A agar at 30°C until the late exponential phase. Specifically, we ensured that the fatty acid analysis was performed under identical conditions across all strains.
Additionally, we conducted the fatty acid analysis in triplicate to confirm the reproducibility and reliability of the results. The MIDI system was used for these analyses, and the results consistently showed differences in C14:0 content and the absence of certain polar lipids in strain 24SJ04S-2ᵀ across all replicates. Although we believe these findings reliably indicate species-level differences, we agree that further studies under varying culture conditions could provide additional confirmation of the stability of these markers.

9. Were the sequence alignments checked for quality before tree construction?

→ (Response) Yes, sequence alignments were thoroughly checked for quality before constructing phylogenetic trees. Multiple sequence alignments were performed using ClustalW and refined with EzEditor2 to ensure accuracy. Regions with ambiguous sequences were manually inspected and adjusted. These alignments were then used to construct trees using neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods.

10. Would the authors consider adding outgroups in the phylogenetic trees to improve taxonomic resolution?

→ (Response) Yes, outgroups were included to improve taxonomic resolution. Specifically, Achromobacter animicus LMG 26690ᵀ was used as an outgroup in the 16S rRNA gene-based phylogenetic tree. This provided a clear reference point for rooting and helped accurately resolve branching patterns. In future studies, we may explore additional outgroups to further validate phylogenetic relationships.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper is well written. Please attend to the following specific comments.

Line 107. Please mention the DNA polymerase used.

Line 108. Please describe the sequencing method and instrument used.

Line 118. Please describe the library construction methodology.

Line 150. Please include the reference for the TYGS tool.

Line 165. Please cite EzBioCloud

Line 236. Figure 1. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation.

Line 260. Figure 2. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation.

Line 283. Figure 3. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation. Please include a color code for COG categories. Please increase the letter size to 9 points.

Line 294. Figure S1. Please explain why the number of features found by category does not correspond to the pie graph. Vg: no photosynthesis features were found, but a ≈5% “slide” is presented in the pie. The same is true for cell division and cell cycling.

Line 318. Figure 4. Please add the word “putative” before “secondary.” Please increase the letter size to 9 points.

Line 328. Please substitute “Orthologous gene cluster analysis using OrthoVenn3” with “Venn diagram”. Please explain and cite OrthoVenn 3 in the M&M section. Please remove this figure and include it as supplementary material.

Line 333. Figure S2. Please take a better microphotograph and include it as a new Figure 5a

Line 335. Figure S2. Please take a clear photograph of an isolated colony showing morphology and include it as a new Figure 5b

Line 368. Please substitute “%” with “percentage”

 

Line 371. Table 1. Please change numbers 1, 2, and 3 from the column labeled with the species name.

Author Response

Dear Reviewer,

We sincerely appreciate your thoughtful and constructive review of our manuscript. Your valuable comments and suggestions have greatly contributed to enhancing the clarity, quality, and scientific rigor of our work. We have carefully considered each of your points and made the necessary revisions to the manuscript, including improvements to figures, additional references, and clarifications in the Materials and Methods and Results sections.

Thank you for your kind words regarding the quality of our study. Your detailed feedback has been invaluable in helping us present our findings on Niveibacterium terrae sp. nov. in a more refined and comprehensive manner. Your suggestions have significantly improved the overall quality and presentation of the manuscript.

We hope that the revisions meet your expectations, and we are grateful for your insightful recommendations and continued support. Thank you once again for your valuable input, which has greatly contributed to strengthening our work.

 

1. Line 107. Please mention the DNA polymerase used.

→ (Response) We have added information regarding the DNA polymerase used during the PCR amplification of the 16S rRNA gene. Specifically, we used Taq DNA polymerase (Thermo Fisher Scientific), which was selected for its high reliability and compatibility with our sequencing approach. The PCR conditions, including denaturation, annealing, and extension cycles, were optimized to maximize amplification success. This addition has been included in the Materials and Methods section.

2. Line 108. Please describe the sequencing method and instrument used.

→ (Response) We have included a description of the sequencing method and instrument. The sequencing was performed using the PacBio Sequel II platform, which offers long-read sequencing with high accuracy. This approach was chosen to obtain a complete genome assembly, particularly in resolving complex and repetitive regions. A 20 kb SMRTbell library was used to generate high-quality data.

3. Line 118. Please describe the library construction methodology.

→ (Response) We have updated the text to include details on the SMRTbell library construction protocol. This process involved DNA shearing, end-repair, adapter ligation, and size selection. These steps were conducted to ensure the generation of a high-quality library suitable for PacBio sequencing. The updated details are provided in the Materials and Methods section.

4. Line 150. Please include the reference for the TYGS tool.

→ (Response) We have added a reference for the Type (Strain) Genome Server (TYGS) tool, following your request.

5. Line 165. Please cite EzBioCloud.

→ (Response) We have added a citation for the EzBioCloud database using the reference provided by Yoon et al. (2017). The reference has been included in both the main text and the bibliography.

6. Line 236. Figure 1. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation.
7. Line 260. Figure 2. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation.
8. Line 283. Figure 3. Please provide a high-resolution image or, better, a vectorial image to avoid pixelation. Please include a color code for COG categories. Please increase the letter size to 9 points.

→ (6-8. Response) We have replaced Figures 1, 2, and 3 with high-resolution vector-based images in SVG format to prevent pixelation and ensure image clarity. Additionally, Figure 3 was updated with a color code for COG categories, and the font size was increased to 9 points to improve readability.

9. Line 294. Figure S1. Please explain why the number of features found by category does not correspond to the pie graph. Vg: no photosynthesis features were found, but a ≈5% “slide” is presented in the pie. The same is true for cell division and cell cycling.

→ (Response) Thank you for pointing out the discrepancy between the number of features found and the pie chart representation in Figure S1. This issue arises from the RAST annotation process, which includes both fully annotated and predicted features in the subsystem categories. As a result, categories such as photosynthesis and cell division may display small percentages in the chart despite the absence of fully annotated genes in these pathways. We have updated the Results and Discussion section, as well as the legend of Figure S1, to clarify this point.

10. Line 318. Figure 4. Please add the word “putative” before “secondary.” Please increase the letter size to 9 points.

→ (Response) We have updated Figure 4 by adding the word "putative" before "secondary" and increasing the font size to 9 points for better readability. The revised figure is included in the updated manuscript.

11. Line 328. Please substitute “Orthologous gene cluster analysis using OrthoVenn3” with “Venn diagram”. Please explain and cite OrthoVenn 3 in the M&M section. Please remove this figure and include it as supplementary material.

→ (Response) We have replaced the phrase “Orthologous gene cluster analysis using OrthoVenn3” with “Venn diagram” in the figure title and moved the figure to the Supplementary Materials section as requested.

A description of the OrthoVenn3 tool has been added to the Materials and Methods section, along with the following explanation and citation:
"Orthologous gene clusters were identified using OrthoVenn3 [Sun et al., 2023], an integrated platform for exploring and visualizing orthologous data across multiple genomes. The tool facilitates the comparison of shared and unique protein clusters, providing insights into functional specialization among related species."

The updated citation is:
Sun, J., Lu, F., Luo, Y., Bie, L., Xu, L., & Wang, Y. (2023). OrthoVenn3: an integrated platform for exploring and visualizing orthologous data across genomes. Nucleic Acids Research, 51(W1), W397–W403.

12. Line 333. Figure S3. Please take a better microphotograph and include it as a new Figure 5a.

→ (Response) We have captured a higher-resolution transmission electron micrograph (TEM) of strain 24SJ04S-2ᵀ and included it as Figure 5a in the main manuscript. This new image provides improved detail of the cellular morphology at high magnification.

13. Line 335. Figure S4. Please take a clear photograph of an isolated colony showing morphology and include it as a new Figure 5b.

→ (Response) A high-quality photograph of an isolated colony of strain 24SJ04S-2ᵀ has been taken and included as Figure 5b. The image clearly displays the colony's morphology, showing circular, smooth, and cream-colored characteristics after growth on R2A agar at 30°C for 3 days.

14. Line 368. Please substitute “%” with “percentage” 

→ (Response) We have replaced all occurrences of “%” with “percentage” throughout the manuscript, including figure legends, tables, and the main text, to maintain consistency.

15. Line 371. Table 1. Please change numbers 1, 2, and 3 from the column labeled with the species name.

→ (Response) We have updated Table 1 by replacing the numerical labels 1, 2, and 3 with the respective species names to improve clarity. This change ensures that the data is directly associated with each species without ambiguity.