Phylogenetic Incongruence of Cyclic di-GMP-Activated Glycosyltransferase nfrB with 16S rRNA Gene Tree Reflects In Silico-Predicted Protein Structural Divergence in Diaphorobacter nitroreducens Isolated from Estero de Paco, Manila, Philippines
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study addresses an interesting and timely topic, exploring phylogenetic incongruence between the nfrB gene and the canonical 16S rRNA gene, and further integrates structural predictions of the NfrB protein using AlphaFold. This combination of phylogenetic and in silico structural analysis provides new perspectives on the evolutionary dynamics of glycosyltransferases and highlights potential implications for microbial ecology and biotechnology. Overall, the manuscript is original, well-structured, and clearly written. However, I noted several areas that require clarification and revision:
12–14: The method of isolation (EMJH medium) is briefly mentioned, but “soil and wastewater” (line 13) contradicts the Methods section, which says soil + water were collected, then filtered. Please ensure consistency. Including such detail (medium type, location) in the abstract may be too specific, better to summarize (“isolated from wastewater samples in Manila, Philippines”).
40-42: “our understanding of the genetic and molecular mechanisms underlying its ecological resilience and functional capabilities remains sparse” the claim feels somewhat general and more specific reference to previous studies highlighting these knowledge gaps should be provided.
43–48: The role of the nfrB gene in EPS secretion and environmental survival is well explained. However, the statement “EPS facilitates pollutant degradation, including hydrocarbons” could be misleading, since EPS alone does not degrade hydrocarbons but supports microbial communities that do. Please clarify.
53–54: The sentence “functional characteristics of nfrB have been studied with phage interactions and operon-level dynamics within Enterobacteriaceae” is not clear. This should be supported with examples and references.
71–79: The authors describe three sampling sites with GPS coordinates. The inclusion of socio-environmental context (near informal settlements and market) is useful. However, this section would benefit from a short justification for why these sites were chosen.
72: “Sampling Site 2: 14.578055, 120993925” — looks like a formatting error; I think “120993925” should be corrected to “120.993925”.
84-85: The manuscript states that the supernatant was filtered through a 0.22 µm pore size filter. This step requires clarification and supporting references. Since Diaphorobacter nitroreducens is a rod-shaped bacterium typically larger than 0.22 µm in diameter, such filtration would be expected to exclude the organism rather than allow its passage.
86: The EMJH medium is not standard for Diaphorobacter. Please provide a rationale for why EMJH was selected, especially since it is conventionally used for Leptospira species.
87: “10X STAFF” is not explained. Please define abbreviation.
87–89: Incubation for “2–3 weeks” is unusually long; authors should justify this duration and provide references.
99-108: Please state whether DNA purity/concentration was checked.
103-105: storage conditions should be accompanied by information about DNA stability/quality checks.
112-113: Primers for nfrB are provided (good detail). However, they seem unusually long. please confirm sequences and provide reference for primer design.
119-123: authors should clarify whether both nfrB and 16S sequences were submitted to GenBank (with accession numbers).
142–145: AlphaFold 3 and PyMol are clearly described. please provide confidence metrics (pTM, pLDDT) up front. Authors mention later in Results but should reference methodology here.
146: All figures (Figures 1–5) referenced in the Results section are missing from the manuscript.
147–152: The description of colony morphology (opaque white, transparent, translucent) is clear, but the identification still seems weak without biochemical confirmation. Colony color/shape is not sufficient to confirm D. nitroreducens. Please add supporting evidence or explicitly acknowledge the limitation.
157–160: It is good that multiple phylogenetic approaches were used. However, the statement “highly consistent topologies” is unclear. Please clarify whether bootstrap/posterior probability values were all high (>90%), and whether any topological differences occurred.
172–191: The comparison between nfrB and 16S trees is the core novelty of this paper. However, the authors should provide a statistical measure of incongruence. Simply stating “notable differences” is too subjective.
183–185: The claim that nfrB phylogeny suggests horizontal gene transfer should be more cautiously phrased unless explicitly supported by recombination/transfer detection analyses.
193–203: The 16S tree legend is clear. However, the comparison in Figure 3 is oversimplified and branch lengths are ignored reduces phylogenetic resolution. Consider providing both full-length and simplified comparisons.
204–226: Some structural features (β-strands, α-helices counts, molecular mass, charge) seem overly detailed and not clearly relevant to functional interpretation. Suggest moving these raw values to supplementary tables and focusing in the main text on structural features with functional implications.
221–223: “50.26% solvent-accessible surface”, please clarify how this value was calculated (PyMol or another tool?).
239–262: The detailed comparison among clades is useful, but too descriptive. Suggest summarizing in a table (residues present/absent, additional helices, RMSD values) instead of long text blocks.
254–258: The claim that structural differences “may facilitate greater interaction with substrates” is speculative unless docking/interaction analysis was performed. Authors should rephrase cautiously.
264–272: the authors restate the ecological and biotechnological importance of D. nitroreducens and the role of nfrB. While this provides context, much of the content repeats from the Introduction. Suggest condensing and focusing on novel insights derived from this study.
273–295: Good explanation of why EMJH medium was effective, but this entire section reads more like methods justification than discussion. The comparison to LB medium (lines 280–282) should be supported with references specific to Diaphorobacter or related taxa.
309–311: Suggest mentioning that multi-gene phylogenies are widely accepted for bacteria (e.g., MLSA approaches), with a recent reference.
324–332: Applications such as pollutant degradation, engineered strains, or metabolite production are exciting but too speculative given only in silico data. Please qualify these statements as potential future directions rather than conclusions.
333–341: This section nicely ties findings to microbial ecology and biotechnology. However, the authors should emphasize the novelty of their specific study (phylogenetic incongruence + structural predictions of nfrB in D. nitroreducens), rather than broad generalities about glycosyltransferases. I suggest to add a concise statement of what this study contributes uniquely, and what needs to be validated experimentally.
343–349: The authors highlight the incongruence between nfrB and 16S phylogenies. This is indeed the main novelty, but the conclusion could be strengthened by restating whether this observation was statistically supported (currently absent). Without quantitative support, the statement about “distinct evolutionary trajectories” should be framed cautiously.
350–356: The emphasis on conserved binding sites and divergent terminal regions is good, but again it is purely in silico. Please explicitly acknowledge that these findings are based on computational predictions and require experimental validation. Also, the claim that features “offer promising applications in biofilm engineering, pollutant degradation, and synthetic biology” is speculative. Suggest rephrasing as “may offer future applications”.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors discussed phylogenetic incongruence between the cyclic di-GMP-activated glycosyltransferase NfrB and 16S rRNA, Reflecting Predicted Structural Divergence in Diaphorobacter nitroreducens from Estero de Paco, Manila, Philippines. Below are my comments/concerns:
- Figures are not there in the manuscript.
- Some sentences are overly long, making it harder for readers to follow. Break them into smaller, more digestible segments.
- Italicise gene names (nfrB) and keep protein names (NfrB) in regular font to maintain standard conventions.
- Phrases like “environmental, industrial, and biotechnological contexts” appear multiple times. Consolidate similar terms to make the text more concise.
- Computational predictions (Alphafold 3 structure, phylogenetic inferences) are strong, but adding a mention of potential experimental confirmation (e.g., functional assays, site-directed mutagenesis) would strengthen the conclusions.
- The study focuses on structural and phylogenetic analysis but does not examine upstream regulation of nfrB or its expression under environmental stress.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for providing the revised copy of your manuscript. I appreciate that all comments have been carefully considered and addressed, either through corrections or appropriate justifications.
