Microbially-mediated halogenation and dehalogenation cycling of organohalides in the ocean

Domain: earth_science
Published: 2025-11-27
Editorial outcome: minor_revision

Abstract
Microbially mediated organohalide cycling in the ocean has profound implications for global biogeochemical cycles and climate, but the geographic distribution and diversity of the halogenation-dehalogenation cycling microorganisms remain unknown. Here, we constructed an organohalide-cycling gene database (HaloCycDB) to explore the global atlas of halogenation-dehalogenation cycling microorganisms and genes from 1473 marine metagenomes. Strikingly, 6204 out of 15,252 metagenome-assembled genomes (MAGs) carry organohalide-cycling genes, of which 84.30% are dehalogenating populations. Microorganisms of Pseudomonadota with even spatial distribution dominate both halogenation and dehalogenation potentials in the ocean, in contrast to lineages of Asgardarchaeota and Thermoproteota solely mediating dehalogenation in the Northern hemisphere. Notably, 80.91% of reductive dehalogenase (RDase) genes and 91.35% of RDase-containing prokaryotes represent uncharacterized lineages, substantially expanding known dehalogenation diversity. Further integration of microbial cultivation, protein structure prediction, and molecular docking revealed four unique “microorganism-RDase-organohalide” patterns for marine dehalogenation and its coupling with carbon/sulfur cycles, being distinctively different from their terrestrial patterns. These results advance our understanding of microbial organohalide cycling by providing insights into the halogenation-dehalogenation microbiomes in the ocean.

Introduction
As the Earth’s largest reservoir of dissolved organic carbon, sulfur, and halogens, the ocean is a hotspot for microbially-mediated biogeochemical element cycling, potentially influencing global food webs and climate1–3. In terms of the organic halogen species, over than 8000 organohalides have been identified to date from natural sources, of which the majority originate from marine environments4,5. These organohalides can serve as antibiotics and signaling molecules, profoundly affecting marine community structure and ecological competition5–7. For example, bromo-furanones produced by marine red alga change bacterial quorum sensing systems to inhibit biofilm formation6, and natural methoxylated polybrominated diphenyl ethers (MeO-PBDEs) disrupt endocrine and immune functions of higher trophic organisms through bioaccumulation in marine food webs8. Moreover, ocean-derived and short-lived organohalides (e.g., CH3Br) contribute up to 50% of ozone loss within the Antarctic springtime ozone hole7, suggesting their pivotal role in affecting global climate change. Nonetheless, in contrast to the extensively studied organic carbon and sulfur cycles9,10, investigation on the biogeochemical cycling of organohalides in the ocean is still in its infancy.

The biogeochemical cycling of organohalides is primarily driven by their biosynthesis (halogenation) and attenuation (dehalogenation) processes11, mediated by microorganisms that have evolved specialized enzyme systems4,12. For the halogenation process, accumulating biochemical evidence has revealed four groups of halogenases from phylogenetically diverse bacteria, algae, and fungi to catalyze this secondary metabolism process in marine and terrestrial ecosystems4,13, i.e., flavin-dependent halogenase (FlaHase), vanadium-dependent halogenase (VanHase), nonheme iron-dependent halogenase (NHFeHase), and S-adenosyl-L-methionine-dependent halogenase (SAM-Hase). By contrast, seven groups of dehalogenases, primarily from a small group of bacterial lineages of terrestrial sources, have been characterized to catalyze the dehalogenation of organohalides and mostly in energy-associated primary metabolic ways, including oxidative dehalogenase (OxDase)14, hydrolytic dehalogenase (HyDase)15, reductive dehalogenase (RDase)16,17, glutathione S-transferase (GSTDase)18, methyltransferase (MetDase)19, dehydrochlorinase (DehyDase)20, and halohydrin dehalogenase (HahyDase)21. These halogenases and dehalogenases, together with their host microorganisms and organohalide substrates, can form an extremely complex “microorganism-enzyme-organohalide” network, especially in the ocean with high salinity, oligotrophy, and extreme habitats22. The heterogeneity of microbial growth niches in the ocean23 can promote specialized and diverse “microorganism-enzyme-organohalide” patterns to drive the marine halogenation and dehalogenation cycling of organohalides. These patterns refer to acclimatization and interactions among niche-specific microorganisms, associated enzymatic machinery, and localized organohalide pools. Driven by redox potential and substrate availability, the “microorganism-enzyme-organohalide” patterns could shape the taxonomic and functional diversification for halogenation and dehalogenation of organohalides in ocean, which represent yet-to-be-explored microbial resources of novel organohalide-cycling microorganisms, enzymes, and active chemical species. Nonetheless, our current understanding of the halogenases/dehalogenases, as well as involved microorganisms and organohalides, is mainly based on cultivation and biochemical characterization studies, being largely hindered by the fact that both the medium-derived cultivation bias24,25 and unculturable microbial dark matter26,27 leave the majority of marine organohalide-cycling microorganisms and their functional genes uncharacterized. Moreover, previous studies mainly focused on the organohalide formation or dehalogenation potential of specific terrestrial regions (e.g., paddy soil28 and polluted urban rivers13) or microbial lineages of specific dehalogenation populations (e.g., organohalide-respiring bacteria of Dehalococcoides and Dehalobacter)29, lacking a comprehensive catalogue and characterization of organohalide-cycling microorganisms across the microbial kingdom and global ocean habitats. These limitations greatly impede our comprehension of the microbially-driven halogenation and dehalogenation cycling of organohalides in the ocean.

The culture-independent metagenomics offers opportunities to surmount these limitations30. Large-scale sampling and sequencing efforts (e.g., the Tara Oceans and Global Ocean Sampling expeditions) have generated a vast expanse of genetic resources to explore novel taxa and functions31. At the taxonomic level, novel microbial lineages continually emerge and expand marine biodiversity, including the recently identified uncultured bacterial phylum Candidatus Eudoremicrobiaceae to harbor an exceptionally high abundance of biosynthetic gene clusters for producing organohalides and other natural products32. At the functional level, metagenomic analyses have uncovered previously unrecognized metabolic capabilities of both known and novel microbial populations, exemplified by the identification of photosynthetic genes (e.g., pufLM) in the predatory Myxococcota33. Therefore, advances in metagenomics hold the potential to reveal a full picture of microbially-mediated halogenation and dehalogenation cycling of organohalides in the ocean, which requires orthology databases to support metagenomic analyses. Nonetheless, despite their unquestionable merits, currently available KEGG34 and similar comprehensive databases tend to be biased toward eukaryotes and the metabolism of model organisms for historical reasons35, and lack partial enzymatic routes for the microbially-mediated organohalide cycling, which often results in suboptimal functional assignment and false-positive outcomes35. Consequently, exploring organohalide-cycling microbiomes urges the development of a function-specific database.

To explore the global-scale microbially-mediated halogenation and dehalogenation cycling of organohalides in the ocean, we developed an organohalide-cycling gene database (HaloCycDB) to support organohalide-cycling metagenomic analyses, and analyzed 1473 metagenomes from 506 marine sampling sites to reveal the geographic distribution of organohalide-cycling microorganisms and functional genes at the global scale. Also, the microorganisms and associated functional genes for halogenation and dehalogenation cycling of organohalides were further catalogued to explore novel taxonomic and genetic resources. Additionally, the “microorganism-enzyme-organohalide” patterns for reductive dehalogenation were analyzed and confirmed with protein structure prediction, molecular docking, and laboratory cultivation experiments. To the best of our knowledge, this study provides insight into the halogenation and dehalogenation cycling of organohalides in the ocean, which provides a roadmap for exploring marine organohalide-associated bioresources.

Results
Database development to profile marine organohalide-cycling potential
To facilitate the metagenome-based estimation of microbially mediated halogenation and dehalogenation potentials and to enable the robust projection of organohalide cycling in natural environments, an organohalide-cycling gene database (HaloCycDB) was manually curated to profile the halogenation-dehalogenation cycling genes (Fig. 1a). The HaloCycDB comprises a meticulously curated core database (coreDB) and a full database (fullDB) (Supplementary Fig. 1), containing sequences of 221 functionally-characterized genes and 187,289 representative homologous genes, respectively, and covering all of the four halogenation and seven dehalogenation processes (Fig. 1a). Compared to canonical KEGG, COG and their derivative databases, the annotation accuracy, coverage and sensitivity of the HaloCycDB were significantly improved in profiling organohalide-cycling genes (Fig. 1b), e.g., an accuracy of 99.97%, 79.20% and 70.89%, as well as a sensitivity of 100%, 58.76% and 42.28%, for the HaloCycDB, KEGG and COG databases, respectively. To ensure the gene annotation accuracy in data analyses, both sequence similarity and conserved regions/motifs (≥50% amino-acid/AA sequence similarity, or ≥30% AA sequence similarity plus conserved regions/motifs) were included as key screening parameters to prevent false annotation of halogenation and dehalogenation genes (Supplementary Fig. 1). Notably, with the strict screening criteria, the total abundance of confirmed halogenation and dehalogenation genes was decreased by 32.26–99.97% and 50.98–83.72%, respectively, relative to the widely employed less rigorous criteria (≥30% AA sequence similarity) (Fig. 1d).

To profile the cross-kingdom microbially-mediated cycling of organohalides in marine environments, we performed a large-scale metagenomic assembly on 1473 marine water and sediment metagenomes across the globe (Fig. 1c; Supplementary Fig. 2; Supplementary Data 1). There were a total of 145,897,437 contigs assembled from the 1473 metagenomes, which were employed to profile the halogenation and dehalogenation genes in marine water and sediment microbiomes by the HaloCycDB (Fig. 1d). In contrast to Ascomycota as the major eukaryotic player in dehalogenation, phylogenetically diverse Prokaryotes harbored 98.89% of all organohalide-cycling genes and were dominant players to mediate both the halogenation and dehalogenation processes of organohalides in the ocean (Fig. 1d; Supplementary Fig. 3a). These prokaryotes predominantly employed FlaHase/VanHase halogenases and HyDase/OxDase/RDase dehalogenases to mediate halogenation and dehalogenation, respectively (Fig. 1d; Supplementary Fig. 3a). Based on the HaloCycDB, we further reconstructed a total of 6204 non-redundant prokaryotic metagenome-assembled genomes (MAGs) with organohalide-cycling potential by harboring halogenation or dehalogenation genes (completeness ≥50% and contamination ≤10%; Supplementary Fig. 3b and 3c). Interestingly, the 6,204 organohalide-cycling MAGs accounted for 40.68% of all 15,252 reconstructed MAGs from global-scale marine metagenomes, of which 84.30% and 15.70% were identified to be dehalogenation and halogenation populations, respectively (Supplementary Fig. 3d). These results suggested the widespread presence and consequent potential critical roles of organohalide-cycling microorganisms in marine biogeochemical cycles, which were neglected by previous studies.

Prokaryote-mediated organohalide cycling and their geographic distribution in the ocean
To examine prokaryote-mediated organohalide cycling in the ocean, the 1473 ocean metagenomes were used to profile their halogenation and dehalogenation potentials, as well as their three-dimensional spatial distribution (Fig. 2). Results showed a core group of bacteria dominate the marine organohalide-cycling metabolisms. For example, Pseudomonadota being widespread in marine water and sediment environments mediated 65.73% and 65.14% potential halogenation and dehalogenation activities, respectively (Fig. 2a, b; Supplementary Fig. 4a). In addition, Chloroflexota mediated 5.98% of potential organohalide-cycling activities (Fig. 2a), and shared a similar geographic distribution pattern in the ocean with the organohalide-cycling Pseudomonadota (Fig. 2b; Supplementary Fig. 4b). Notably, Dehalococcoidia of the Chloroflexota mainly involved in FlaHase- and HyDase-mediated halogenation and dehalogenation, respectively, and the well characterized terrestrial obligate organohalide-respiring bacteria (OHRB) of this class (i.e., Dehalococcoides and Dehalogenimonas) for RDase-mediated reductive dehalogenation were absent in the marine water and sediment niches (Fig. 2a). In contrast, Anaerolineae instead of the Dehalococcoidia were major players of the phylum Chloroflexota to host RDase genes for reductive dehalogenation in the ocean29.

There were phylogenetically diverse prokaryotic groups solely mediating dehalogenation in the ocean (Fig. 2a, b), including the sediment-inhabiting and HyDase/RDase-harboring archaea of Lokiarchaeia (Asgardarchaeota) and Bathyarchaeia (Thermoproteota) classes (Fig. 2a, b). Interestingly, compared to their even distribution along the longitude (Supplementary Fig. 4d), these HyDase/RDase gene-harboring Asgardarchaeota and Thermoproteota mainly (>80.73%) gathered in sediment of the Northern hemisphere (0–90°N) (Fig. 2b). In contrast to the sediment-inhabiting and HyDase/RDase-harboring archaea, microorganisms (PCC-6307 order) of Cyanobacteriota were the only halogenating prokaryotes without dehalogenation capability (Fig. 2a) and mainly colonized at the surface ocean (Fig. 2b; Supplementary Fig. 4c), particularly the tropical and temperate regions (45°S–45°N) with appropriate temperature and illumination conditions for their cell growth (Fig. 2b).

Of the organohalide-cycling gene distribution in the overall marine cross-sections, FlaHase and HyDase/OxDase/RDase were predominant halogenation and dehalogenation genes, respectively, accounting for 9.72% and 64.20%/12.61%/6.32% (totally 92.85%) of all potential organohalide-cycling activities (Fig. 2c; Supplementary Data 2). Specifically, in contrast to FlaHase as a dominant halogenase accounting for 76.20% of all halogenase genes, HyDase, OxDase, and RDase as major dehalogenases reached 73.59%, 14.45%, and 7.25% of total dehalogenase genes. The vertical distribution patterns of these organohalide-cycling genes were in accord with their increasing relative abundance along with marine water depth, i.e., 8.63%, 14.38%, 17.81% and 21.29% of the total organohalide-cycling genes in microbiomes of epipelagic (EPI), mesopelagic (MES), bathypelagic (BAT) and abyssopelagic (ABY) layers, respectively (Fig. 2c). In contrast, heterogeneous distribution patterns of these organohalide-cycling genes were observed in the cold seep, hydrothermal vent, trench and other sediment, e.g., 43.04% and 14.31% RDase genes, as well as 10.16% and 39.88% HyDase genes, were present in the cold seep and trench, respectively (Fig. 2c; Supplementary Data 2). Due to the very different biomass of microbiomes in the four marine water layers and the four sedimentary environments, the distribution of these organohalide-cycling genes varied distinctively from the above-described patterns when incorporating the biomass abundance (Fig. 2d). Particularly, in the epipelagic and mesopelagic layers, where micro-biomass abundance accounted for 70.03% of total marine microbial mass36,37, the HyDase and OxDase genes achieved 41.59% and 36.64% of their total abundance in the ocean, respectively (Fig. 2d; Supplementary Data 2). In contrast to the concentration of potential HyDase and OxDase activities in the surface ocean, 49.28% of total RDase genes were gathered in sedimentary environments (Fig. 2d; Supplementary Data 2), which might support the establishment of microbiomes in oligotrophic sediment by converting recalcitrant organohalides into comparatively labile organic matter for downstream microbially-mediated organic metabolisms.

Expanded catalog of organohalide-cycling genes and genomes from global marine water and sediment microbiomes
To uncover the halogenation/dehalogenation gene diversity, we clustered genes encoding major halogenases (FlaHase) and dehalogenases (HyDase and RDase) in marine water and sediment microbiomes. The FlaHase, HyDase, and RDase genes were clustered at 90%, 75%, and 90% amino acid identity, respectively, which were commonly used standards for grouping their functional-like genes38. Based on the functional gene annotations from the strict criteria of HaloCycDB, 32.73%, 53.01% and 80.91% were identified to be unknown FlaHase, HyDase and RDase genes, respectively, greatly expanding the current diversity of halogenation and dehalogenation genes (Fig. 3a; Supplementary Fig. 5). Notably, the ocean-derived FlaHase and HyDase genes were mainly clustered based on their substrate specificities, and showed high coverages of their phylogenetic tree lineages: (1) the ocean-sourced FlaHases evenly covered all major FlaHase groups, including group-2 (PltA/HrmQ/Mpy16-like), group-3/4 (PrnA/KtzQ/SpmH-like) and group-5 (BrvH-like) FlaHases to specifically catalyze halogenation of pyrrole-containing natural products, L-tryptophan and indole derivatives, respectively (Supplementary Fig. 5a); (2) marine HyDases covered all major groups, but were mainly clustered into the group-6 (62.74%) HyDases that degraded haloalkanes (Supplementary Fig. 5b), which together with predominance of HyDases in the overall dehalogenation potential suggested the critical role of halogenated alkanes in sustaining microbiomes by conversion of persistent organohalides into labile organic matter in oligotrophic marine environments39. In contrast to the high coverage of ocean-sourced FlaHases and HyDases, marine RDases formed 6 new clusters (groups 6–11) that were separated from previously reported terrestrial RDases (groups 1–5; Supplementary Fig. 5c). This observation indicated the different evolutionary processes of the terrestrial and marine RDases, which could be partially due to the varied RDase-hosting organohalide-respiring microorganisms in terrestrial and marine environments. Moreover, 95.20% of the marine RDases, lacking a highly conserved twin-arginine motif (Tat) and a small associated membrane anchor protein (RdhB), were different from the terrestrial RDases, which mostly possess Tat and RdhB (Fig. 3a), suggesting that the RDase-based dehalogenation of organohalides proceeds in the cytoplasm of marine OHRB rather than in the periplasm of terrestrial OHRB.

To reveal new microbial populations with halogenation/dehalogenation potential, we clustered the 4430 non-redundant prokaryotic MAGs with a quality score (completeness − 5 × contamination) of ≥50 containing halogenation/dehalogenation genes at multiple taxonomic levels (from genus to phylum; Fig. 3b; Supplementary Fig. 6). The 4430 prokaryotic MAGs were assigned to 60 bacterial and 5 archaeal phyla, with Pseudomonadota (n=1784), Chloroflexota (n=422), Actinomycetota (n=408), Bacteroidota (n=395) and Planctomycetota (n=230) as predominant bacterial phyla, and Asgardarchaeota (n=37) and Thermoproteota (n=29) as dominant archaeal phyla (Fig. 3b). Notably, at the genus level, 48.73%, 82.01% and 91.35% of FlaHase-, HyDase- and RDase-hosting prokaryotes, respectively, were unknown halogenation/dehalogenation populations (functionally novel organohalide-cycling microorganisms) identified as mediating organohalide cycling (Fig. 3c). Especially, the largely expanded diversity of RDase-containing microorganisms represented a vast yet-to-be-explored resource in ocean for potential bioremediation applications. Compared to the HyDase- and RDase-containing dehalogenation prokaryotes, FlaHase-based halogenation microorganisms had larger genome sizes, lower protein-coding density, and a higher proportion of fast growers (Supplementary Fig. 7), which suggested their distinctively different niche-colonizing capabilities and ecological roles40. Interestingly, in contrast to Pseudomonadota as predominant host microorganisms of FlaHase and HyDase with a comparatively small size range (<1000 amino acids; Fig. 3a), both RDases and their host microorganisms were phylogenetically diverse, and the marine RDases were clustered based on the taxonomy and redox-potential-related metabolisms of their host microorganisms: (1) obligate anaerobes, these anaerobic OHRB were capable of sulfate reduction and/or fermentation, and contained small-size RDases (<500 amino acids) that accounted for 78.01% of all anaerobic-OHRB RDases; (2) facultative anaerobes and aerobes, these OHRB were major hosts of the medium- and large-size RDases accounting for 51.14% and 42.72% of all facultative-anaerobic and aerobic OHRB RDases, respectively (Supplementary Data 3).

Structure-based phylogeny of RDases and associated “microorganism-enzyme-organohalide” patterns
Due to the high percentages of novel RDases and functionally novel RDase-hosting microorganisms in ocean, the RDase was selected to investigate the phylogeny of AlphaFold2-predicted structures of marine RDases and associated “microorganism-enzyme-organohalide” patterns (Fig. 4). Notably, marine RDase structures can be clustered into 7 distinct evolutionary groups (groups 1–7, or Gstr 1–7) with varied preferences of host microorganisms and associated growth niches, of which phylogeny is aligned with RDase sequence-based phylogenetic clustering (Gseq), i.e., Gstr 1, 2, 3, 4, 5, 6 and 7 mainly derived from Gseq 11, 5, 10, 4, 1/2/3/6, 7/8 and 9, respectively (Fig. 4a; Supplementary Data 4). For example, group-1 and group-2 RDases are preferentially hosted by aerobic and seawater-originated populations of α/γ-Proteobacteria (Fig. 4b). Moreover, the group-1 and group-2 RDases exhibit larger channel volumes (Fig. 4c; ANOVA, p<0.05) and greater accessible vertices (Fig. 4d; ANOVA, p<0.05) relative to other RDase groups. In contrast, group-3 and group-4 RDases are mainly employed by anaerobic microorganisms (e.g., sulfate-reducing bacteria, SRB) from low-redox sedimentary environments, including the cold seep (Fig. 4b). The groups 5–7 RDases have a comparatively more diverse range of host OHRB relative to the groups 1–4 RDases, and consequently present in all marine habitats (Fig. 4b).

Based on a ligand library comprising 66 representative ocean-sourced organohalides (Supplementary Data 5), the binding energies for 6600 protein-ligand (RDase-organohalide) pairs were calculated to range from 0.59 to −8.51 kcal/mol (Fig. 4e; Supplementary Fig. 8; Supplementary Data 6). The ocean-sourced RDases generally have high affinity for prevalent marine organohalides, including halogenated pyrroles, phenols, and benzenes (Fig. 4e; Supplementary Fig. 8). Moreover, tetrachloroethylene (PCE), as a common dechlorination substrate of diverse RDases has a mean RDase-PCE binding energy of −3.24 kcal/mol, within the dechlorination-active range (Fig. 4e). In contrast, the lower binding affinity of RDases to halomethanes (Fig. 4e) suggests that the dehalogenation of halomethanes in the ocean is probably mediated by other processes rather than RDase-based reductive dehalogenation. Further exploration of the substrate specificity shows an intriguing observation that the molecular size of organohalides significantly affects the RDase-organohalide binding specificity. Specifically, small molecular organohalides (e.g., PCE and halomethane) exhibit RDase-independent binding energies (Fig. 4e; ANOVA, p>0.05). Conversely, the RDase-organohalide binding energy of large molecular organohalides (e.g., halogenated pyrroles) is RDase-specific (ANOVA, p<0.05), e.g., groups 1, 2, and 5 RDases exhibit higher affinities for the large molecular organohalides than other RDase groups (Fig. 4e). To elucidate the underlying mechanism, structural features of RDases have been examined to show that binding energy difference may arise from variations in max accessible vertices of these RDases. Specifically, binding energies of small molecular organohalides (PCE and halomethanes) show no significant correlation with protein max accessible vertices (p>0.05), whereas the binding energies of large molecular organohalides (hexabromo-2,2’-bipyrrole) exhibit a significant positive correlation with the protein max accessible vertices (p<0.001) (Fig. 4f). These observations indicate that the binding energy of large molecules is more easily affected by the enzyme’s channel structure and active site accessibility relative to small molecules.

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_3": "This manuscript presents a comprehensive analysis of the global distribution and diversity of organohalide-cycling microorganisms in the ocean. By combining a newly developed curated database (HaloCycDB), large-scale metagenomic analysis, protein structural modeling, and laboratory cultivation experiments, the authors build a compelling narrative around the microbially mediated halogenation and dehalogenation cycles in marine environments.\nNoteworthy contributions include:\n- The construction of HaloCycDB, a highly specific and validated functional database, which outperforms existing resources (e.g., KEGG, COG) in identifying organohalide-cycling genes.\n- The identification of over 6,000 MAGs with halogenation/dehalogenation potential from 1,473 marine metagenomes, significantly expanding known functional diversity.\n- The demonstration that marine organohalide-cycling is dominated by Pseudomonadota and Chloroflexota, and distinct from terrestrial systems.\n- The use of AlphaFold2-based structure prediction and molecular docking to propose seven RDase structural groups, linked to four ecological models of dehalogenation.\n- The experimental validation of RDase activity through targeted PCE-dechlorinating sediment cultures.\nThese findings are novel and impactful. They offer a foundational reference for future work on microbial contributions to halogen biogeochemistry, and provide a valuable resource for the broader microbial ecology community.\nThat said, I would like to offer a few points for clarification and improvement:\n1. Definition of novelty in MAGs and genes:\nThe manuscript refers frequently to “novel MAGs” or “novel lineages,” but it is unclear whether novelty refers to unclassified taxonomic groups or to newly identified functions within known taxa. Please clarify this distinction throughout, especially in relation to Figure 3.\n2. Functional vs. active roles:\nThe study rightly focuses on genomic potential, but it would be helpful to acknowledge the limitations of this approach. A brief discussion of how future work (e.g., transcriptomics, proteomics, or biogeochemical assays) could address gene expression and ecosystem-level function would enhance the framing of the study.\n3. Ecological integration:\nThe results would benefit from a deeper discussion of how organohalide-cycling genes and microorganisms influence broader marine processes—such as carbon remineralization, sulfur cycling, or microbial food webs. While such integration may be beyond the scope of the present study, recognizing this potential would help position the findings within a larger ecological context.\n4. Data and code availability:\nThe development of HaloCycDB is a major contribution and one of the strengths of the manuscript. However, the database and associated code are currently inaccessible—the link provided appears to be disabled or leads to a non-functional resource. This is a significant concern, as the reproducibility of the analyses and the broader utility of the database depend on open and verifiable access. I strongly encourage the authors to ensure that HaloCycDB, along with the scripts used for gene screening and annotation, are made publicly and reliably available prior to publication.\n5. Figure clarity:\nSome figures—particularly Figure 2a and Figure 3—are visually dense and could benefit from simplified layouts, improved color coding, or alternative formats. Enhancing readability would make the complex results more accessible.\nMethodology and standards: The methodology is sound and well executed. The authors use established approaches for metagenomic binning, functional annotation, and phylogenetic inference. The addition of structure-function modeling (via AlphaFold2) and experimental validation is particularly commendable and uncommon in studies of this scale. The methods section appears sufficiently detailed for reproduction of the core analyses, though confirmation of access to the HaloCycDB pipeline would be essential.\nConclusion: This is an original, high-quality manuscript that makes a meaningful contribution to marine microbial ecology, microbial genomics, and biogeochemistry. With a few clarifications and refinements, it should be suitable for publication in Nature Communications.", "reviewer_4": "REVIEW for: Microbially-mediated halogenation and dehalogenation cycling of organohalides in the ocean\nThe manuscript by Zhou et al. offers the first comprehensive overview of global ocean microbial organohalide cycling, emphasizing previously unrecognized biological resources that influence essential biogeochemical processes. They (1) developed an organohalide-cycling gene database (HaloCycDB), and used this to (2) evaluate cross-kingdom microbially-driven halogenation and dehalogenation abilities in the ocean by analyzing 1,473 marine metagenomes from around the world. I commend the authors for their considerable effort in consolidating data from multiple studies (inc. data from five big marine expeditions) and using robust analytical methods for the publicly available metagenomes. The availability of open-access metagenomic data marks a significant advancement, enabling exploration of microbial processes in ways that were previously impossible. I also praise the authors for providing additional analyses that confirm the distinctive “microbe-enzyme-organohalide” interaction patterns for reductive dehalogenation by combining microbial cultivation, protein structure modeling, and molecular docking. These integrative methods not only improve our mechanistic understanding but also lay the groundwork for developing targeted strategies that exploit these specific interactions. Overall, this work is a valuable resource for the field and clearly demonstrates the power of metagenomic approaches in advancing our understanding of microbially-mediated marine biogeochemical cycles.\nThe manuscript is well-written and references the relevant literature thoroughly. However, I have several suggestions to enhance the manuscript. For an assessment of cross-kingdom microbially-mediated cycling of organohalides in marine environments, the authors include non-microbial eukaryotes (i.e., arthropods) in this categorization. The authors should provide a clear rationale for including genes from non-microbial hosts in their database and global assessment. This clarification is needed to prevent misrepresentation in the study. Also, although the results section contains essential information, its density might be overwhelming for readers. I recommend further summarizing certain parts. Additionally, while I have some concerns about the methodology (i.e., metagenomics processing), they are not major flaws and do not affect the overall presentation of the study.\nI ask the authors to address these points and provide additional rationale for clarification. Below are additional comments/suggestions for each section:\nAbstract:\nLine 32: Please correct “microbiome” to microorganisms.\nI recommend rephrasing the words/phrases “revolutionize” and “a first-ever picture” in the last sentence. Such bold and compelling words are unnecessary to emphasize the novelty of the study.\nMain:\nThe introduction section was well-written. However, I still recommend improving/revising specific presentations, as they could create a more direct narrative and make it easier to read. In the statement, “These organohalides can serve as antibiotics and signaling molecules in allelopathy, and consequently have a profound impact on the marine ecology.” I recommend providing more and wider examples of how organohalides affect marine ecology. Also, the current example, “For example, bromo-furanones produced by the marine red alga Delisea pulchra effectively disrupt bacterial quorum sensing systems to inhibit biofilm formation on algal surfaces”, is too specific and not representative of a more global impact. I recommend substituting this sentence with a more effective example.\nLines 50-52: I suggest referencing more recent studies in addition to the research highlight of York (2018: Nat. Rev. Microbiol.) for this opening statement.\nLine 79-83: “The heterogeneity of microbial growth niches in the ocean can promote specialized and diverse “microorganism-enzyme-organohalide” patterns to drive the marine halogenation and dehalogenation cycling of organohalides....” is an important part of the introduction as it emphasizes the need to understand these specialized and diverse “microorganism-enzyme-organohalide” global patterns. I recommend the authors provide a clearer definition of these patterns to strengthen this point.\nLine 115: “organohalide-cycling gene database (HaloCycDB)” but shown as “organohalide-cycling database (HaloCycDB)” in the abstract. Please use the same name consistently across the manuscript.\nLine 117: The authors should provide a more precise definition of what they mean by \"oceanic sites\". Please revise this.\nResults:\nLines 128-129: The subheading can further be shortened.\nLines 142-144: The use of >50% amino-acid/AA sequence similarity threshold was not mentioned in the methods. Please add.\nLines 149-159: I suggest moving the environmental metagenomics data information to the methods.\nLine 171: What about the high-quality MAGs (i.e., >90% completeness)? Of the 6,204 organohalide-cycling MAGs, how many are high-quality?\nIs it possible that the high detection of organohalide-cycling microorganisms in the study resulted from the inclusion of mid-quality MAGs (i.e., >50% completeness), unlike other studies that typically use only high-quality MAGs?\nLines 187-216: This section should be revised and reorganised. The narrative currently reads more like a list of taxa and functions than a discussion of the findings for each environment.\nDiscussion:\nLines 439-457: The comparison between the ocean and terrestrial environments, although insightful, is somewhat misleading. I suggest the authors begin the discussion by elaborating on the results and how they relate to the motivations for assessing the halogenation and dehalogenation cycling of organohalides in the ocean.\nI suggest the authors further expound on the “… the greatly expanded diversity of organohalide-cycling genes and microorganisms provides valuable resources for future biomedical and bioremediation applications”.\nMaterials and methods:\nFor the core database, the authors compiled 221 functionally characterized organohalide-cycling genes, catalyzing reactions and substrates/products. Only 194 of these belonged to bacteria, while two were from a marine worm, and one from a virus. Please clarify this in the methods section, especially since the authors refer to the core database coming from “host microorganisms” to avoid misrepresentation.\nMoreover, some of the genes were identified from eukaryotic hosts (e.g., kelp), which should not be considered microbial. I advise the authors to provide a reason for keeping these non-microbial eukaryotes in the core database.\nLines 508-510: Please add a reference on the choice of a global identity threshold of >30. Sequence clustering is complex, and while a 30% identity threshold is commonly used, different protein motifs often require prior assessment to determine the most appropriate similarity cutoff.\nSince the sequences were taxonomically assigned before dereplication at 100% using CD-HIT (v4.8.1), how did the authors handle gene-host microorganism taxonomic identification of clustered sequences with different identifications? Should the assignments be done after clustering?\nLines 517-518: “Finally, all representative sequences and associated host taxonomic information were checked for accuracy and used for full database construction.”, how was this done? I suggest the authors provide the details on this step.\nLines 555-564: For the metagenomics data processing, please indicate the necessary information on each step. For example, how were the contigs assembled? Individual or by group? Why use both CAT and Kaiju for tax ID?\nLines 576-577: Did the authors explore model tree optimization methods? Why use the WAG+GAMMA model for the tree?\nLine 610: Please specify how the normalization to RPKM was performed. Also, please make sure to add the definition of the abbreviation on first use.\nLine 616: Please correct “cd-hit” to CD-HIT.\nLines 634-635: Please expound on how the abundance and sample habitat normalization were performed.\nLine 697: For the 16S data, the authors could have used the GTDB database for taxonomic identification rather than the SILVA database. This also ensures that the taxonomic assignment nomenclature would be comparable with the MAGs’ IDs and the metagenomics data, especially since GTDB has rank-normalized names compared to the conventional naming scheme by SILVA.\nThe HalCycDB and analysis codes deposited in GitHub (i.e., https://github.com/metabiolab-wang/HaloCycDB) were not publicly accessible and were scheduled to be available in December 2026. In accordance with the Nature Communications requirements for research papers, authors are required to make the code/scripts used for their analysis openly available.\nFigures and tables:\nI suggest separating Figure 1 panels into two Figures (e.g., Fig. 1a and b, and Fig. 1c and d) for an easier visualization. Please include the definition of RPKM in Fig. 1d’s legend.\nAlso, Figure 4 panels (g and h) can be separated.\nPlease revise the legend of Supplementary Table 2 to “Distribution of halogenation-dehalogenation cycling genes in diverse marine water and sediment environments with and without biomass correction”."}, "author_response": "Response to the Reviewer’s comments\nIn this response, reviewers’ comments are in black; authors’ responses are in blue.\nRevised text excerpts are shown in bold. Line numbers refer to the clean version of the\nrevised manuscript. References from the manuscript use numbers; those cited only here\nuse author-year format. All modifications in the revised manuscript are highlighted in\nyellow.\nReviewer #3:\nThis manuscript presents a comprehensive analysis of the global distribution\nand diversity of organohalide-cycling microorganisms in the ocean. By combining\na newly developed curated database (HaloCycDB), large-scale metagenomic\nanalysis, protein structural modeling, and laboratory cultivation experiments, the\nauthors build a compelling narrative around the microbially mediated\nhalogenation and dehalogenation cycles in marine environments.\nNoteworthy contributions include:\n- The construction of HaloCycDB, a highly specific and validated functional\ndatabase, which outperforms existing resources (e.g., KEGG, COG) in identifying\norganohalide-cycling genes.\n- The identification of over 6,000 MAGs with halogenation/dehalogenation\npotential from 1,473 marine metagenomes, significantly expanding known\nfunctional diversity.\n- The demonstration that marine organohalide-cycling is dominated by\nPseudomonadota and Chloroflexota, and distinct from terrestrial systems.\n- The use of AlphaFold2-based structure prediction and molecular docking to\npropose seven RDase structural groups, linked to four ecological models of\ndehalogenation.\n- The experimental validation of RDase activity through targeted PCE-\ndechlorinating sediment cultures.\nThese findings are novel and impactful. They offer a foundational reference for\nfuture work on microbial contributions to halogen biogeochemistry, and provide\na valuable resource for the broader microbial ecology community.\n- We are grateful to the reviewer for acknowledging the scientific merit of this work.\nThat said, I would like to offer a few points for clarification and improvement:\n1. Definition of novelty in MAGs and genes:\nThe manuscript refers frequently to “novel MAGs” or “novel lineages,” but it\nis unclear whether novelty refers to unclassified taxonomic groups or to newly\nidentified functions within known taxa. Please clarify this distinction throughout,\nespecially in relation to Figure 3.\n- We thank the reviewer for the insightful comment. We agree that the term “novelty”\nencompasses multiple dimensions and must be precisely defined to avoid confusion\n1\nbetween taxonomically and functionally novel MAGs or lineages. In this study, the\nnovel MAGs/lineages refer to the taxa that were previously unknown to involve in\norganohalide cycling and were identified for the first time in this study to have the\nhalogenation/dehalogenation potential by carrying organohalide-cycling genes (Fig.\n3c). In contrast, the taxonomic novel MAGs/lineages refer to the microorganisms that\nwere taxonomically uncharacterized and referred as “microbial dark matter” (Fig. S6).\nWe have clarified this point in the revised manuscript (lines 289-290, 311-312, 316).\nFig. 3c. The percentages of functionally novel organohalide-cycling MAGs at\ndifferent taxonomic levels.\nFig. S6. Taxonomically novel organohalide-cycling MAGs in the ocean at different\ntaxonomic levels.\n2. Functional vs. active roles:\nThe study rightly focuses on genomic potential, but it would be helpful to\nacknowledge the limitations of this approach. A brief discussion of how future\nwork (e.g., transcriptomics, proteomics, or biogeochemical assays) could address\ngene expression and ecosystem-level function would enhance the framing of the\nstudy.\n- We agree and thank the reviewer for the constructive comment. As suggested, we have\nexpanded the Discussion section to explicitly acknowledge this limitation for future\nresearch (lines 486-491):\n“To further explore these bioresources, following studies are warranted: (1)\n2\nmetagenomics analyses can only predict the potential of gene functions and microbial\nactivities, of which validation requires multi-omics data (e.g., transcriptomics and\nproteomics) 46 and biogeochemical assays; nonetheless, current acquisition of marine\nmeta-transcriptomics and proteomics data is challenged by sample collection,\ntransportation, and treatment during ocean expeditions47”\nReference\n46. Tarazona, S., Arzalluz-Luque, A. & Conesa, A. Undisclosed, unmet and neglected\nchallenges in multi-omics studies. Nat. Comput. Sci. 1, 395-402 (2021).\n47. Pinto, Y. & Bhatt, A. S. Sequencing-based analysis of microbiomes. Nat. Rev. Genet.\n25, 829-845 (2024).\n3. Ecological integration:\nThe results would benefit from a deeper discussion of how organohalide-cycling\ngenes and microorganisms influence broader marine processes—such as carbon\nremineralization, sulfur cycling, or microbial food webs. While such integration\nmay be beyond the scope of the present study, recognizing this potential would\nhelp position the findings within a larger ecological context.\n- We sincerely appreciate the reviewer’s insightful suggestion regarding the broader\necological implications of our findings. As suggested, we have expanded the Discussion\nsection in the revised manuscript (lines 441-457):\n“The microbial-driven cycling of organohalides in marine environments\nsignificantly impacts the global biogeochemical cycles and climate systems, which\nmay generate far-reaching ecological consequences. For example, marine\nmicroorganisms employ halogenases to synthesize a diverse range of recalcitrant\norganohalides for varied biological functions (e.g., signal transduction and\nchemical defense5-7), playing an important role in marine cycling of organohalides\nand organic carbon. Subsequently, these recalcitrant organohalides can be\nconverted into labile organic carbon through dehalogeases-catalyzed carbon-\nhalogen bond cleavage, providing both energy and carbon sources for marine\nmicrobiomes. In oligotrophic oceans, organohalides have comparable\nconcentrations to other organic compounds41, enabling a variety of marine\nmicroorganisms to function as facultative populations that employ organohalide-\ncycling activities to complement other metabolic processes (e.g., fermentation and\nsulfate reduction) and to enhance competitiveness17. Take the marine\nmicroorganism - Desulfoluna as an example, this facultative dehalogenator can\ncouple bromobenzene-to-phenol conversion with sulfate reduction, linking\norganohalide respiration with sulfur cycling17. These dehalogenators can also act\nas metabolic hubs and provide carbon sources and reducing equivalents for\nmethanogens and other microorganisms, which is particularly important to\nsustain marine microbiomes in oligotrophic oceans.”\n5. Gribble, G. W. Naturally occurring organohalogen compounds—a comprehensive\nreview. Prog. Chem. Org. Nat. Prod. 121, 1-546 (2023).\n6. Borchardt, S. et al. Reaction of acylated homoserine lactone bacterial signaling\nmolecules with oxidized halogen antimicrobials. Appl. Environ. Microbiol. 67, 3174-\n3179 (2001).\n7. Hossaini, R. et al. Efficiency of short-lived halogens at influencing climate through\ndepletion of stratospheric ozone. Nat. Geosci. 8, 186-190 (2015).\n17. Peng, P. et al. Organohalide-respiring Desulfoluna species isolated from marine\nenvironments. ISME J. 14, 815-827 (2020).\n41. Gribble, G. W. Naturally occurring organohalogen compounds. Acc. Chem. Res. 31,\n141-152 (1998).\n4. Data and code availability:\nThe development of HaloCycDB is a major contribution and one of the strengths\nof the manuscript. However, the database and associated code are currently\ninaccessible—the link provided appears to be disabled or leads to a non-functional\nresource. This is a significant concern, as the reproducibility of the analyses and\nthe broader utility of the database depend on open and verifiable access. I strongly\nencourage the authors to ensure that HaloCycDB, along with the scripts used for\ngene screening and annotation, are made publicly and reliably available prior to\npublication.\n- We sincerely appreciate the reviewer’s recognition of the significance of HaloCycDB.\nThe HaloCycDB database, together with associated scripts for gene screening and\nannotation, is now publicly available on GitHub at https://github.com/metabiolab-\nwang/HaloCycDB. The repository includes all relevant documents to facilitate easy\naccess and use.\n5. Figure clarity:\nSome figures—particularly Figure 2a and Figure 3—are visually dense and\ncould benefit from simplified layouts, improved color coding, or alternative\nformats. Enhancing readability would make the complex results more accessible.\n- We thank the reviewer for the suggestion. To improve the figure clarity, we have made\nthe following changes:\n1. In Fig. 2a, we have changed the line color from multicolor into light gray to\nimprove contrast and reduce visual complexity, while preserving data clarity.\n2. In Fig. 3, we streamlined the figure by merging the legends of panels (a) and (b)\nfor a simplified and intuitive layout. In addition, the color of MAG-based tree branch\nwas changed from multicolor into either red (functionally novel taxa) or gray color\n(known organohalide-cycling taxa).\nocean. a. Rooted maximum likelihood phylogenetic trees of FlaHase, HyDase, and\nRDase genes identified from marine samples and HaloCycDB. b. Rooted maximum\nlikelihood phylogenetic tree of organohalide-cycling MAGs. The color of the internal\nbranches indicates whether the organohalide-cycling MAGs were reported for the first\ntime (functionally novel; red) or reported previously (known; light gray). c. The\npercentages of functionally novel organohalide-cycling MAGs at different taxonomic\nlevels. Bootstrap of >70% in the phylogenetic trees are indicated as gray circles.\nMethodology and standards: The methodology is sound and well executed. The\nauthors use established approaches for metagenomic binning, functional\nannotation, and phylogenetic inference. The addition of structure-function\nmodeling (via AlphaFold2) and experimental validation is particularly\ncommendable and uncommon in studies of this scale. The methods section appears\n6\nsufficiently detailed for reproduction of the core analyses, though confirmation of\naccess to the HaloCycDB pipeline would be essential.\n- We appreciate the reviewer’s assessment of our methodology and recognition of the\nstructural modeling and experimental validation components. As mentioned in our\nrevised manuscript, we have addressed the accessibility of the HaloCycDB by making\nboth the database and corresponding analysis scripts publicly available on GitHub\n(https://github.com/metabiolab-wang/HaloCycDB) (lines 754-755). This repository\ncontains all associated documents to facilitate reproduction of our analyses.\nConclusion: This is an original, high-quality manuscript that makes a meaningful\ncontribution to marine microbial ecology, microbial genomics, and\nbiogeochemistry. With a few clarifications and refinements, it should be suitable\nfor publication in Nature Communications.\n- We sincerely thank the reviewer for the constructive suggestions, and the recognition\nof the scientific contribution of our study.\nThe manuscript by Zhou et al. offers the first comprehensive overview of global\nocean microbial organohalide cycling, emphasizing previously unrecognized\nbiological resources that influence essential biogeochemical processes. They (1)\ndeveloped an organohalide-cycling gene database (HaloCycDB), and used this to\n(2) evaluate cross-kingdom microbially-driven halogenation and dehalogenation\nabilities in the ocean by analyzing 1,473 marine metagenomes from around the\nworld. I commend the authors for their considerable effort in consolidating data\nfrom multiple studies (inc. data from five big marine expeditions) and using robust\nanalytical methods for the publicly available metagenomes. The availability of\nopen-access metagenomic data marks a significant advancement, enabling\nexploration of microbial processes in ways that were previously impossible. I also\npraise the authors for providing additional analyses that confirm the distinctive\n“microbe-enzyme-organohalide” interaction patterns for reductive\ndehalogenation by combining microbial cultivation, protein structure modeling,\nand molecular docking. These integrative methods not only improve our\nmechanistic understanding but also lay the groundwork for developing targeted\nstrategies that exploit these specific interactions. Overall, this work is a valuable\nresource for the field and clearly demonstrates the power of metagenomic\napproaches in advancing our understanding of microbially-mediated marine\nbiogeochemical cycles.\n- We sincerely thank the reviewer for the recognition of our work. We are especially\ngrateful to the reviewer for the acknowledgment of this study’s value in understanding\nthe marine biogeochemical cycles.\nThe manuscript is well-written and references the relevant literature thoroughly.\nHowever, I have several suggestions to enhance the manuscript. For an assessment\nof cross-kingdom microbially-mediated cycling of organohalides in marine\nenvironments, the authors include non-microbial eukaryotes (i.e., arthropods) in\nthis categorization. The authors should provide a clear rationale for including\ngenes from non-microbial hosts in their database and global assessment. This\nclarification is needed to prevent misrepresentation in the study.\n- We thank the reviewer for the valuable comment regarding the rationale for including\ngenes from non-microbial hosts in our database and global assessment.\nRationale for including eukaryotic and viral sequences in HaloCycDB:\nThe reviewer is right that the majority of sequences in HaloCycDB are of prokaryotic\norigin; the database also contains genes from eukaryotic microorganisms, a limited\nnumber from non-microbial eukaryotes, and one viral gene. We initially chose to\ninclude genes from eukaryotic and viral hosts in HaloCycDB for the following reasons:\nFirst, functional genes can be transferred horizontally from eukaryotes to prokaryotes.\nsignificant contribution to bacterial evolution (Keeling et al., Nat. Rev. Genet., 2008;\nYuan et al., Nucleic Acids Res., 2024). For halogenase and dehalogenase genes, sponge-\nmicrobial symbiotic communities with close interactions may potentially facilitate\nhorizontal gene transfer from sponge to microbiota, possibly enabling microbial\ncommunities to enhance metabolic diversity and ecological adaptability by acquiring\nadditional organohalide cycling genes (Hentschel et al., Nat. Rev. Microbiol., 2012;\nZilber-Rosenberg et al., FEMS Microbiol. Rev., 2021).\nSecond, to ensure a comprehensive analysis of organohalide cycling processes, we\ninitially included these genes in our database construction to avoid potential\ninformation loss that could result from excluding non-prokaryotic sequences, which\ncould facilitate a complete assessment of the genetic potential for organohalide cycling\nacross kingdoms in marine environments.\nTo clarify this approach, we have revised the methods section to explicitly state the\ntaxonomic origins of genes included in HaloCycDB and replaced the term “host\nmicroorganisms” with “host organisms and viruses” throughout the revised manuscript\n(lines 505-506, 515-516, 525, 536).\nRemoval of non-microbial eukaryotes in the global assessment:\nWe agree with the reviewer’s comment that the inclusion of arthropods was\ninappropriate for this microbially-focused study. We thank the reviewer for pointing out\nthis inconsistency.\nIn the revised manuscript, we have removed non-microbial eukaryotes (arthropods),\nand updated the corresponding figures, including Fig. 1d and Supplementary Fig. 3a.\nAccordingly, we have also revised the main text to replace “Arthropoda” with\n“Ascomycota” as the representative eukaryotic microorganism in our discussion of\norganohalide cycling patterns (line 159). This revision ensures that our assessment\nfocuses on microbially-mediated processes. We hope these revisions address the\nreviewer’s concern while maintaining the scientific rigor and accuracy of our study.\nReference\nKeeling, P. J. & Palmer, J. D. Horizontal gene transfer in eukaryotic evolution. Nat. Rev.\nGenet. 9, 605-618 (2008).\nYuan, W., Yu, J. & Li, Z. Rapid functional activation of horizontally transferred\neukaryotic intron-containing genes in the bacterial recipient. Nucleic Acids Res. 52,\n8344-8355 (2024).\nHentschel, U., Piel, J., Degnan, S. M. & Taylor, M. W. Genomic insights into the marine\nsponge microbiome. Nat. Rev. Microbiol. 10, 641-654 (2012).\nZilber-Rosenberg, I. & Rosenberg, E. Microbial-driven genetic variation in holobionts.\nFEMS Microbiol. Rev. 45, fuab022 (2021).\nAlso, although the results section contains essential information, its density\n9\nmight be overwhelming for readers. I recommend further summarizing certain\nparts.\n- We appreciate the reviewer’s suggestion regarding the density of the results section.\nAs suggested, we have streamlined sections by condensing redundant information,\nsummarizing key findings more concisely (lines 189-200), and moving detailed\ntechnical data to Materials and Methods (lines 578-585).\nAdditionally, while I have some concerns about the methodology (i.e.,\nmetagenomics processing), they are not major flaws and do not affect the overall\npresentation of the study.\n- We appreciate the reviewer’s constructive comment regarding the methodology. We\nhave carefully reviewed each methodological concern raised by the reviewer,\nparticularly those related to metagenomics processing. In our following detailed\nresponse, we provide point-by-point clarifications and corrections addressing these\nissues. Where appropriate, we have revised our methods and updated the manuscript to\nensure clarity and methodological rigor.\nWe believe these revisions strengthen the manuscript and address the concerns of the\nreviewer. We are grateful for the thorough review and the opportunity to improve our\nwork.\nI ask the authors to address these points and provide additional rationale for\nclarification. Below are additional comments/suggestions for each section:\nAbstract:\nLine 32: Please correct “microbiome” to microorganisms.\n- Thanks for the suggestion. We have revised it accordingly (line 32).\nI recommend rephrasing the words/phrases “revolutionize” and “a first-ever\npicture” in the last sentence. Such bold and compelling words are unnecessary to\nemphasize the novelty of the study.\n- We agree and have deleted these words from the text as suggested (lines 45-46).\nMain:\nThe introduction section was well-written. However, I still recommend\nimproving/revising specific presentations, as they could create a more direct\nnarrative and make it easier to read. In the statement, “These organohalides can\nserve as antibiotics and signaling molecules in allelopathy, and consequently have\na profound impact on the marine ecology.” I recommend providing more and\n10\nwider examples of how organohalides affect marine ecology. Also, the current\nexample, “For example, bromo-furanones produced by the marine red alga\nDelisea pulchra effectively disrupt bacterial quorum sensing systems to inhibit\nbiofilm formation on algal surfaces”, is too specific and not representative of a\nmore global impact. I recommend substituting this sentence with a more effective\nexample.\n- We appreciate the reviewer for the constructive suggestion. We have revised the\nintroduction section accordingly (lines 54-59):\n“These organohalides can serve as antibiotics and signaling molecules,\nprofoundly\naffecting marine community structure and ecological competition5-7. For example,\nbromo-furanones produced by marine red alga change bacterial quorum sensing\nsystems to inhibit biofilm formation6, and natural methoxylated polybrominated\ndiphenyl ethers (MeO-PBDEs) disrupt endocrine and immune functions of higher\ntrophic organisms through bioaccumulation in marine food webs8.”\nReference\n5. Gribble, G. W. Naturally occurring organohalogen compounds—a comprehensive\nreview. Prog. Chem. Org. Nat. Prod. 121, 1-546 (2023).\n6. Borchardt, S. et al. Reaction of acylated homoserine lactone bacterial signaling\nmolecules with oxidized halogen antimicrobials. Appl. Environ. Microbiol. 67, 3174-\n3179 (2001).\n7. Hossaini, R. et al. Efficiency of short-lived halogens at influencing climate through\ndepletion of stratospheric ozone. Nat. Geosci. 8, 186-190 (2015).\n8. Teuten, E. L., Xu, L. & Reddy, C. M. Two abundant bioaccumulated halogenated\ncompounds are natural products. Science 307, 917-920 (2005).\nLines 50-52: I suggest referencing more recent studies in addition to the research\nhighlight of York (2018: Nat. Rev. Microbiol.) for this opening statement.\n- Thanks for the comment. As suggested, two recent references have been added in the\nmanuscript accordingly (lines 50-52):\n“As the Earth’s largest reservoir of dissolved organic carbon, sulfur, and halogen\nspecies, the ocean is a hotspot for microbially-mediated biogeochemical element\ncycling, potentially influencing global food webs and climate1-3.”\nReference\n1. York, A. Marine biogeochemical cycles in a changing world. Nat. Rev. Microbiol. 16,\n259-259 (2018).\n2. Danovaro, R., Levin, L. A., Fanelli, G., Scenna, L. & Corinaldesi, C. Microbes as\nmarine habitat formers and ecosystem engineers. Nat. Ecol. Evol. 8, 1407-1419 (2024).\n3. Heneghan, R. F. et al. The global distribution and climate resilience of marine\nheterotrophic prokaryotes. Nat. Commun. 15, 6943 (2024).\npromote specialized and diverse “microorganism-enzyme-organohalide” patterns\nto drive the marine halogenation and dehalogenation cycling of organohalides....”\nis an important part of the introduction as it emphasizes the need to understand\nthese specialized and diverse “microorganism-enzyme-organohalide” global\npatterns. I recommend the authors provide a clearer definition of these patterns\nto strengthen this point.\n- We sincerely thank the reviewer for this constructive comment. The suggestion to\nclarify the definition of “microorganism-enzyme-organohalide” patterns is valuable for\nimproving the manuscript’s clarity. To provide a clearer definition, we have revised the\ntext accordingly (lines 80-88):\n“The heterogeneity of microbial growth niches in the ocean23 can promote\nspecialized and diverse “microorganism-enzyme-organohalide” patterns to drive the\nmarine halogenation and dehalogenation cycling of organohalides. These patterns\nrefer to acclimatization and interactions among niche-specific microorganisms,\nassociated enzymatic machinery, and localized organohalide pools. Driven by\nredox potential and substrate availability, the “microorganism-enzyme-\norganohalide” patterns could shape the taxonomic and functional diversification\nfor halogenation and dehalogenation of organohalides in ocean, which represent\nyet-to-be-explored microbial resources of novel organohalide-cycling microorganisms,\nenzymes, and active chemical species.”\nReference\n23. Gralka, M., Szabo, R., Stocker, R. & Cordero, O. X. Trophic interactions and the\ndrivers of microbial community assembly. Curr. Biol. 30, R1176-R1188 (2020).\nLine 115: “organohalide-cycling gene database (HaloCycDB)” but shown as\n“organohalide-cycling database (HaloCycDB)” in the abstract. Please use the\nsame name consistently across the manuscript.\n- Thanks for the suggestion. We have revised the manuscript to consistently use\n“organohalide-cycling gene database (HaloCycDB)” throughout the manuscript (line\n33).\nLine 117: The authors should provide a more precise definition of what they mean\nby \"oceanic sites\". Please revise this.\n- Thanks for the comment. To avoid ambiguity, we have replaced the term “oceanic\nsites” with “marine sampling sites” in the revised manuscript (line 122). Specifically,\nthe phrase referred to geographical locations with different latitudes and longitudes for\n12\nsample collection.\nResults:\nLines 128-129: The subheading can further be shortened.\n- Thanks for the suggestion. We have changed the subheading into “Database\ndevelopment to profile marine organohalide-cycling potential” (lines 134).\nLines 142-144: The use of >50% amino-acid/AA sequence similarity threshold was\nnot mentioned in the methods. Please add.\n- Thanks for the comment. The corresponding sentences have been added accordingly\nin the revised manuscript (lines 637-639):\n“The organohalide-cycling genes without motifs were filtered with criteria of\nsequence similarity of ≥50% with confirmed organohalide-cycling gene sequences.”\nLines 149-159: I suggest moving the environmental metagenomics data\ninformation to the methods.\n- Thanks for the suggestion. The section describing the environmental metagenomics\ndata has been moved to the Methods section (lines 578-585).\nLine 171: What about the high-quality MAGs (i.e., >90% completeness)? Of the\n6,204 organohalide-cycling MAGs, how many are high-quality?\n- Thanks for the comment. Among the 6,204 organohalide-cycling MAGs, we identified\n1,687 high-quality MAGs (completeness ≥90% and contamination ≤5%). In addition,\n4,430 of the 6,204 MAGs had a quality score ≥ 50 (completeness − 5 × contamination;\nlines 281-283).\nIs it possible that the high detection of organohalide-cycling microorganisms in the\nstudy resulted from the inclusion of mid-quality MAGs (i.e., >50% completeness),\nunlike other studies that typically use only high-quality MAGs?\n- We thank the reviewer for the comment. In this study, we utilized MAGs with quality\nscores greater than 50 in downstream analyses (as mentioned in our manuscript; lines\n281-283), a criterion between high-quality MAGs (≥90% completeness, ≤5%\ncontamination) and mid-quality MAGs (≥50% completeness, ≤10% contamination).\nThis criterion (quality score ≥50) has been widely employed in large-scale\nmetagenomic studies, including sulfur oxidation genes (Baltar et al., Nat. Microbiol.,\n2023), and CRISPR-Cas systems and antimicrobial peptides (Chen et al., Nature, 2024)\n13\nin marine environments. In addition, we have actually conducted a comparative analysis\nacross the three categories of MAGs quality thresholds to identify their impact on the\nproportions of organohalide-cycling microorganisms (as shown in Response Fig. 1):\nResponse Fig. 1. The proportion of halogenation and dehalogenation populations in\nmarine metagenomes based on the presence or absence of organohalide-cycling genes\nin high-quality MAGs (a), mid-quality MAGs (b), and MAGs with a quality score ≥50\nemployed in this study (c).\nAs shown in the figure, the proportions of organohalide-cycling populations account\nfor 55.37%, 40.68% and 42.33% of total microbial communities based on high-quality\nMAGs, mid-quality MAGs, and MAGs with the quality score of ≥50 (employed in this\nstudy), respectively. The organohalide-cycling populations of the high-quality MAGs\nremained a comparatively higher proportion relative to the other two criteria. To\nprevent overestimation of the organohalide-cycling microorganisms in marine\nenvironments, together with the consideration that the marine microbial complexity\nnecessitates slightly relaxed completeness thresholds to capture broader functional\ndiversity, we finally chose the MAGs with the quality score of ≥50 in this study.\nReference\nBaltar, F. et al. A ubiquitous gammaproteobacterial clade dominates expression of sulfur\noxidation genes across the mesopelagic ocean. Nat. Microbiol. 8, 1137-1148 (2023).\nChen, J. et al. Global marine microbial diversity and its potential in bioprospecting.\nNature 633, 371-379 (2024).\nLines 187-216: This section should be revised and reorganised. The narrative\ncurrently reads more like a list of taxa and functions than a discussion of the\nfindings for each environment.\n- Thanks for the comment. We have thoroughly revised this part to focus on the findings\nas suggested by the reviewer (lines 189-200):\n“To examine prokaryote-mediated organohalide cycling in the ocean, the 1,473 ocean\nmetagenomes were used to profile their halogenation and dehalogenation potentials, as\nwell as their three-dimension spatial distribution (Fig. 2). Results showed a core group\nof bacteria dominate the marine organohalide-cycling metabolisms. For example,\nPseudomonadota being widespread in marine water and sediment environments\nmediated 65.73% and 65.14% potential halogenation and dehalogenation\n14\nactivities, respectively (Fig. 2a and b; Supplementary Fig. 4a). In addition,\nChloroflexota mediated 5.98% of potential organohalide-cycling activities (Fig.\n2a), and shared a similar geographic distribution pattern in the ocean with the\norganohalide-cycling Pseudomonadota (Fig. 2b; Supplementary Fig. 4b). Notably,\nDehalococcoidia of the Chloroflexota mainly involved in FlaHase- and HyDase-\nmediated halogenation and dehalogenation, respectively, and the well characterized\nterrestrial obligate organohalide-respiring bacteria (OHRB) of this class (i.e.,\nDehalococcoides and Dehalogenimonas) for RDase-mediated reductive\ndehalogenation were absent in the marine water and sediment niches (Fig. 2a). In\ncontrast, Anaerolineae instead of the Dehalococcoidia were major players of the\nphylum Chloroflexota to host RDase genes for reductive dehalogenation in the ocean29.”\nReference\n29. Adrian, L. & Löffler, F. E. Organohalide-respiring bacteria. Vol. 85 (Springer, 2016).\nDiscussion:\nLines 439-457: The comparison between the ocean and terrestrial environments,\nalthough insightful, is somewhat misleading. I suggest the authors begin the\ndiscussion by elaborating on the results and how they relate to the motivations for\nassessing the halogenation and dehalogenation cycling of organohalides in the\nocean.\n- We thank the reviewer for the constructive comment. To better elaborate on our\nfindings, we have revised this part as suggested (lines 441-458):\n“This study provides the first insight into the global-scale microbially mediated\norganohalide cycling in the ocean, which greatly expands the diversity of organohalide-\ncycling genes and microorganisms. The microbial-driven cycling of organohalides\nin marine environments significantly impacts the global biogeochemical cycles and\nclimate systems, which may generate far-reaching ecological consequences. For\nexample, marine microorganisms employ halogenases to synthesize a diverse\nrange of recalcitrant organohalides for varied biological functions (e.g., signal\ntransduction and chemical defense5-7), playing an important role in marine cycling\nof organohalides and organic carbon. Subsequently, these recalcitrant\norganohalides can be\nconverted into labile organic carbon through dehalogeases-\ncatalyzed carbon-halogen bond cleavage, providing both energy and carbon\nsources for marine microbiomes. In oligotrophic oceans, organohalides have\ncomparable concentrations to other organic compounds41, enabling a variety of\nmarine microorganisms to function as facultative populations that employ\norganohalide-cycling activities to complement other metabolic processes (e.g.,\nfermentation and sulfate reduction) and to enhance competitiveness17. Take the\nmarine microorganism - Desulfoluna as an example, this facultative dehalogenator\ncan couple bromobenzene-to-phenol conversion with sulfate reduction, linking\norganohalide respiration with sulfur cycling17. These dehalogenators can also act\nas metabolic hubs and provide carbon sources and reducing equivalents for\n15\nmethanogens and other microorganisms, which is particularly important to\nsustain marine microbiomes in oligotrophic oceans. Notably, scenarios are\ndifferent for the organohalide cycling in terrestrial and marine environments.”\nReference\n5. Gribble, G. W. Naturally occurring organohalogen compounds-a comprehensive\nreview. Prog. Chem. Org. Nat. Prod. 121, 1-546 (2023).\n6. Borchardt, S. et al. Reaction of acylated homoserine lactone bacterial signaling\nmolecules with oxidized halogen antimicrobials. Appl. Environ. Microbiol. 67, 3174-\n3179 (2001).\n7. Hossaini, R. et al. Efficiency of short-lived halogens at influencing climate through\ndepletion of stratospheric ozone. Nat. Geosci. 8, 186-190 (2015).\n17. Peng, P. et al. Organohalide-respiring Desulfoluna species isolated from marine\nenvironments. ISME J. 14, 815-827 (2020).\n41. Gribble, G. W. Naturally occurring organohalogen compounds. Acc. Chem. Res. 31,\n141-152 (1998).\nI suggest the authors further expound on the “… the greatly expanded diversity of\norganohalide-cycling genes and microorganisms provides valuable resources for\nfuture biomedical and bioremediation applications”.\n- Thanks for the suggestion. We have revised the statement to provide detailed\nexplanations about the potential applications (lines 483-486):\n“In this study, the greatly expanded diversity of organohalide-cycling genes and\nmicroorganisms provides valuable resources for future biomedical and bioremediation\napplications. For example, through synthetic biology approaches, the novel RDase\nidentified in this study could be further integrated into engineered strains such as\nVCOD-1545, broadening their substrate range and enhancing bioremediation\nefficacy.”\nReference\n45. Su, C. et al. Bioremediation of complex organic pollutants by engineered Vibrio\nnatriegens. Nature 642, 1024-1033 (2025).\nMaterials and methods:\nFor the core database, the authors compiled 221 functionally characterized\norganohalide-cycling genes, catalyzing reactions and substrates/products. Only\n194 of these belonged to bacteria, while two were from a marine worm, and one\nfrom a virus. Please clarify this in the methods section, especially since the authors\nrefer to the core database coming from “host microorganisms” to avoid\nmisrepresentation.\nMoreover, some of the genes were identified from eukaryotic hosts (e.g., kelp),\n16\nwhich should not be considered microbial. I advise the authors to provide a reason\nfor keeping these non-microbial eukaryotes in the core database.\n- Thanks for the constructive suggestion. We agree that the majority of HaloCycDB\nsequences are of prokaryotic origin, and a very small portion from virus and eukaryotes\nincluding non-microbial eukaryotes. We included the non-microbial eukaryotes-\nderived organohalide-cycling genes in the HaloCycDB database due to the following\nreasons: functional genes can be transferred horizontally from eukaryotes to\nprokaryotes, which makes a great contribution to drive bacterial evolution (Keeling et\nal., Nat. Rev. Genet., 2008; Yuan et al., Nucleic Acids Res., 2024). For\nhalogenase/dehalogenase genes, for example, sponge-bacteria symbiotic communities\nwith close interactions potentially facilitate horizontal transfer of\nhalogenase/dehalogenase genes from sponge to microbiota, enabling microbial\ncommunities to enhance metabolic diversity and ecological adaptability by acquiring\norganohalide cycling genes (Hentschel et al., Nat. Rev. Microbiol., 2012; Zilber-\nRosenberg et al., FEMS Microbiol. Rev., 2021); Therefore, to ensure comprehensive\nanalysis of organohalide cycling processes and to avoid potential information loss, we\nincluded non-microbial eukaryotes-derived organohalide-cycling genes in the database,\nwhich facilitate cross-kingdom assessment of the organohalide cycling in marine\nenvironments. To clarify this consideration, we have revised the method section to\nexplicitly state the taxonomic origins of genes included in HaloCycDB and replaced\nthe term “host microorganisms” with “host organisms and virus” in the revised\nmanuscript (lines 505-506, 515-516, 525, 536).\nReference\nKeeling, P. J. & Palmer, J. D. Horizontal gene transfer in eukaryotic evolution. Nat.\nRev. Genet. 9, 605-618 (2008).\nYuan, W., Yu, J. & Li, Z. Rapid functional activation of horizontally transferred\neukaryotic intron-containing genes in the bacterial recipient. Nucleic Acids Res. 52,\n8344-8355 (2024).\nHentschel, U., Piel, J., Degnan, S. M. & Taylor, M. W. Genomic insights into the marine\nsponge microbiome. Nat. Rev. Microbiol. 10, 641-654 (2012).\nZilber-Rosenberg, I. & Rosenberg, E. Microbial-driven genetic variation in holobionts.\nFEMS Microbiol. Rev. 45, fuab022 (2021).\nLines 508-510: Please add a reference on the choice of a global identity threshold\nof >30. Sequence clustering is complex, and while a 30% identity threshold is\ncommonly used, different protein motifs often require prior assessment to\ndetermine the most appropriate similarity cutoff.\n- We thank the reviewer for the helpful comment. We have added the references on the\nchoice of a global identity threshold of >30 in the manuscript (lines 530-531).\nSpecifically, our selection of a 30% global identity threshold was based on following\nconsiderations:\n17\n1. Precedent in the annotation of metabolic genes\nOur choice follows established practices for the annotation of functionally diverse\ngenes. A 30% identity threshold is a commonly used standard for inferring homology\nand functional similarity, particularly when dealing with large, functionally diverse\nprotein superfamilies for element cycles, including the N- (Tu et al., Bioinformatics,\n2019; Ji et al., Nat. Commun., 2024), P- (Zeng et al., Microbiome, 2022), S-cycles (Yu\net al., Mol. Ecol. Resour., 2021; Song et al., Nat. Commun., 2025).\n2. Specific considerations of halogenation and dehalogenation genes\nFor the halogenation genes, a 30% identity threshold is a commonly used standard\nfor defining sequence clusters and identifying homologous proteins (Ni et al., Nat.\nCommun., 2024). This threshold strikes a balance between capturing functional\nsimilarities and allowing sequence divergence in the enzyme family.\nFor the dehalgoenation genes, we used the 30% identity threshold based on the\nconsideration of their vast diversity. For instance, hydrolytic dehalogenases belong to\ntwo structurally distinct superfamilies, i.e., the α/β-hydrolase (ABH) and the haloacid\ndehalogenase (HAD) superfamily, which share a minimal sequence similarity of 31.00%\n(Chan et al., Microb Biotechnol, 2010). A stringent identity cutoff may fail to capture\nthis broad evolutionary divergence. Similarly, reductive dehalogenases like PcbA5 and\nCprA exhibit sequence identities as low as 30.04%, underscoring the necessity of a\nrelaxed threshold to encompass functionally relevant but distantly related sequences.\n3. Balancing sensitivity and specificity in gene identification\nTo avoid the limitations of identifying halogenase/dehalogenase genes solely based\non the sequence identity, we complemented this approach with conserved domain/motif\nanalysis. The 30% cutoff allowed us to capture a broad range of\nhalogenase/dehalogenase gene sequences, while subsequent gene filtering based on\nconserved domains/motifs ensured the functional relevance of the identified sequences.\nThis integrated and complemented strategy minimizes false negatives—a critical\nconcern given the functional diversity of these enzyme families—while ensuring\nannotation confidence.\nReference\nTu, Q., Lin, L., Cheng, L., Deng, Y. & He, Z. NCycDB: a curated integrative database\nfor fast and accurate metagenomic profiling of nitrogen cycling genes. Bioinformatics\n35, 1040-1048 (2019).\nJi, M. et al. Biodiversity of mudflat intertidal viromes along the Chinese coasts. Nat.\nCommun. 15, 8611 (2024).\nZeng, J. et al. PCycDB: a comprehensive and accurate database for fast analysis of\nphosphorus cycling genes. Microbiome 10, 101 (2022).\nYu, X. et al. SCycDB: a curated functional gene database for metagenomic profiling of\nsulphur cycling pathways. Mol. Ecol. Resour. 21, 924-940 (2021).\nSong, X. et al. Rhizosphere-triggered viral lysogeny mediates microbial metabolic\nreprogramming to enhance arsenic oxidation. Nat. Commun. 16, 4048 (2025).\nspecificity engineering of nucleotide halogenases. Nat. Commun. 15, 5254 (2024).\nChan, W. Y. et al. Sequence‐and activity‐based screening of microbial genomes for\nnovel dehalogenases. Microb. Biotechnol. 3, 107-120 (2010).\nSince the sequences were taxonomically assigned before dereplication at 100%\nusing CD-HIT (v4.8.1), how did the authors handle gene-host microorganism\ntaxonomic identification of clustered sequences with different identifications?\nShould the assignments be done after clustering?\n- Thanks for the constructive suggestion. To remove completely identical gene\nsequences and avoid the sequence redundancy of HaloCycDB, we used CD-HIT with\n100% identity for dereplication with parameters ‘-c 1.0 -aS 1.0 -aL 1.0’, which ensures\nthat only completely identical sequences are removed. To prevent misunderstanding,\nwe have added the purpose and specific parameters of dereplication by CD-HIT in the\nrevised manuscript (lines 537-538):\n“All gene sequences from the pre-full database were further clustered by CD-HIT64\n(v4.8.1) at 100% identity with parameters ‘-c 1.0 aS 1.0 -aL 1.0’ to remove\ncompletely identical gene sequences and avoid the database sequence redundancy.”\nSince only completely identical gene sequences were clustered together after\nperforming dereplication and clustering with the above parameters using CD-HIT, the\ntaxonomic assignment for the gene-host microorganisms of sequences from the same\ncluster were also completely identical. Therefore, the taxonomic annotation performed\nbefore clustering could be directly applied without causing conflicts in host assignment\nfrom the same clustered sequences.\nReference\n64. Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the\nnext-generation sequencing data. Bioinformatics 28, 3150-3152 (2012).\nLines 517-518: “Finally, all representative sequences and associated host\ntaxonomic information were checked for accuracy and used for full database\nconstruction.”, how was this done? I suggest the authors provide the details on this\nstep.\n- Thanks for the suggestion. After performing taxonomic annotation using the NCBI\nRefSeq databases, corresponding NCBI taxonomy identifier (TaxIds) of the\nrepresentative sequences in HaloCycDB were obtained. To acquire accurate and\nstandardized taxonomic information of representative sequences by TaxIds, we used the\nsoftware TaxonKit (Shen, W. et al., J. Genet. Genomics, 2021). Taxonkit could identify\nold TaxIds that have been deleted and merged in NCBI and automatically convert them\ninto new TaxIds to prevent annotation inaccuracies caused by outdated taxonomic\ninformation. The new TaxIds could be further transformed into complete taxonomic\nlineages and standardized into seven levels (i.e., kingdom/domain, phylum, class, order,\nfamily, genus, and species). As suggested, we have added these details in the revised\nmanuscript (lines 539-544).\nReference\nShen, W. & Ren, H. TaxonKit: A practical and efficient NCBI taxonomy toolkit. J.\nGenet. Genomics 48, 844-850 (2021).\nLines 555-564: For the metagenomics data processing, please indicate the\nnecessary information on each step. For example, how were the contigs assembled?\nIndividual or by group? Why use both CAT and Kaiju for tax ID?\n- We thank the reviewer for the helpful comment. We have added the necessary details\non metagenomic data processing regarding assembly and taxonomic annotation of\ncontigs in the revised manuscript (lines 589–594).\nSpecifically, for the assembly of contigs, we used MEGAHIT with multiple k-mer\nstrategies to individually assemble each sample. We have added the information in the\ntext accordingly (lines 589-592).\nFor taxonomic annotation of contigs, we used CAT and Kaiju to combine their\nstrengths and improve both the accuracy and breadth of taxonomic classification. CAT\nis a pipeline designed for the taxonomic classification of long DNA sequences from\nboth known and (highly) unknown microorganisms (Von Meijenfeldt et al., Genome\nBiol, 2019), which involves ORF (open reading frame) prediction, mapping of\npredicted ORFs against a protein database, and voting-based classification of the entire\ncontig based on classification of the ORFs. The taxonomic classification using CAT is\nbased on the GTDB database and provides high accuracy for bacterial and archaeal\nannotations of contigs. In contrast, Kaiju is used to directly align contigs against the\nNCBI NR database (Menzel et al., Nat. Commun., 2016), which includes sequences\nfrom bacteria, archaea, viruses, fungi, and other eukaryotic microorganisms, thus\nproviding broad taxonomic coverage. The integration of CAT and Kaiju enables\ncomprehensive annotation of diverse microbial communities We have added the\ninformation on why we used both CAT and Kaiju for taxonomic identification in the\nrevised manuscript accordingly (lines 592-594).\nReference\nVon Meijenfeldt, F. B., Arkhipova, K., Cambuy, D. D., Coutinho, F. H. & Dutilh, B. E.\nRobust taxonomic classification of uncharted microbial sequences and bins with CAT\nand BAT. Genome. Biol. 20, 1-14 (2019).\nMenzel, P., Ng, K. L. & Krogh, A. Fast and sensitive taxonomic classification for\nmetagenomics with Kaiju. Nat. commun. 7, 11257 (2016).\nLines 576-577: Did the authors explore model tree optimization methods? Why\n20\nuse the WAG+GAMMA model for the tree?\n- Thanks for the comment. We constructed the phylogenetic tree of MAGs based on\nwidely used methods in GTDB-tk. Specifically, GTDB-Tk builds a protein-sequence-\nbased phylogenetic tree of MAGs using FastTree, which supports three amino-acid\nsubstitution models—JTT (Jones-Taylor-Thornton), LG (Le & Gascuel), and WAG\n(Whelan & Goldman; default). WAG model with gamma-distributed rate (+GAMMA)\nwas performed to better capture the variation in evolutionary rates across sites, which\ncould enhance the fit and accuracy of the phylogenetic tree, especially with the large-\nscale datasets (Bourquin et al., Nat. Commun., 2025; Parks et al., Nat. Microbiol., 2017).\nTherefore, considering the default parameters in GTDB-tk and the common use of this\nmethod in other studies (Bourquin et al., Nat. Commun., 2025; Parks et al., Nat.\nMicrobiol., 2017; Liu et al., Nature, 2021; Wu et al., Sci. Adv., 2025), we used the\nWAG+GAMMA model with FastTree to construct the phylogenetic tree for the 4,430\nMAGs.\nReference\nBourquin, M. et al. Predicting climate-change impacts on the global glacier-fed stream\nmicrobiome. Nat. Commun. 16, 1264 (2025).\nParks, D. H. et al. Recovery of nearly 8,000 metagenome-assembled genomes\nsubstantially expands the tree of life. Nat. Microbiol. 2, 1533-1542 (2017).\nLiu, Y. et al. Expanded diversity of Asgard archaea and their relationships with\neukaryotes. Nature 593, 553-557 (2021).\nWu, D., Seshadri, R., Kyrpides, N. C. & Ivanova, N. N. A metagenomic perspective on\nthe microbial prokaryotic genome census. Sci. Adv. 11, eadq2166 (2025).\nLine 610: Please specify how the normalization to RPKM was performed. Also,\nplease make sure to add the definition of the abbreviation on first use.\n- We appreciate the reviewer for the helpful suggestion. As suggested, we have added a\ndetailed description of the RPKM (Reads Per Kilobase per Million mapped reads)\nnormalization procedure in the revised manuscript (lines 646-649). Specifically, The\nRPKM were calculated using the equation:\nnumReads × 109\nRPKM=\nseqLength × totalNumReads\nWhere numReads is the number of reads mapped to a sequence; seqLength is the\nlength of the sequence; totalNumReads is the total number of mapped reads of a sample.\nAdditionally, as suggested, we have added the full name of RPKM on first use in the\nrevised manuscript (lines 183-184): “The heatmap illustrates the gene abundances\nbased on RPKM (Reads Per Kilobase per Million mapped reads) values.”\n- Thanks for the suggestion. We have changed the “cd-hit” to “CD-HIT” in the revised\nmanuscript (line 655).\nLines 634-635: Please expound on how the abundance and sample habitat\nnormalization were performed.\n- Thanks for the comment. We added the description on how the abundance and sample\nhabitat normalization were performed in the revised manuscript (lines 673-675):\n“Finally, to minimize unequal-sampling-size derived bias, the total abundance of\norganohalide cycling genes in each habitat was normalized by dividing the sample\nnumbers in corresponding habitats.”\nLine 697: For the 16S data, the authors could have used the GTDB database for\ntaxonomic identification rather than the SILVA database. This also ensures that\nthe taxonomic assignment nomenclature would be comparable with the MAGs’\nIDs and the metagenomics data, especially since GTDB has rank-normalized\nnames compared to the conventional naming scheme by SILVA.\n- We agree with the reviewer that both the 16S rRNA gene and metagenomic analyses\nbetter to use the same database (GTDB in this study) for the taxonomic classification.\nTherefore, we have re-performed the 16S rRNA gene-based taxonomic identification\nusing the GTDB database instead of the SILVA database as suggested. Results showed\ncomparable profiles of the organohalide-respiring microorganisms in our cultures, i.e.,\nthe presence of facultative organohalide-respiring bacteria (OHRB) and absence of\nobligate OHRB (Response Fig. 2). Additionally, the GTDB database-based 16S rRNA\ngene community profiling showed the presence of three new groups of facultative\nOHRB (i.e., Carboxylicivirga, Marinifilum, and Scatomorpha), being in line with the\nGTDB-based metagenomic data (Fig. 5b in the manuscript). This result showed\nadvantages of the GTDB database over than the SILVA database, especially when\ncomparing the 16S rRNA gene-based community results with the metagenomic data.\nWe have added a discussion of these findings in the revised manuscript (lines 407-410),\nand changed SILVA-based result to the GTDB-based figure (Fig. 5; line 428).\ncultures, and the relative abundance of OHRB based on 16S rRNA analysis using\nGTDB (a) and SILVA (b) databases.\nThe HalCycDB and analysis codes deposited in GitHub (i.e.,\nhttps://github.com/metabiolab-wang/HaloCycDB) were not publicly accessible\nand were scheduled to be available in December 2026. In accordance with the\nNature Communications requirements for research papers, authors are required\nto make the code/scripts used for their analysis openly available.\n- We thank the reviewer for the suggestion, and fully acknowledge Nature\nCommunications’ requirements for code accessibility. As suggested and in accordance\nwith journal policy, HaloCycDB and all associated scripts are now released and publicly\navailable on GitHub (https://github.com/metabiolab-wang/HaloCycDB).\nFigures and tables:\nI suggest separating Figure 1 panels into two Figures (e.g., Fig. 1a and b, and Fig.\n1c and d) for an easier visualization. Please include the definition of RPKM in Fig.\n1d’s legend.\n- We appreciate the reviewer for the suggestions to improve the clarity and presentation\nof our figures. We have carefully considered both recommendations regarding Fig. 1:\n1. Splitting Fig. 1 into two separate figures:\nWe greatly appreciate this suggestion and have given it thorough consideration.\nWhile we fully understand the reviewer’s suggestion on improving visualization, we\nwould like to maintain the four panels (a-d) as a unified figure based on the\nconsideration that these panels collectively represent the comprehensive workflow\ndescribed in our result section 1 “Database development to profile marine organohalide-\ncycling potential”:\n• Panel (a) illustrates HaloCycDB construction;\n• Panel (b) evaluates the HaloCycDB using artificial datasets;\n• Panel (c) describes metagenomic sample collection;\n23\n• Panel (d) demonstrates the impact of filtering gene data annotated by HaloCycDB\non the distribution of organohalide-cycling genes and taxa.\nWe believe these panels of Fig. 1 together describe a cohesive methodological\ndevelopment process for HaloCycDB, and we are concerned that separating them might\ninadvertently disrupt the narrative continuity. To better address the reviewer’s valuable\nconcern about visualization clarity, we have made improvements by enlarging font sizes\nacross all panels and optimizing the overall layout to enhance readability.\n2. Definition of RPKM in Fig. 1d’s legend\nAs suggested, we have added the definition of RPKM (Reads Per Kilobase per\nMillion mapped reads) to the legend of Fig. 1d in the revised manuscript (line 183-184).\nAlso, Figure 4 panels (g and h) can be separated.\n- Thanks for the suggestion. We agree with the reviewer and have moved the Fig. 4h to\nthe supplementary materials (now presented as Supplementary Fig. 10). Accordingly,\nFig. 4 has been updated as shown below (line 378):\nPlease revise the legend of Supplementary Table 2 to “Distribution of\nhalogenation-dehalogenation cycling genes in diverse marine water and sediment\nenvironments with and without biomass correction”.\n- Thanks for the suggestion. We have revised the table legend accordingly.\n24"}, {"round": 2, "reviewer_comments": {"reviewer_3": "The authors have satisfactorily addressed all reviewer concerns. The revised manuscript is significantly strengthened through clarifications on novelty definitions, methodological transparency, ecological framing, figure improvements, and importantly the public release of the HaloCycDB database and scripts. I have no further concerns and recommend acceptance for publication.\n(Remarks on code availability)\nI did not test the code directly, but the repository is publicly available and includes README and scripts, which ensures transparency and reproducibility.", "reviewer_4": "For this round, I commend the authors for addressing all of the reviewers' comments thoroughly. The improvements to the manuscript and the main figures are clear, and the authors have done an excellent job of revising it.\n(Remarks on code availability)"}, "author_response": "Response to the Reviewer’s comments\nReviewer #3 (Remarks to the Author):\nThe authors have satisfactorily addressed all reviewer concerns. The revised\nmanuscript is significantly strengthened through clarifications on novelty\ndefinitions, methodological transparency, ecological framing, figure\nimprovements, and importantly the public release of the HaloCycDB database and\nscripts. I have no further concerns and recommend acceptance for publication.\n-We sincerely thank the Referee #3 for the thoughtful comments and kind suggestions,\nthe manuscript would not be significantly improved without the reviewer. Thank you\nagain for your great efforts during the whole review process.\nReviewer #4 (Remarks to the Author):\nFor this round, I commend the authors for addressing all of the reviewers'\ncomments thoroughly. The improvements to the manuscript and the main figures\nare clear, and the authors have done an excellent job of revising it.\n-We are very pleased that Referee #4 finds that our revisions thoroughly address the\nissues raised during the review process. We thank Referee #4 for the insightful\ncomments and suggestions, which have helped to strengthen the manuscript.\n1"}]

Decision letter