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Genome-centric insight into metabolically active microbial population in shallow-sea hydrothermal vents

Background Geothermal systems have contributed greatly to both our understanding of the functions of extreme life and the evolutionary history of life itself. Shallow-sea hydrothermal systems are ecological intermediates of deep-sea systems and terrestrial springs, harboring unique and complexed ecosystems, which are well-lit and present physicochemical gradients. The microbial communities of deep-sea and terrestrial geothermal systems have been well-studied at the population genome level, yet little is known about the communities inhabiting the shallow-sea hydrothermal systems and how they compare to those inhabiting other geothermal systems. Results Here, we used genome-resolved metagenomic and metaproteomic approaches to probe into the genetic potential and protein expression of microorganisms from the shallow-sea vent fluids off Kueishantao Island. The families Nautiliaceae and Campylobacteraceae within the Epsilonbacteraeota and the Thiomicrospiraceae within the Gammaproteobacteria were prevalent in vent fluids over a 3-year sampling period. We successfully reconstructed the in situ metabolic modules of the predominant populations within the Epsilonbacteraeota and Gammaproteobacteria by mapping the metaproteomic data back to metagenome-assembled genomes. Those active bacteria could use the reductive tricarboxylic acid cycle or Calvin-Benson-Bassham cycle for autotrophic carbon fixation, with the ability to use reduced sulfur species, hydrogen or formate as electron donors, and oxygen as a terminal electron acceptor via cytochrome bd oxidase or cytochrome bb3 oxidase. Comparative metagenomic and genomic analyses revealed dramatic differences between submarine and terrestrial geothermal systems, including microbial functional potentials for carbon fixation and energy conversion. Furthermore, shallow-sea hydrothermal systems shared many of the major microbial genera that were first isolated from deep-sea and terrestrial geothermal systems, while deep-sea and terrestrial geothermal systems shared few genera. Conclusions The metabolic machinery of the active populations within Epsilonbacteraeota and Gammaproteobacteria at shallow-sea vents can mirror those living at deep-sea vents. With respect to specific taxa and metabolic potentials, the microbial realm in the shallow-sea hydrothermal system presented ecological linkage to both deep-sea and terrestrial geothermal systems. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-022-01351-7.

regions or near seamounts located in the center of tectonic plates [1]. The discovery of geothermal systems, harboring spectacular communities, has greatly expanded our knowledge and understanding of the habitat range for and the origin of life [2,3]. Deep-sea alkaline hydrothermal vents [4] and terrestrial hot springs [3] were proposed as locations that may have provided suitable conditions for prebiotic syntheses and acted as potential cradles of the very first life. Shallow-sea hydrothermal vents (depth < 200 m) not only present fluid geochemistry with temperature and redox gradients created by complexed physicochemical steps like those of deep-sea vents, but also experience exposure to sunlight as terrestrial hot springs do, thus harboring unique ecosystems that host a great biodiversity [1]. Recent emerging evidence strongly supports the view that life would have formed in a well-lit environment undergoing wetdry cycles with a limited amount of water [5][6][7] and then would have subsequently expanded into the oceanic environment [5]. Investigations of shallow-sea hydrothermal systems, the physicochemical intermediate of terrestrial and deep-sea geothermal systems, should reveal additional clues for the origin or evolution of life. Moreover, it is well known that the heterogeneity and similarity in microbial populations and functional potentials among distinct geothermal systems to date are not completely elucidated.
Compared to their deep-sea counterparts, shallow-sea hydrothermal vents are more accessible and less expensive to sample by scuba divers. Therefore, shallow-sea vents are good sources for studying genetic adaptation in the hostile environments. Previous studies have confirmed high compositional and functional diversity of chemosynthetic ecosystems in diverse shallow-sea hydrothermal systems using sequencing-based metagenomic technologies [8][9][10][11][12][13]. The shallow-sea vents, like their deep-sea counterparts, may contain a reservoir of physiologically diverse microorganisms, many of which are endemic to submarine hydrothermal systems at the genus level, such as the Nautilia and Caminibacter within the class Epsilonproteobacteria (reclassified to a new phylum Epsilonbacteraeota; [14]), and the gammaproteobacterial Thiomicrospiraceae [15]. Transcriptomic and metaproteomic studies of shallow-sea hydrothermal microbiomes noted potential connections between the metabolic capacity of the microbial population and chemical characteristics of the vent fluids [16,17]. These studies have focused on characterizing community-wide metabolic networks in shallow-sea hydrothermal vents; however, our knowledge of population-level networks still remains limited, partly due to the absence of cultivated isolates [18]. Genome-resolved metagenomic analysis of microbial communities enables the reconstruction of individual genomes, providing the necessary information to examine how well they adapt to hydrothermal systems. Accordingly, enormous efforts over the past few years have generated a vast number of metagenome-assembled genomes (MAGs) from deepsea vents and terrestrial hot springs, some of which may represent putative novel species and contain a wealth of information on their metabolic potential [19][20][21][22][23][24]. Yet, microbial MAGs from shallow-sea hydrothermal systems are still scarce [17]. Ideally, metagenome-assembled and metaproteomic approaches should be combined to identify the microbial population-level metabolic network, thereby bridging the gap between a microbe's taxonomy and its functional traits.
In this study, we sampled the fluids being emitted from vent orifices at an andesite-hosted shallow-sea hydrothermal system off the eastern shore of Kueishantao Island, Taiwan, over a 3-year period (Fig. 1). Metagenomic shotgun sequencing and recovery of MAGs was used to investigate the metabolic function potentials of the collected microorganisms that allow them to survive in the extreme and dynamic vent environment. A MAG-centric metaproteomic approach was used to construct the metabolic modules of those active and predominant populations in the shallow-sea venting fluids. Furthermore, we characterized the functional features of the microbes from geochemically distinct vent fields via metagenomic and genomic analyses. This study not only provides insights into the metabolic machinery of ventassociated microbes living in situ, but also represents a major step forward towards a comprehensive understanding of the biogeographic pattern of microbial metabolic function for different geothermal systems.

Sites and sample collection
Time-series monitoring sampling was performed at two different types of shallow-sea hydrothermal vents, namely yellow vent (HQ) and white vent (BQ), located near Kueishantao Island, offshore of northeast Taiwan 3) was added to each filter tube, which was then sealed at both ends with parafilm. Filters were placed into 50-mL centrifuge tubes and then frozen by liquid nitrogen immersion and stored at -80 °C until further use. A detailed filtration protocol can be found online at http:// www. jove. com/ video/ 1159 [25].
Fluid samples were split into aliquots onboard. Unfiltered fluids were transferred to glass and plastic containers, for carbonate chemistry and nutrient analysis, respectively. Both types of samples were immediately treated with a saturated mercury chloride (HgCl 2 ) solution at a volume ratio of 1000:1, and frozen at -20 °C. For prokaryotic abundance, triplicate fluid samples (2 mL, prefiltered by 20 μm) were fixed with glutaraldehyde at a final concentration of 0.5% and stored in the dark at -20 °C. Additional fluid samples (0.5-0.8 L) were filtered using Whatman GF/F glass fiber filters (47 mm), which

Biogeochemical analysis
The in situ temperature of vent fluids was determined by scuba divers using a thermocouple. Salinity values were measured onboard shortly after collection with an Autosal 8400B salinometer calibrated with IAPSO standard seawater. pH values were measured with a pH meter (Radiometer PHM-85, Denmark) at 25 °C, with a precision better than 0.003 pH unit. Dissolved oxygen was measured using a gas chromatographic method. Nitrate, nitrite, and ammonium nitrogen concentrations were colorimetrically analyzed with a continuous flow autoanalyzer (QuAAtro, SEAL Analytical Inc.). Dissolved sulfide concentrations were assayed by the methylene blue method [26] (the precision was 0.7% at 20 μM, n = 6), immediately after returning to the landbased laboratory. Dissolved CH 4 was measured by gas chromatography using the gas-stripping method [27]. A dissolved inorganic analyzer (AS-C3, Apollo SciTech, United States) was employed for the analysis of dissolved inorganic carbon values, with a precision of 0.1%. Chl a was extracted from the filters using the acetone extraction method and measured with a fluorometer according to the procedure summarized in [28]. Prokaryotic abundances were determined with an Accuri C6 flow cytometer (BD Biosciences).

DNA extraction, metagenomic sequencing, and assembly
Total genomic DNA was extracted from Sterivex filters as described in [29]. Briefly, lysozyme (0.125 mg mL −1 ) was added to the thawed Sterivex filters and then incubated at 37 °C for 1 h with intermittent mixing, followed by the addition of Proteinase K (Qiagen) and 20% SDS and incubated at 55 °C for 2 h with intermittent mixing. Cell lysate was removed using a syringe, and the filters were then rinsed with lysis buffer that was combined with the original lysate. The lysate was extracted by phenol: chloroform: IAA (25:24:1, pH 8.0) and the aqueous layer collected by centrifugation, then loaded onto a 10 K Amicon Ultra filter cartridge (Millipore), washed three times with 2 mL TE buffer, and concentrated by centrifugation to a final volume of 200-500 μL. The concentration and the quality of all DNA samples were evaluated using the NanoDrop-1000 (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. DNA was fragmented via Covaris M220 sonicator (Covaris, Woburn, MA, USA), and the library was prepared with the NEBNext DNA Library Prep Kit (Illumina) following the manufacturer's instructions. Paired-end (2 × 150 bp) metagenomes were sequenced on the Illumina Hiseq platform. Sequence quality was assessed with FastQC v0.11.2 [30], and sequences were then trimmed using Trimmomatic v0.36 [31] to remove adaptors and low-quality bases. A minimum quality score of 20 and a read length higher than 35 were required. The clean reads were assembled using the assembler IDBA-UD v1.1.2 [32].

Metagenomic binning and annotation
Genome reconstruction utilizing the metagenomic sequencing data was performed with the function modules of metaWRAP v1.1.1 [33] to recover individual genomes. The module MetaWRAP Bin_refinement was used for further refinement on the sets of bins. Genome quality was estimated using CheckM v1.0.12 [34] to calculate genome completeness and contamination (Table  S1). The Metagenome-Assembled Genome standards set up by the Genomic Standards Consortium [35] were also used to evaluate the quality of the MAGs (Table S1).
Open reading frames of the assembled contigs from the metagenomes and MAGS were predicted using Prodigal v2.60 [36] for those sequences longer than 100 bp. The sequences were clustered using CD-HIT [37], with 85% coverage and 90% identity. Gene sets were aligned to NCBI non-redundant database using DIA-MOND BLASTP [38] for taxonomic annotations, with an e-value cutoff of 1 × 10 −5 . The predicted protein coding sequences were blasted (BLASTP, e-value < 10 −5 ) against eggNOG using DIAMOND to obtain Clusters of Orthologous Groups of proteins (COG) annotation [38,39]. GhostKOALA servers were used to obtain Kyoto Encyclopedia of Genes and Genomes annotations for the gene set [40]. To determine the relative abundances of predicted genes both for metagenomes and MAGs, Bow-tie2 v2.1.0 (local alignment, default settings) was used to perform alignments between the clean reads with the non-redundant gene set [41]. SAMtools v0.1.18 was used to count the number of annotated sequences matched to each gene [42]. Reads per kilobase per million mapped reads (RPKM) calculations were carried out to facilitate transparent comparison of gene abundance between samples [43]. Sequencing and analysis summary statistics for the number of generated reads and the quality of all the metagenomes are provided in Supplementary Table  S2.

Phylogenetic and comparative genomic analysis
Taxonomic lineages of MAGs were classified using the recently developed database GTDB release v95 [44] and the relevant Genome Taxonomy Database Toolkit GTDB-Tk v1.2.0 [45]. A phylogenetic tree using a concatenated alignment of four single-copy orthologous genes shared by all bins (COG0016, COG0532, COG0533, and COG0541) was constructed by the Maximum Likelihood method using MEGA X with LG+F model plus a gamma distribution with eight categories [46]. Bootstrap resampling was performed for 1000 replications. Phylogenetic trees were visualized with Evolview v3 [47]. Comparative genome analyses were carried out on all MAGs in this study, combined with publicly available microbial genomes from marine and terrestrial geothermal systems in the Integrated Microbial Genomes (IMG) database [48]. To retrieve the gene information, searches of the IMG database were performed using both keywords and enzyme codes, and the selected sequences were filtered manually. The average nucleotide identity (ANI) and amino acid identity (AAI) between two genome sequences were calculated by enveomics tools [49]. The previously defined six types (I-VI) of 92 sulfide quinone oxidoreductase (Sqr) amino acid sequences [50] were used as seeds to find sqr homologs within the metagenomic and genomic datasets by BLASTP, and a phylogenetic tree of Sqr protein sequences was created by the Neighbor-Joining method using MEGA X for classification of Sqr [46]. Putative hydrogenase classification was confirmed and classified using HydDB [51].

Environmental protein extraction and identification
The extraction of total protein from Sterivex filters was performed as described in [29]. Briefly, BugBuster (Novagen) was added to the Sterivex filters to lyse cells, and the lysate was then recovered using a syringe. Buffer exchange was implemented with 100 mM NH 4 HCO 3 on a 10K Amicon (Millipore). Samples were amended with urea and dithiothreitol and then subject to overnight trypsin digestion, followed by purification with μ-C18 Zip Tips (Millipore) and vacuum dried. The peptides were resuspended in solution A (0.1% formic acid in 2% acetonitrile) and separated by Nano HPLC (Easy-nLC 1000 HPLC system, Thermo Fisher Scientific) then analyzed by tandem mass spectrometry (MS/MS) in a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Separation was achieved using a self-packed reversed-phase analytical column (C18, 15-cm length, 75 μm i.d.) with a segmented linear gradient at 350 nL min −1 : 5-8% solution B (0.1% formic acid in 90% acetonitrile) for 2 min, 8-24% solution B for 40 min, 24-36% solution B for 12 min, 36-80% solution B for 3 min, and finally maintained at 80% solution B for 3 min. The electrospray voltage applied was 2.0 kV. Mass spectrometry data were acquired using a data-dependent procedure with a full scan (350-1600 m/z, resolution 60,000) followed by MS/MS (100 m/z, resolution 15,000; HCD relative collision energy 28%) on the 10 most intense ions with a dynamic exclusion duration of 30 s. The acquired spectral data were searched using the MaxQuant search engine (v.1.5.2.8) [53] to a target-decoy database, containing the peptide sequences of the corresponding metagenome (ORFs counts: 74,611, 48,331, and 673,029 for 2018 BQ and HQ, and 2019 BQ metagenome, respectively), as well as reverse decoy database and common laboratory contaminants. The following parameters were used for protein identification: enzyme type, trypsin; maximum missed cleavage, 2; precursor ion tolerance: 5 ppm; fragment ion tolerance: 0.02 Da; fixed modification: carbamidomethyl cysteine; variable modifications: oxidation on methionine, N-terminal protein acetylation). Minimum score for peptides was set to more than 40 and false discovery rate was adjusted to less than 1%. The normalized spectral abundance factor (NSAF) values were calculated [54], normalizing spectral count values to protein size and to the sum of all spectral counts in each sample, thus giving the relative abundance of a given protein or protein group that could be compared between samples.

Environmental parameters
The two shallow-sea vents fields, HQ and BQ, expelled elemental sulfur-rich yellowish and whitish fluids, respectively [16]. The emitted vent fluids were rich in dissolved inorganic carbon (2738-5087 μM), acidic (pH 5-6 on average, lowest recoded pH was 1.6), and hot (temperatures between 90 and 102 °C in HQ, between 38 and 80 °C in BQ) (Fig. 1). The fluids were rich in oxygen and presented high variation in the actual concentrations between distinct sites (Fig. 1). Microbial abundances decreased as temperature increased in the shallow-sea hydrothermal vent environment, ranging from 7.01 × 10 5 to 7.61 × 10 6 cells mL −1 , with cell abundances in BQ higher than in HQ (Fig. 1). In addition, the microbial abundance reached undetectable levels at the HQ site when extremely hot and acidic fluids (102 °C, pH 1.6) were present in 2019 (Fig. 1), showing a natural limit to life in this shallow-sea hydrothermal system. In contrast to the abundance of microbial photosynthetic organisms in terrestrial hot springs, these organisms were scarce in the shallow vents with extremely low or undetectable Chl a contents (Fig. 1).

Metagenomic analysis of shallow vents
The metagenomes of microbial communities over the 3-year period contained highly abundant reads assigned to the genes from Epsilonbacteraeota, of which the majority of matching hits were to Nautiliaceae (up to 71.3% of the total functional gene sequences in an HQ metagenomic dataset from 2017), followed by Campylobacteraceae ( Fig. 2A). The sequences assigned to genes from the gammaproteobacterial Thiomicrospiraceae were also prevalent, but their relative abundance varied markedly in the metagenomic datasets (0.1-15.9% of all annotated sequences) ( Fig. 2A). Epsilonbacteraeota have the ability to tolerate a much higher sulfide concentration than Gammaproteobacteria, which possibly facilitates their competitiveness in colonizing sulfidic environments [17]. During our sampling timeframe, sequences matching genes from Epsilonbacteraeota occupied a higher percentage in HQ metagenomes than BQ, while those matching genes from Thiomicrospiraceae displayed the opposite trend ( Fig. 2A), consistent with the higher sulfide concentrations and lower pH in HQ (Fig. 1). Of the archaeal matches, the most common hits were Crenarchaeota (class Thermoprotei) and Euryarchaeota (class Thermococci) ( Fig. 2A). They accounted for < 1% of the total assigned sequences in metagenomic datasets from 2017 and 2018, whereas in a BQ metagenomic dataset from 2019, they comprised 4.1% and 2.5% of the total annotated sequences, respectively. Approximately 0.1-1.3% of the total gene sequences were assigned to cyanobacteria in datasets (contained within the "others" group in Fig. 2A).
The relative abundances of genes involved in aerobic respiration, H 2 , carbon, nitrogen, sulfur and phosphorus metabolisms from the metagenomic datasets are available in Table S4. The genes involved in carbon fixation, sulfur metabolism, hydrogen oxidation, oxygen utilization, and denitrification were similar in each metagenome, but their abundances varied (Fig. 2B). The relative abundances of the genes encoding ATPcitrate lyase (aclAB, RPKM ranged from 777.22 to 2345.20), as markers for the reductive tricarboxylic acid cycle (rTCA) [55], as well as key genes encoding fumarate reductase (frdAB, 881.69-1117.45 RPKM) and 2-oxoglutarate: ferredoxin oxidoreductase (korAB, 485.84-1630.51 RPKM) were abundant in the metaproteomes (Table S4). Their relative abundances were higher than those encoding ribulose-1,5-bisphosphate carboxylase (rbcLS, < 100 RPKM; Table S4), the markers for the Calvin-Benson-Bassham cycle (CBB) [55]. This result suggested that the rTCA cycle was potentially a major CO 2 fixation pathway in these shallow-sea hydrothermal vents. Investigation of the genes related to sulfur oxidation showed that type IV and type VI sulfite:quinone oxidoreductase (sqr) genes [50] were numerically dominant in all datasets, with RPKM values of up to 2587.85 and 965.77, respectively (Fig. 2B), while genes encoding flavocytochrome c sulfide dehydrogenase (fccAB), from the truncated sox multienzyme system (sox) genes for sulfur oxidation [56], were found at lower abundances among the metagenomic datasets (< 200 RPKM; Fig. 2B and Table S4). FccAB mediates the initial step of H 2 S oxidation to elemental sulfur (S 0 ), which has been observed to function under low sulfide conditions [56]. All metagenomes contained a gene encoding for polysulfide reductase (psr; Fig. 2B), which is responsible for the quinone-coupled reduction of polysulfide to H 2 [57]. Genes encoding formate dehydrogenase (fdh) for the oxidation of formate [58] were enriched in the metagenomes (473.45-1180.78 RPKM).
[NiFe]-hydrogenases-encoding sequences for the oxidation of H 2 [59] were abundant in the metagenomes (306. .07 RPKM; Fig. 2B and Table S4). Metagenomes revealed the presence of genes encoding for high-affinity cytochrome bd-type (cydAB, averaged RPKM of 733.99) and cbb3-type oxygen reductases (ccoNOP, up to 120.52 RPKM), as well as low-affinity cytochrome c oxidases (coxABC, less than 100 RPKM) [60]. Genes involved in dissimilatory (napAB and nirBD) and assimilatory (narB and nirA) nitrate reduction to ammonium [61] were also present in the metagenomes ( Fig. 2B; Table S4). Based on the genes participating in the redox reactions mentioned above, it could be hypothesized that microorganisms from these vent communities had the genetic potential to gain energy using sulfide, hydrogen, and formate as electron donors, while utilizing oxygen, polysulfide and nitrate as electron acceptors.
Each sample was binned individually, allowing us to evaluate the abundance and persistence of the recovered MAGs over the course of 3 years using metagenome fragment recruitment against the MAGs. One Nautiliaceae MAG (bin 2017-4) represented the most prevalent population accounting for approximately 2.32-11.57% in all metagenomes (Fig. S1). The two Lebetimonas MAGs (bins 2018-5 and 2018-6) accounted for approximately 13.41% and 2.63% of the BQ metagenomes reads in 2018, respectively, but they were only a small fraction (< 1%) in other samples (Fig. S1). The Thiomicrorhabdus MAGs (bins 2017-3, 2018-9, and 2019-2) were consistently observed each year and contributed up to approximately 12.78% of the BQ metagenome reads in 2018 (Fig. S1).  (Fig. S1).

Metaproteomic analysis of shallow-vent-associated microbes
A metaproteomic approach provides insight into the in situ expression of metabolic pathways of microbial communities at a given point in time. We performed protein mass spectrometry of samples from BQ and HQ in 2018 and BQ in 2019, yielding 23,680, 14,464, and 21,260 unique peptides, corresponding to 6286, 5164, and 7559 protein groups, respectively (Table S6). At the COG category level, proteins involved in energy production, translation, amino acid, and nucleotide metabolism as well as cell wall and membrane biogenesis, together, accounted for at least 50% of the relative abundances of peptides per metaproteome (Fig. 4). Meanwhile, proteins associated with carbohydrate metabolism were expressed at abundances of only 3-4% (Fig. 4). In addition, transporter proteins were present in the vent metaproteomes at approximately 1%, 5%, and 9% of total identified proteins in the 2018 HQ, 2018 BQ and 2019 BQ samples, respectively (Fig. S2), while those previously found in metaproteomes of surface and deep seawater heterotrophic communities generally account for 23-39% [62]. The diverse organic nutrient transporters that are generally rich in heterotrophic ecosystems, such as transporters for carbohydrates (including monosaccharides, disaccharides, and oligosaccharides), dicarboxylates, taurine, and  Table S6 urea [62], were not detected in the 2018 HQ metaproteome (Table S6).
The homologous proteins assigned to the recovered 20 MAGs, together, occupied to 77%, 59%, and 59% of total detected proteins in 2018 BQ and HQ, and 2019 BQ metaproteome, respectively. The information of homologous proteins assigned to each MAG in metaproteomes is shown in Table S7. The majority of identified proteins were affiliated with the Epsilonbacteraeota, followed by Gammaproteobacteria, which is in line with the observation in metagenomes. The organism with the highest overall abundance in the metaproteomes was Nautiliaceae bin 2017-4, accounting for 29% of the total identified proteins on average. Lebetimonas MAGs (on average 19% and 14% of metaproteomes for bins 2018-5 and 2018-6, respectively) and Thiomicrorhabdus MAGs (13% of metaproteome on average) were highly expressed as well. These data indicated that these bacterial populations represented the core and active taxa of the microbial communities, whose metabolic functions would reflect how the resident organisms could tolerate a hydrothermal environment.

Reconstruction of metabolic modules of predominant and active shallow-vent populations
Based on the protein profiles of individual MAGs, mapped back to their respective genomic data, we reconstructed and examined the core metabolic functions responsible for adaption to thermally and geochemically dynamic habitats. All enzymes required for a complete rTCA cycle and gluconeogenesis, as well as phosphoenolpyruvate carboxykinase for the removal of intermediates from the rTCA cycle [63], were expressed in Epsilonbacteraeota bins 2017-4, 2018-5 and 2018-6 ( Fig. 5A and Table S8). Enzymes for the non-oxidative pentose phosphate pathway that synthesizes ribose 5-phosphate for use in nucleotide biosynthesis [64] were also expressed ( Fig. 5A and Table S8). Nautiliaceae bin 2017-4 and Lebetimonas bin 2018-5 expressed Sqr for the oxidation of H 2 S, which would provide energy for their chemolithoautotrophic growth (Fig. 5A). Lebetimonas bin 2018-6 lacked Sqr, but possessed a [NiFe] hydrogenase (Fig. 5A), enabling it to use H 2 as an energy source. All three Epsilonbacteraeota MAGs had the ability to use formate as electron donors in the presence of formate dehydrogenase (Fig. 5A). They could use the cytochrome bd complex for oxidizing ubiquinol-reducing oxygen as part of the aerobic respiratory electron transport chain (Fig. 5A). Their type I NAD(P)H dehydrogenases [65] could contribute to electron transfer either through forward electron transfer (NAD(P)H to ubiquinone) or reverse electron transfer (ubiquinol to NAD + ) (Fig. 5A). The presence of nitrate reductase and polysulfide reductase (Psr) would allow all three Epsilonbacteraeota MAGs to use nitrate and polysulfide as the respective electron acceptors (Fig. 5A). Psr might conserve energy by proton translocation while balancing the reduced quinone pool [57]. The expression of multiple energetic metabolic pathways simultaneously for chemosynthesis may enable these populations to flourish across diverse redox gradients.
All enzymes involved in the CBB cycle and gluconeogenesis/glycolysis were identified in the gammaproteobacterial Thiomicrorhabdus MAGs (Fig. 5B and Table  S8). Succinate dehydrogenase that catalyzes the oxidation of succinate into fumarate [66] was absent in these MAGs, indicating an incomplete TCA cycle (Fig. 5B). These MAGs expressed malic enzyme and phosphoenolpyruvate carboxylase for the entry of intermediates (pyruvate and phosphoenolpyruvate, respectively) into the TCA cycle [67] (Fig. 5B), ensuring their ability to convert metabolites from the CBB cycle to biosynthetic precursors as well as generate reducing equivalents. Besides Sqr, their protein profiles revealed that the oxidation of reduced sulfur compounds could proceed via the FccAB and Sox pathways (Fig. 5B). Together, these two pathways are proposed to produce energy and reducing power via a reverse and forward electron transfer of sulfur oxidation, including cytochrome c proteins, cytochrome bc1 complex, and type I NADH dehydrogenase (or NADPH)-quinone oxidoreductases (Fig. 5B). Proteomic data indicated that these Thiomicrorhabdus respired oxygen with a cytochrome bb3 terminal oxidase ( Fig. 5B and Table S8). The metaproteomic results indicated that these MAGs had the ability to couple chemosynthesis with multiple energy metabolisms.
Two Epsilonbacteraeota (bins 2017-4 and 2018-5) and all three gammaproteobacterial MAGs could express carbonic anhydrase enzyme to sequester CO 2 [68], which could be subsequently incorporated into organic material (Table S8). Proteins involved in assimilatory nitrate reduction to ammonium were present in Nautiliaceae bin 2017-4 and all of the gammaproteobacterial Thiomicrorhabdus MAGs (Fig. 5). Further, glutamine synthetase for the assimilation of ammonium [69] was found in all these microorganisms (Fig. 5). The results indicated that these microorganisms are capable of meeting their nitrogen requirement via endogenous synthesis, whereas heterotrophs showed high expression of proteins for the uptake of organic nitrogen substrates [70]. Additionally, Thiomicrorhabdus microorganisms could use methylphosphonic acid as a phosphorus source if the transporter and enzymes involved in methylphosphonate utilization were present [71] (Fig. 5B). There were between 59 and 77 transporter genes in the Epsilonbacteraeota MAGs, less than the number observed in previously sequenced  (Table S8). Continuous lines represent complete metabolic processes for which all the relevant proteins were detected in the metaproteomes, while dashed lines represent pathways where only some of the proteins involved were detected (Table S8). Q, ubiquinone; QH 2 , ubiquinol heterotrophic Epsilonbacteraeota (75 to 124 genes) [70]. The autotrophic Thiomicrorhabdus MAGs possessed 87 to 113 genes coding for transporters, fewer than the number commonly found in heterotrophic gammaproteobacterial genomes (approximately 110 to 250 genes). Additionally, approximately 25.3-59.2% of the total transporter genes in these MAGs were expressed (Fig. S3), while generally 54.0-85.4% of the total transporter genes could be expressed in heterotrophic bacteria [72]. This is an apparent difference between these autotrophs and heterotrophs.

Metagenomic and genomic comparison of microbes from geothermal systems
We performed an in-depth comparative genomic analysis, using both metagenomes and genomes of microbes originating from three types of geothermal systems. The aclAB genes were enriched in microbial communities living in marine hydrothermal systems, especially for shallow-sea hydrothermal systems (average of 0.45 genes per cell; Fig. 6). The gene abundances of alcAB in deep-sea hydrothermal systems varied in a wide range, with the highest abundance up to 1.27 genes per cell in microbial communities from the East Pacific Rise, Pacific Ocean (Table S3). In contrast, terrestrial hot spring-associated microbial communities possessed a significantly lower aclAB abundance (average of 0.03 genes per cell, p < 0.0001; Fig. 6). In addition to Epsilonbacteraeota, the rTCA cycle has recently been identified as the CO 2 fixation mechanism used by members of Aquificae, which are often the dominant species in high-temperature and near-neutral terrestrial hot springs [73]. Consistently, all Epsilonbacteraeota and Aquifex MAGs recovered in this study possessed genes involved in the rTCA cycle (Fig. 7). Microbial rbcLS genes did not reveal any significant differences among distinct hydrothermal systems (Fig. 6). The CBB cycle ensures carbon fixation not only in autotrophic Gammaproteobacteria but also in thermophilic cyanobacteria (Fig. 7).
The average gene frequencies of sqr, fccB, and psrA genes were significantly more abundant in shallow-sea hydrothermal systems than other geothermal systems (p < 0.001; Fig. 6). The genes encoding [NiFe] hydrogenase group 1 were more abundant in oceanic sites than terrestrial ones (p < 0.05; Fig. 6). Furthermore, many Epsilonbacteraeota and Gammaproteobacteria, both those that have been cultured and the MAGs recovered in this study, harbored multiple sqr gene families (Fig. 7). Within the culturable genera from submarine vents and terrestrial springs, more than two-thirds of which possess sqr genes, while more than half contain multiple sets of hydrogenases ( Fig. 7 and Table S9), indicating the importance of sulfide and H 2 oxidation for energy generation. Notably, most hydrogen utilizers also possess genes encoding Sqr, both for the culturable genera and MAGs recovered in this study (Fig. 7), indicating their ability to generate energy via multiple redox reactions. Additionally, formate oxidation, which is commonly coupled with H 2 evolution mediated by H 2 -evolving hydrogenases for energy [59], appears to be prevalent in vent-or spring-inhabiting chemoautotrophs, including all of the Epsilonbacteraeota MAGs recovered from the Kueishantao vents ( Fig. 7 and Table  S10). With respect to aerobic respiratory, microbial communities living in shallow-sea hydrothermal systems were enriched in cydAB genes, with the abundance of average 0.59 genes per cell (Fig. 6). While the low-affinity cytochrome c oxidase genes coxABC were enriched in microbes inhabiting terrestrial springs (averaged 0.54 genes per cell; Fig. 6), where oxygen is easily available due to the rapid atmospheric exchange and products from photosynthesis release [74]. A majority of aerobic autotrophs possesses multiple oxidases genes, including three Epsilonbacteraeota MAGs found in this study (Fig. 7), which could facilitate their growth under different oxygen tensions in a wide range of ecological niches within geothermal systems. Genes encoding the high-affinity nitrate reductase (napAB) presented a significantly higher abundance in oceanic than terrestrial geothermal systems (averaging 0.18, 0.31, and 0.05 genes per cell for shallow-sea, deep-sea and terrestrial systems, respectively, p < 0.0001; Fig. 6), and were prevalent in Epsilonbacteraeota (Fig. 7). Genes narGHI and nitrite reductase gene (nirK) displayed significantly lower abundances in shallow-sea hydrothermal microbial communities than other geothermal systems, whereas the ferredoxin-nitrate reductase coding gene (narB) was more abundant in shallow-sea hydrothermal systems (p < 0.0001; Fig. 6). Though gene nasA was also present in shallow-sea hydrothermal systems (Fig. 6), only rare sequences of this gene have been found in the Kueishantao hydrothermal vent sites (Fig. 2B). The complete denitrification and dissimilatory reduction of nitrate appeared to be uncommon among prokaryotes from geothermal systems (Fig. 7).

Endemicity of microbial populations in shallow-sea hydrothermal systems
Hydrothermal systems present a variety of ecological niches enabling the colonization of diverse microorganisms, many of which are endemic to these systems. A total of 20 of the 57 culturable genera had a restricted distribution, appearing only in one category of geothermal system (Table S10). Shallow-sea hydrothermal systems shared a total of 28 genera (15 bacterial and 13 archaeal) and 14 genera (5 bacterial and 9 archaeal) with deep-sea hydrothermal systems and terrestrial hot springs, respectively (Fig. 7). While only 5 genera (3 bacterial and 2 archaeal) were common to deep-sea hydrothermal systems and terrestrial hot springs, which were also found in shallow-sea hydrothermal systems (Fig. 7).
Thermophilic cyanobacteria are endemic to terrestrial hot springs (Fig. 7) and have successfully colonized this extreme environment [75]. Culturable genera of Epsilonbacteraeota and Gammaproteobacterial isolates have been retrieved from marine hydrothermal systems; however, none of those genera have been observed in Fig. 6 The gene frequency of key metabolic genes in the metagenomes of shallow-sea, deep-sea, and terrestrial geothermal systems. More detail on datasets is shown in Table S3. The average gene frequencies are presented as mean values ± standard error of mean. Differences were considered significant if the p-value of an ANOVA was less than 0.05 (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001). Green, shallow-sea hydrothermal systems; red, deep-sea hydrothermal systems; purple, thermal spring terrestrial hot springs (Fig. 7), suggesting a high degree of endemicity in marine hydrothermal vents.
Members of the Nautiliaceae within Epsilonbacteraeota and Thiomicrospiraceae within Gammaproteobacteria have frequently been found to be the major active bacterial groups in the hydrothermal systems of Kueishantao Island [9,16,76]. Here we consistently recovered MAGs belonging to Nautiliaceae and Thiomicrospiraceae during our 3-year investigation period and successfully resolved their in situ protein expression at the population level for the first time. The genera of Nautiliaceae and Thiomicrospiraceae are also prevalent at deep-sea hydrothermal vents; however, their metabolic processes operating in deep-sea vents in situ at the individual population level are poorly described [77,78]. Cultured Nautiliaceae species can grow autotrophically via the rTCA cycle for carbon fixation, using S 0 reduction coupled with the oxidation of H 2 S, H 2 , or formate [79], and these processes were found to operate in situ in their relatives thriving at the Kueishantao vents (Fig. 5A). Thiomicrorhabdus MAGs within Thiomicrospiraceae possessed the potential ability to generate energy via the oxidation of multiple reduced sulfur compounds and transfer electrons to oxygen or nitrate (Fig. 5B). This result is in line with previous studies of deep-sea vent-associated Thiomicrorhabdus species which noted their metabolic versatility as aerobic chemoautotrophs [80][81][82]. Thus, the metabolic machinery of the active and predominant Nautiliaceae and Thiomicrospiraceae populations at shallow-sea vents can mirror those living at deep-sea vents.

Linking microbial community functional potential to shallow-sea hydrothermal environmental settings Autotrophy
The physical and chemical characteristics of geothermal systems play a significant role in constraining microbial functional metabolisms. Phototrophs could inhabit not only terrestrial hot springs but also shallow-sea hydrothermal systems where sunlight is present, including the Kueishantao system [9,76], which represents a striking difference with deep-sea vent systems [1]. Generally, the rTCA cycle is the major pathway for chemosynthesis in marine hydrothermal systems, including the Kueishantao vents (Figs. 2 and 6). However, the in situ chemosynthetic carbon fixation rates suggested only a minor contribution to the primary production of terrestrial hot spring ecosystem, accounting for approximately 3% of the local photosynthetic carbon fixation rates [83]. Many autotrophic hydrothermal microbes, similar to the gammaproteobacterial Thiomicrorhabdus, appear to have an incomplete oxidative TCA cycle, including but not limited to members of Thermodesulfobacteria and methanogens (Fig. 7). Generating energy chemosynthetically from reduced chemicals, instead of by the complete oxidation of exogenous organic carbon, might give an advantage to the living hydrothermal microorganisms to thrive in such extreme environments. The primary function of the incomplete TCA cycle might be responsible for producing metabolites for growth [84], reflecting adaptation to life in the vents. In contrast to a heterotrophic community, with its high expression of proteins involved in carbohydrate metabolism and organic matter transporter functions [60,70,72], the chemolithoautotrophs-dominant Kueishantao community could meet their growth requirements mainly via endogenous synthesis.

Energy conversion
Geothermal fluids often contain reduced sulfur species (mainly H 2 S and S 0 ), though the actual composition and concentration can vary dramatically depending on the geological setting [85,86]. The andesite-hosted Kueishantao hydrothermal system contained μM level concentrations of H 2 S and were rich in S 0 [87]. The metagenomic and metaproteomic results revealed that oxidation of reduced sulfur species, especially H 2 S, constituted the most abundant chemolithotrophic energy metabolism in all Kueishantao hydrothermal vents (Tables S4 and S6). Fluids from shallow-sea hydrothermal vents are often   characterized by low H 2 concentrations, as compared to that of deep-sea hydrothermal vents, and trace H 2 was found in Kueishantao hydrothermal fluids [87,88]. The Kueishantao vent metagenomes contained a much lower proportion of putative hydrogenase sequences compared to those from H 2 -rich environments (Table S3), such as serpentinizing deep-sea hydrothermal systems [89]. Nevertheless, hydrogenases were actively expressed in situ by microbes inhabiting this hydrothermal system (Table  S6), enabling them to utilize the available H 2 for energy generation.
Geothermal autotrophs that possess the capability of aerobic respiration have a competitive advantage in microbial communities, due to the greater energy yields from the oxidation of reducing substrates with oxygen compared to nitrate or sulfate respiration [90]. It is noteworthy that the uncultured Nautiliaceae members recovered within this study could utilize O 2 as an electron acceptor (Fig. 5A) and were persistently abundant in the O 2 -rich Kueishantao hydrothermal fluids ( Fig. 2A), while all cultured Nautiliaceae species could only grow under anaerobic conditions [79]. Given that a bacterial phenotype is the result of multiple factors, there would be distinctions in observations between laboratory or shipboard incubations and in situ processes. The culturable species of Nautiliaceae that possessed cydAB genes (Fig. 7), may present a tolerance for oxygen in submarine hydrothermal systems. Furthermore, the bd-type oxidase were the most abundant terminal oxidases in the metagenomes and metaproteomes of all Kueishantao vent colonizing microbial communities (Tables S4 and  S6), suggesting their ability to grow in even low-oxygen habitats as well as maintain redox balance [91]. Under anaerobic or microaerobic conditions of geothermal habitats, several microorganisms that possess nitrate or nitrite reductase genes, mainly Epsilonbacteraeota and Gammaproteobacteria (Fig. 7), could utilize nitrate or nitrite as an electron acceptor. Epsilonbacteraeota could simultaneously express nitrate reductase and cytochrome oxidases, such as in the prevalent Nautiliaceae populations of the Kueishantao vents (Fig. 5A), which may enable them to flourish across diverse redox gradients. High rates of sulfate reduction are frequently measured in terrestrial hot springs, enhanced by the presence of photosynthesis products [92][93][94], whereas the sulfate reduction rates at marine hydrothermal systems are much lower [95,96]. Although sulfate is plentiful in Kueishantao hydrothermal fluids, with concentrations of approximately 24.5-30.4 mM [87], only a small number of sulfate-reducing bacteria [16] and few gene sequences involved in sulfate reduction (this study; Table S4) were present in the Kueishantao bacterial community.
The functional potentials analyses results suggested the metabolic versatility of microbial communities as chemolithoautotrophs in the Kueishantao shallow-sea vents, generating energy via multiple redox reactions for adapting to low H 2 S, H 2 -poor, and O 2 -rich environments.

Rare taxa of shallow-sea vents as future research hotspots
The steep geochemical (oxic to anoxic) gradients at shallow-sea vents not only contains aerobic microorganisms but also harbors anaerobic ones that occupy a narrow range of ecological niches and may include rare populations within Thermotogae, Caldiserica, Thermococci, and Thermoprotei (Fig. 7). These taxa could serve as a reservoir of special functional potentials, like that in deep-sea vents [97,98]. It was recently shown that high CO 2 levels drive the TCA cycle backward (namely reversed oxidative tricarboxylic acid cycle) [99], allowing carbon fixation in bacteria inhabiting geothermal systems, such as the hot spring-associated Desulfurella acetivorans [100], deepsea vent-associated Thermosulfidibacter takaii [101], and shallow-sea hydrothermal-associated Hippea martima [102]. Such a carbon fixation strategy was thought to operate in microorganisms on CO 2 -rich ancient Earth [102]. Here, we found that autotrophs from Kueishantao Island submarine vents harbored a similar capability for carbon fixation. For example, two bins (2017-1 and 2018-7) belonging to the thermophilic anaerobic bacterial genus Hippea (Fig. 7). Additionally, shallow-sea vents contain special and rare species, including thermophilic nitrogen fixing bacteria (such as Methanotorris), a radiation-resistant bacterium (in Truepera) [103], the only currently known hyperthermophilic archaeal host (in Ignicoccus) [104] and a hyperthermophilic and neutrophilic archaeon (in Hyperthermus) [105] (Fig. 7). These results indicated that the rare taxa of shallow-sea hydrothermal systems might curate a seed bank of functional genes involved in ancient metabolic pathways that have survived from when such microbes throve in the early anoxic Earth's geothermal environments. Hence, shallowsea hydrothermal rare taxa are worth further exploration to expand our knowledge of microbial metabolic functions in extreme environments and provide clues about the microbial life of early Earth.

Conclusions
Here, genome-centric metagenomic analysis of ventassociated microbiomes over a 3-year period allowed the recovery of 20 MAGs, substantially increasing the number of genomes sequenced from shallow-sea hydrothermal environments. The metabolic modules of the active and predominant populations within Epsilonbacteraeota and Gammaproteobacteria in shallow-sea vents could mirror those living in deep-sea vents, as suggested by the metagenomic and metaproteomic data. Although hydrothermal microbiomes exhibit different facets of functional traits including the adaption to regional environmental conditions, the microbial functional signatures in the shallow-sea hydrothermal system shed light on a linkage to deep-sea and terrestrial counterparts. Future investigations on the intrinsic functions of rare species from shallow-sea hydrothermal systems could reveal undiscovered metabolic capabilities and facilitate our understanding of microbial ecology and evolution in extreme environments.