Novel isolates of hydrogen-oxidizing chemolithoautotrophic Sulfurospirillum provide insight to the functions and adaptation mechanisms of Campylobacteria in shallow-water hydrothermal vents

ABSTRACT Enhancing the availability of representative isolates from hydrothermal vents (HTVs) is imperative for comprehending the microbial processes that propel the vent ecosystem. In recent years, Campylobacteria have emerged as the predominant and ubiquitous taxon across both shallow and deep-sea vent systems. Nevertheless, only a few isolates have been cultured, primarily originating from deep-sea HTVs. Presently, no cultivable isolates of Campylobacteria are accessible in shallow water vent systems (<200 m), which exhibit markedly distinct environmental conditions from their deep-sea counterparts. In this study, we enriched a novel isolate (genus Sulfurospirillum, Campylobacteria) from shallow-water HTVs of Kueishan Island. Genomic and physiological analysis revealed that this novel Campylobacteria species grows on a variety of substrate and carbon/energy sources. The pan-genome and phenotypic comparisons with 12 previously isolated Sulfurospirillum species from different environments supported the identification of functional features in Sulfurospirillum genomes crucial for adaptation to vent environments, such as sulfur oxidation, carbon fixation, biofilm formation, and benzoate/toluene degradation, as well as diverse genes related with signal transportation. To conclude, the metabolic characteristics of this novel Campylobacteria augment our understanding of Campylobacteria spanning from deep-sea to shallow-water vent systems. IMPORTANCE Campylobacteria emerge as the dominant and ubiquitous taxa within vent systems, playing important roles in the vent ecosystems. However, isolated representatives of Campylobacteria have been mainly from the deep-sea hydrothermal fields, leaving a significant knowledge gap regarding the functions, activities, and adaptation strategies of the vent microorganisms in shallow-water hydrothermal vents (HTVs). This study bridges this gap by providing insights into the phenomics and genomic diversity of genus Sulfurospirillum (order Campylobacterales, class Campylobacteria) based on data derived from a novel isolate obtained from shallow-water HTVs. Our mesophilic isolate of Sulfurospirillum not only augments the genus diversity of Campylobacteria pure cultures derived from vent systems but also serves as the inaugural reference isolate for Campylobacteria in shallow-water environments.


Enrichment and isolation
Sulfur-rich sediments of the Kueishan Island SW-HTV (121.96232°E,24.83420°N), at depth of 21 m, were used as the inoculum in our study.Detailed sampling information and environmental parameters can be found in our previous studies (41,42).The surface portion of each sediment core was discarded and the remaining portion was stored in sterile polypropylene bags and kept on ice during transportation to the laboratory.
In the laboratory, 25 g sediment sample was mixed with 20 mL MJ synthetic seawater (44) in a 120 mL serum bottle (Whatman) under a gas phase of pure N 2 (200 kPa) at 28°C.After 48 h enrichment, 0.5 mL of the sediment slurry was transferred to a new 20 mL MMJHS medium for longer culture under a gas phase of N 2 /H 2 /CO 2 (50:40:10, 200 kPa).Microbial pure cultures were obtained using a dilution-to-extinction approach.MMJHS medium was prepared by dissolving 1 g each of NaHCO 3 , Na 2 S 2 O 3 •5H 2 O and NaNO 3 , 3 g S 0 , and 10 mL trace vitamin solution (45) in 1 L of MJ synthetic seawater (44).All the ingredients except sulfur were mixed, and the pH was adjusted to 6.5 with NaOH.Then, the medium was filter-sterilized with 0.2 µm filter (Whatman, United Kingdom).Next, 20 mL medium was distributed into 120 mL serum bottle, which was autoclaved together with sulfur power.Resazurin was added as a redox indicator to monitor bacterial growth.However, we did not add resazurin to the medium during subsequent substrate and growth factor tests to avoid potential disturbances to the optical density (OD) values.

Morphology and growth factors
The morphology of the cells was examined using fluorescence microscope and transmission electron microscopy.To assess potential growth controlling factors such as pH, temperature, and NaCl concentration, changes in OD at 600 nm were monitored.Sensitivity to oxygen was determined by adding different concentrations of an oxygen scavenger to the medium under the gas phase composed of N 2 /H 2 /CO 2 = 50:40:10, at 200 kPa.The oxygen scavenger utilized was a mixture of 2.5% Na 2 S•9H 2 O and 2.5% cysteine HCl.methylated using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0) and identified by using the RTSBA6.0database of the Microbial Identification System (46).Polar lipids of freeze-dried cells were extracted and separated on silica gel 60F254 aluminum-backed thin-layer plates (10 × 10 cm 2 ; Merk 5554), which were dried for 30 min at 55°C and further analyzed according to Minnikin et al. (47).The first dimension of the solvent system was chloroform/methanol/water (65:24:4, by volume) and the second dimension was chloroform/glacial acetic acid/methanol/water (80:15:12:4, by volume).Other reagents such as α-naphthol, ninhydrin, and molybdenum blue (Sigma) were used to detect glycolipids, amino lipids, and phospholipids, according to Tindall (48).Phosphomolybdic acid (5%, wt/vol, dissolved in alcohol) was sprayed on the plates, which were then heated at 160°C for 10-15 min, to identify total lipids.Respiratory quinones were extracted using the method described by Minnikin et al. (47) and analyzed by HPLC as described by Tindall (48).

Molecular analysis
The 16S rRNA gene was amplified via PCR using primers Eubac 27F/1492R (49) and sequenced using Sanger dideoxynucleotide chain-termination method by Sangon Biotech Co. Ltd. (Shanghai, China).Unambiguously nucleotide sequences were used for phylogenetic analysis with MEGA package (50).
Genomic DNA was extracted using MiniBEST bacteria genomic DNA extraction kit Ver.3.0 (Takara BIO INC.) according to the manufacturer's protocol.The purified genomic DNA was quantified using a TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA).High-quality DNA (OD260/280 = 1.8-2.0,>20 µg) was sequenced using a combination of PacBio RS II Single MoleculeReal Time (SMRT) and Illumina sequencing platforms by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).The Illumina data were employed to evaluate the genome complexity.After quality trimming, the reads were assembled into contigs using hierarchical genome assembly process (51).Subsequently, error correction of the PacBio assembly results was performed using the Illumina reads with Pilon.Glimmer (52) was utilized for CDS prediction, tRNA-scan-SE (53) for tRNA prediction, and Barrnap for rRNA prediction.Predicted CDSs were annotated from NR, Swiss-Prot, Pfam, GO (Gene Ontology), COG (Clusters of Orthologous Groups), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases using sequence alignment tools such as BLAST, Diamond, and HMMER.Each set of query proteins was aligned with the databases, and annotations of best-matched subjects (e-value < 10 −5 ) were obtained for gene annotation.A phylogenomic tree of Campylobacteria genomes was reconstructed based on 400 universal marker genes using PhyloPhlAn (54).
[FeFe]-and [NiFe]-hydrogenase homologs were discretely distributed at the domain, phylum, and order levels of taxonomic classifications (58).Based on the phylogeny of the large subunits, [NiFe] hydrogenase was further classified into different groups according to a previous study (59).The annotated hydrogenases of our isolates' genome were aligned with the reference hydrogenase sequences (59) by MUSCLE (60), and a phylogenetic tree was constructed in MEGA (50).
The software was executed with default settings unless other specified.

Computing the pangenome
The anvi'o pangenome workflow (https://merenlab.org/2016/11/08/pangenomics-v2) was employed to compute the pangenome with default settings (61), involving several steps: (i) generating an anvi'o genome database of Sulfurospirillum (anvi-gengenomes-storage) to store DNA and amino acid sequences (ii), computing the pan genome (anvi-pan-genome) from a genome database by identifying "gene clusters" (iii), displaying the pangenome (anvi-display-pan) to visualize the distribution of gene clusters across genomes and interactively bin gene clusters into logical groups such as "core genes, " "unique genes of 1612, " and "accessory genes absenting in S. arcachonens and 1612" bins (iv), reporting summary output as a profile database including the names of gene clusters and their numbers in each bin (anvi-summarize) (v), obtaining the FASTA file with sequences for each bin (anvi-get-sequences-for-gene-clusters), followed by annotation by KEGG or COG.In this context, a "gene cluster" represents sequences of one or more predicted open reading frames grouped together based on their homology at the translated DNA sequence level.Gene clusters with more than one sequence may contain orthologous or paralogous sequences, or both, from one or more genomes analyzed in the pangenome.The figure of pan-genome profile was generated by BPGA with default parameters (62).

Data description
The

Morphology, physiology, and growth characteristics of the isolate
Cells of strain 1612 were observed to be slightly curved rods, with lengths ranging from 1.0 to 2.5 µm, and each cell possessed a polar flagellum (Supporting materials, Fig. S2).
The cell was stained Gram-negative.Strain 1612 exhibited growth at 4-50°C, with an optimum growth temperature of 28°C.No growth was observed below 4°C or above 50°C (Supporting materials, Fig. S3A).The growing pH of the bacteria ranged from 5.5 to 8.0, with an optimum growth at pH 6.5 (Supporting materials, Fig. S3B).NaCl requirement for growth was determined by using various concentrations of NaCl (1.0-6.0%,wt/vol) in the medium.The isolate grew in concentration ranging from 1.0% to 5.0% (wt/vol) NaCl, showing optimum growth at 3.0% (wt/vol) NaCl (Supporting materials, Fig. S3C).

Classification based on phylogenomic analysis
A phylogenomic tree of existing Campylobacteria genomes was constructed to illustrate the taxonomic relatedness of the isolates (Fig. 1).Notably, Sulfurospirillum species are distinct from the other related genera like Campylobacter, Arcobacter, Helicobacter, and Wolinella, which are mostly host-associated.In contrast, reported pure cultures of Sulfurospirillum have been from diverse environments, including soil, freshwater sediment, sewage, and hydrocarbon-rich sites.In the phylogenetic tree, the newly isolated 1612 formed a clade with S. arcachonense, which was isolated from marine sediment.
We compared the ANI, AAI, and POCP values among the genomes of genus Sul furospirillum.The ANI values between 1612 and other Sulfurospirillum genomes were around 71% (Supporting materials, Fig. S4; Table S3), far below the hypothesized species demarcation threshold of 95% (63), indicating that 1612 represents a distinct species.The ANI value (Fig. S4; Table S3) was also lower than the threshold for genus definition (73.98%) ( 64), but AAI and POCP values were around the threshold to assign 1612 into a novel genus (Supporting materials, Fig. S4; Table S4).The 63-65% of AAI value between 1612 and other strains (Supporting materials, Fig. S4; Table S4) was below the suggested 65% threshold to set a novel genus (65), but a little higher than the suggested 60% boundary mentioned in another study (66).The results of POCP values (55-63%, in Supporting materials, Table S5) were higher than 50%, a genus boundary for prokaryotic lineages (57).Considering the uncertainty of POCP value in delineating a new genus, we proposed strain 1612 to be a novel species within the genus Sulfurospirillum, named as Sulfurospirillum kueishanense sp.nov.However, the potential to establish a new genus out of genus Sulfurospirillum needs to be considered in the future.

Genomic features of the novel Campylobacteria isolate from SW-HTVs
The genome of strain 1612 (accession no.CP140614) comprised one circular chromo some with 2,377,931 bp containing 2365 open reading frames (Supporting materials, Table S6; Fig. S5).The total length of all the identified genes was 2,228,148, accounting for 93.7% of the total genomic length.The total GC-content was 37.84%.A total of 6 rRNAs (5S + 16S + 23S) and 39 tRNAs were detected.Approximately 90.19% of all identified genes, about 2,133 genes, could be annotated by the COG database.Excluding "function unknown, " the categories "energy production and conversion" and "amino acid transport and metabolism" were the most abundant, represented by about 227 and 200 genes, respectively (Supporting materials, Fig. S6).Using the KEGG database, approximately 58.01% of all identified genes, about 1372 gene, could be annotated.Most of the annotated genes were related to "metabolism" pathway (green histogram in Supporting materials, Fig. S7).

Metabolisms and potential functions of the Campylobacteria isolate from SW-HTVs
By combining genome-scale analyses with known physiological traits, the metabolic network of strain 1612 was reconstructed (Fig. 2).The result showed that the bacte ria may live a chemolithoautotrophic lifestyle with high plasticity on metabolisms, potentially contributing to several essential biogeochemical processes such as carbon fixation, hydrogen oxidation, sulfur reduction/oxidation and nitrate reduction.
In addition to carbon fixation, strain 1612 also showed potential for using organic carbon.While glycolysis was incomplete due to the lack of some irreversible enzymes (such as phosphofructokinase in catalyzing phosphorylate fructose-6-phosphate to fructose-1,6-bisphosphate), the potential to degrade glucose was supplemented by the Entner-Doudoroff pathway (KEGG module M00008) in 1612.
Adenosine triphosphate (ATP) production in strain 1612 occurs through substratelevel phosphorylation and oxidative phosphorylation.Substrate level phosphoryla tion (KEGG module M00579) involves enzymes like phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate.Additionally, strain 1612 possesses an F-type ATPase to synthesize ATP through oxidative phosphorylation, utilizing a series of enzyme complexes in the respiratory chain and menaquinone to shuttle electrons along the electron transfer chain.The detected genes of respiratory complexes included the Complex I (NADH:quinone oxidoreductases I, nuoA ~N), Complex II (Succinate dehydro genase/Fumarate reductase, sdhBC and frdABC), Complex III (Cytochrome bc1 reductase), Complex IV (Cytochrome caa 3 -type oxidase, coxABCD; and the cbb3-type, ccoNOPQ), and the Cyt bd oxidoreductase (cydAB) (Fig. 2).Both cbb 3 -type cytochrome c oxidase (67) and Cyt bd oxidoreductase (68) have a high affinity for oxygen and typically function under low-oxygen conditions.In contrast, cytochrome caa 3 -type oxidase has a low affinity for oxygen and is usually expressed and functional under high oxygen conditions in many bacterial species, such as Paracoccus denitrificans, Bradyrhizobium japonicum, and Rhodobacter sphaeroides (69)(70)(71).
Strain 1612 also possesses genes encoding enzymes involved in oxidizing (Sqr, Sox, Ttr, and TST) or reducing (Psr) sulfur compound (Fig. 2).It also included genes for dissimilatory nitrate reduction to ammonium (DNRA) (such as nap and nrf genes involving in reducing nitrate and nitrite reduction to ammonium) and part of denitrification pathway (nos gene involving in N 2 O to nitrite and dinitrogen), suggesting a role in nitrogen cycling.

Physiological experiments validating the metabolic capabilities of strain 1612
The physiological experiments performed aimed to validate the annotated chemotro phic lifestyle of the isolated Campylobacteria species.The results demonstrated that strain 1612 exhibited robust growth when utilizing CO 2 as the sole carbon source in conjunction with H 2 as the electron donor, and either nitrate, thiosulfate or S 0 as electron acceptors (Fig. 4A and B).However, no growth was observed when sulfate, sulfite or nitrite was employed as electron acceptors in conjunction with hydrogen as the sole electron donor and CO 2 as the only carbon source (Fig. 4A and B).Strain 1612 also displayed the ability to utilize simple organic carbon sources, including formate, lactate, pyruvate, fumarate, succinate, and malate, as the electron donors (Fig. 4C, D and E).Each test was conducted with a single type of organic carbon added, while S 0 served as the sole electron acceptor and CO 2 as the only carbon source.Interestingly, when acetate was added, strain 1612 exhibited growth on formate as the sole electron donor in conjunction with nitrate, thiosulfate, or S 0 as the sole electron acceptors (Supporting materials, Fig. S8A and B).Moreover, strain 1612 appeared growing better in the MMJHS medium containing both S 0 and S 2 O 3 2− (Supporting materials, Fig. S8D).However, no growth was observed when sucrose was used as electron donor and S 0 as the electron acceptor (data not shown).The sensitivity to oxygen in strain 1612 was also tested.As shown in Fig. 4F, strain 1612 was not a strict anaerobe and can tolerate a certain level of oxygen, as evidenced by better growth in the absence of an oxygen scavenger.

Pan-genome analysis and functional traits for bacterial adaptation in SW-HTVs
We analyzed 13 complete genomes of Sulfurospirillum strains, including the newly isolated 1612 in this study.These strains were sourced from diverse origins.All the genomes were utilized to construct the genomic phylogenetic tree (Fig. 1).The anvi'o pan-genomic pipeline distinguished 7,553 gene clusters (GCs) out of a total of 35,485 genes across the 13 genomes.The highest numbers of GCs were observed in the core genes shared among all genomes, followed by the unique genes of S. arcachonense, FIG 4 The growth curves for the substrate testing.The spectrum of electron acceptor testing when using H 2 as the sole electron donor (A&B).The electron donor testing was proceeded by adding lactate, pyruvate, fumarate, succinate, malate, or formate when S 0 was the sole electron acceptor (C-E).The growth profile of 1612 under different concentrations of oxygen scavenger (F).All the "control" in the figure were  1612, S. multivorans, and S. cavolei (Fig. 5A).Most of the core genes were associated with essential functions, such as energy production/conversion, translation, and amino acid transport/metabolism (Fig. 5C).The pan-genome profile illustrated that as the number of sequenced genomes increased, the pan-genome size expanded rather than reaching a plateau (Fig. 5B).Consequently, it can be inferred that the pan-genome of the genus Sulfurospirillum followed an open type, indicating a strong potential for horizontal gene transfer events.Considering the potential of 1612 to be a novel genus, we recalculated the pan-genome profile of genus Sulfurospirillum without including strain 1612.The result still indicated an open-type pan-genome (Supporting materials, Fig. S10).
To discern the functional distinction between 1612 and other strains, we identified five bins including the core gene bin, two accessory gene bins, and two unique genes bins using the visualization platform of the Anvi'o pan-genomic workflow (Fig. 6A).The "core genes" comprised 12,889 genes belonging to 957 GCs (Fig. 6A).Most of its genes could be annotated by KEGG (79%) or COG (96%), while the ratio of annotated genes was much lower for the other bins (Supporting materials, Table S8).The GCs number of the "unique genes of 1612" bin and "unique genes of S. arcachonense" bin was comparable to the "core genes" (Fig. 6A).Protein families involved in "protein families: signaling and cellular processes, " "signal transduction, " "membrane transport, " and some "poorly characterized" genes had higher ratios in the unique genes of 1612 and S. arcachonense compared to the core genes (Fig. 6B).At a deeper functional annotation level, signal-related processes including "transporters, " "two-component system, " and "ABC transporters" were much more prevalent in the unique and accessory genes than the core genes (Supporting materials, Fig. S11).At the gene level, we also analyzed the KEGG orthology (KO) detected only in 1612.It was found that 1612 had specific ABC transporters of dipeptide, phosphate, and thiamine (Fig. 6B, purple block).Furthermore, several genes related to biofilm formation and benzoate/toluene degradation were exclusively detected in the unique genes of 1612 (Fig. 6B, blue and green blocks).Regarding energy metabolism, some genes associated with sulfur, nitrogen, methane, oxygen, and hydrogen were only detected in the "unique genes of 1612" (Fig. 6B, orange block, supporting materials, Table S9).

Comparison of key energy metabolic genes
Genes involved in energy metabolisms were compared among genomes, including 13 genomes of Sulfurospirillum and 6 genomes of mesophilic Campylobacterial isolates from deep-sea HTVs (Fig. 7).The genome of strain 1612 harbored characteristic enzyme genes of rTCA cycle for CO 2 fixation (encoded by genes aclAB and oorABCD), sulfur Genes without annotated information were not shown in the figure but used in the calculation of relative abundance.Some annotation categories of "unique genes of 1612" bin were extended to show the information of its unique KOs.
oxidation system SOX, hydrogenase, and nitrate reductase, supporting the potential of chemoautotrophy.The genes of CO 2 fixation (aclAB) and sulfur oxidation system (soxABCDYZ) were presented in the genomes of all collected isolates from deep-sea HTVs and 1612.Except for 1612, the potential of rTCA cycle for CO 2 fixation (aclAB) was also detected in S. cavolei, but was absent in other Sulfurospirillum species.

DISCUSSION
Our novel isolate 1612 from the Kueishan Island SW-HTVs enriched the species diversity of Campylobacteria.The two major orders, Nautiliales and Campylobacter ales, within Campylobacteria, exhibited clear differences in their metabolic potential and core bioenergetics (6).Genera belonging to the order Nautiliales, such as Nau tilia, Caminibacter, Lebetimonas, Cetia, Nitrosophilus, and Nitratiruptor, are commonly found in deep-sea HTVs, according to data from LPSN (List of Prokaryotic names with Standing in Nomenclature).In contrast, most cultured representatives of the order FIG 7 Color-coded table indicating major functional genes and their abundance in 13 genomes of Sulfurospirillum and 6 genomes of mesophilic chemolitho trophic Campylobacteria.The y-axis indicated the genes likely involved in energy metabolism, and the x-axis indicated the strain designations.Red color corresponded to the complete genes identified in carbon, sulfur, and nitrogen nutrient cycles; pink indicated the partial identified in the gene cluster; white indicated an absence of relevant genes.
Campylobacterales thrive at lower temperatures and can typically tolerate a much greater range of oxygen concentrations (15).Genera Sulfurovum and Sulfuromonas are the frequently studied Campylobacterales in vent area, with few reports of gen era Nitratifractor and Hydrogenimonas, based on data from LPSN.Although the initial enrichments of Campylobacteria isolated from deep-sea HTVs (including Am-H and Ex-18.2) (73) are recognized as Sulfurospirillum and have been tested to be moderately thermophilic sulfur-reducing heterotrophs or sulfur lithoautrotrophs using hydrogen as the electron donor, their draft genomes do not provide a basis for deeper comparison with other typical strains of Sulfurospirillum.In this study, our novel isolate (strain 1612) of genus Sulfurospirillum expands the strain diversity of Campylobacterales in hydrother mal vent areas.Through systematic genomic and phenotypic study, these results may contribute to a better understanding of mesophilic Campylobacteria in hydrothermal vent systems.
The ability to fix CO 2 was only detected in strain 1612, but not in other Sulfurospiril lum species.Genus Sulfurospirillum consists of versatile, often microaerophilic bacteria, capable of growing with various growth substrates (74), including hydrogen, formate, nitrate, sulfur compounds, and many toxic compounds (e.g., PCE, toxic arsenate, and selenate).Upon thorough comparison of phenotypic and genomic features within genus Sulfurospirillum, we found that the capacity for chemoautotrophy appears to be a unique metabolism for the novel Sulfurospirillum strain from the Kueishan Island SW-HTVs (strain 1612) (Table 1; Fig. 7).Although chemoautotrophic potential has been partially shown by detection of the aclAB gene in the genome of S. cavolei (75), our study is the first to clearly demonstrate the chemolithoautrophic capacity of the genus Sulfurospirillum via combined results of genomic features and experimental validations.Similar to the prevalent Campylobacterial isolated from deep-sea HTVs (76), such as the mesophilic Sulfurovum and Sulfurimonas, chemolithoautotrophy also appears to be a common feature of Campylobacteria in SW-HTVs.On the other side, considering the unique ability to fix CO 2 in genus Sulfurospirillum and the monophyly formed by strain 1612 and S. arcachonense in the phylogenomic tree (Fig. 1), the potential to establish strain 1612 and our other isolates into a new genus out of genus Sulfurospirillum needs to be considered in future studies.
Hydrogen oxidation is widely distributed in Campylobacteria (58).As demonstrated in Wolinella succinogenes (86), most Campylobacteria only contain Group 1b hydrogenase genes (58).The newly isolated strain 1612 harbors both subgroup 1a, 1b and cytosolic H 2 -uptake subgroup 2d hydrogenase (Fig. 3).Subgroup 2d hydrogenase inferred to generate reductant for carbon fixation via rTCA (87).The observed diverse phylotypes of H 2 utilization hydrogenase genes might be essential for strain 1612's survival in hydrothermal vent ecosystem, as hydrogen oxidation is among the chemosynthetic reactions providing the greatest energy yields (88).Moreover, hydrogen oxidation seems to be a favorable energy source for autotrophic carbon fixation compared to the oxidation of sulfide/thiosulfate, despite considerably more energy being yielded through the oxidation of sulfide/thiosulfate than through hydrogen oxidation (free standard enthalpies are −797 kJ/mol H 2 S vs −237 kJ/mol H 2 with O 2 as electron acceptor) (89).This is because the redox potential of hydrogen is more negative than that of the reducing equivalent NAD(P)/H; in contrast to sulfide, a reverse electron transport is not required  in conjunction with hydrogen oxidation.Thus, only a third of the energy is required for fixing 1 mol of carbon when oxidizing hydrogen compared to sulfide (1,060 kJ for hydrogen vs 3,500 kJ for sulfide) (90).The Group 4a (Hyf) and its homologous Group 3 (Hyc) hydrogenase both function in the FHL complex during fermentation, which produces H 2 in Escherichia coli (85).Similar with Group 4e (Ech) [NiFe]-hydrogenase, they are absent in genomes of typical Campylobacteria strains isolated from deep-sea HTVs, such as those belonging to genera Sulfurovum, Sulfuromonas, and Nitratifractor (Fig. 7).In contrast, the existence of hyf genes (Group 4a) have been reported in the most genomes of Sulfurospirillum, such as S. multivorans (91) and Sulfurospirillum diekertiae (92).The H 2 production activities have also been demonstrated in Sulfurospirillum species, that is, S. multivorans, S. cavolei, S. deleyianum, and S. arsenophilum, which can fermentative grow on pyruvate and produce H 2 after about 20 generations of transfer from the initial cultures (93).The similar structures of hyf gene clusters between strain 1612 and Sulfurospirillum species (93) support the possibilities of H 2 production in strain 1612, yet the detailed mechanisms and in-situ activities of H 2 production need to be further studied in the future.
Diverse respiratory oxygen reductases (terminal oxidases) are found in Campylo bacteria isolated from SW-HTVs.In vent areas, Campylobacteria occupy high-sulfide, low-oxygen niche compared to chemoautotrophic Gammaproteobacteria (94,95).Considering the sharp chemical and physical gradients formed during the mixing of reduced hydrothermal fluids with oxic seawater, the importance of oxygen for Campylo bacteria is underscored.Recent short-term incubations have indicated the importance of oxygen in structuring natural communities and affecting carbon fixation efficiency for Campylobacteria (96).There are three enzyme superfamilies capable of acting as terminal respiratory oxygen reductases-heme-copper oxygen reductases, alternative oxidases, and Cyt bd oxidoreductases (97).Most terminal oxidases belong to the heme-copper oxygen reductases superfamily, such as cytochrome c oxidase (Cox), which uses a c-type cytochrome as its electron donor.Cox emzyme are universal in aerobic and facultative aerobic organisms, represented by caa 3 -type and cbb 3 -type oxidases (98).For our strain 1612 isolated from SW-HTVs, genes for respiratory oxygen reductases with both high (cbb 3 -type Cox and Cyt bd oxidoreductase) and low (caa 3 -type Cox) affinity for oxygen were identified in its genome (Fig. 2).This indicates that strain 1612 is a facultative aerobe, a conclusion supported by the physiological test with oxygen scavenger addition (Fig. 4F).In comparison, most isolates of genera Sulfurovum and Sulfurimonas from deep-sea HTVs only have the genes ccoNOPQ of cbb 3 -type Cox (Fig. 7).Higher concentrations of oxygen are reported in SW-HTVs than in their deep-sea counterparts (4,99).This may be due to more effective mixing with oxygen-rich surface waters and the influence of tidal and wave actions.In Kueishan Island, high temporal variations in water temperature are attributable to diurnal tides (100).It is, therefore, reasonable to expect a similar variation in other parameters within SW-HTVs (99).The presence of more types of oxygen reductases in strain 1612 confirms the higher variable in oxygen concentration in SW-HTVs compared to the deep-sea environment.
Through our study, we identified unique genes in strain 1612 by comparing its genome with the ones of non-vent strains of Sulfurospirillum (Fig. 6B).These unique functional traits of strain 1612, possibly gained via genome extension events (e.g., gene horizontal transfer).Indeed, at least three genomic islands in genomes of 1612 (Supporting materials, Table S6) were found to correspond to the presence of horizon tal gene transfer events.Such genomic plasticity increases micro-diversity and confers a competitive advantage enabling this lineage to thrive in changing physical-chem ical gradients of hydrothermal systems (101).Reflecting the complex environmental conditions within SW-HTVs, the diverse signal genes identified in unique genes of strain 1612 may facilitate its adapting and dominance in vent area.This finding prompts us to focus on specific sources (e.g., dipeptide, phosphate, and thiamine) within the vent area to gain a deeper understanding of Campylobacteria.However, due to limitations in genome numbers and functional annotation, the results still lack a refined selection of target genes.Further physiological experiments involving transcriptome analysis and gene knockout techniques are necessary to elucidate the details of the adaptation mechanisms.

FIG 1
FIG 1 Phylogenomic tree of Campylobacetial isolates.A total of 105 Campylobacetial reference genomes with the complete length were downloaded from NCBI.Combined with some other Sulfurospirillum isolates' genomes, this phylogenetic tree was reconstructed using PhyloPhlAn software.Different colors correspond to the sources of isolates.The root was composed of three genomes of genus Hippea.The scale bar indicates 0.1 substitutions per amino acid position.

FIG 3
FIG 3Classification and phylogeny of hydrogenases identified from 1612 genome.(A) The neighbor-joining skeleton trees showed the phylogenetic relationships of hydrogenases structural gene large subunit identified from strain 1612 and previously reported bacteria(59).The central circle trees were colored by [NiFe]-hydrogenase subgroups.The black nodes indicated the well-supported nodes (bootstrap values >80).Detailed information was shown in Fig.S9; (B) The six hydrogenase clusters identified from the genome of 1612.The color of genes corresponded to the COG categories.The gene names of structural genes of hydrogenase were colored in red.The large subunit used to construct the phylogenetic tree was marked with a gene number.Each cluster included one or two groups of hydrogenases.The hydrogenase subgroups of each cluster were shown in the brackets below the cluster name.The gene hyfG of cluster 6 was annotated as hycE in KEGG, but we renamed it to "hyfG" as it belonged to group 4a hydrogenase in the phylogenetic tree.

FIG 5
FIG 5 Pan-genome analysis results.(A) Upset plot to highlight the number of genes sharing among genomes.The variable component of genomes contributing to a collection of gene clusters was marked by black dots.(B) The Pan-genome profile trends of the genus Sulfurospirillum.(C) The COG annotation results for the shared core genes.

FIG 6
FIG 6 Pan-genome analysis for 13 Sulfurospirillum genomes using the visualization platform of Anvi'o.(A) The first 13 layers, in which dark color indicated the presence of a gene cluster and light color indicated its absence, represented individual genomes.ANI values among different genomes were represented on heatmap determined from the high similarity (red) and low similarity (white).Five specific groups of gene clusters were selected into bins such as the "core genes, " which were indicated in different colors in the outmost layer.The numbers of gene clusters and responding genes included in each bin were listed under the bin names, and separated with "/." The dendrograms on the top represented the hierarchical clustering of genomes based on the frequencies of gene clusters.(B) The breakdown of gene annotation results shows the component of different KEGG level 1 categories in total gene numbers for each selected bin.

TABLE 1
(84)erential phenotypic characteristics of 1612 and other Sulfurospirillum species b,c Its sensitivity to oxygen is evaluated by a simple test involving the addition of oxygen scavenger to the medium (results see Fig.4F).S. cavolei DSM 18149 (75); and 10, 'S.carboxydovorans' DSM 16295(84).+, positive; −, negative; ND, not data.All strains are able to utilize the following as electron donors: formate (+ acetate), hydrogen (+ acetate) and pyruvate.All strains are positive for the fermentation of fumarate.