Geochemical features of hydrothermal fluids
The measured temperature of the hydrothermal fluid discharged from the L-vent and M-vent chimney were 231 °C and 35 ºC, respectively. Measured concentrations of dissolved chemical species in individual vent fluid samples are provided in Additional file 2: Table S1. For the L-vent, the composition of the four IGT fluid samples showed significant variability in the concentration of Mg, indicative of variable degrees of inadvertent seawater entrainment during sample collection. The composition of the endmember fluid at L-vent (Table 1) was calculated by plotting the concentration of a given chemical species against the measured Mg concentration in the same sample and extrapolating to zero-Mg, as is typically done for high temperature submarine vent fluids . Measured concentrations of Mg in the two fluids collected at M-vent showed little variation, consistent with entrainment of seawater in subseafloor environments prior to venting. Because we are interested in the chemical composition of fluids accessible to vent communities living within the chimney structures, the composition of M-vent fluids reported in Table 1 are not extrapolated to zero-Mg endmember values. At both vents, reported pH (25°C) is the lowest measured value in the samples and is not extrapolated to zero-Mg.
Endmember fluids venting through the L-vent structure were characterized by dissolved sulfide and hydrogen concentrations of 6.6 and 0.83 mM, respectively. In contrast, fluids exiting M-vent contained 0.00042 mM sulfide and hydrogen was below the detection limit of 2 μM. Methane was also detected in both the L-vent and M-vent fluids at concentrations of 83 and 24 μM, respectively. Despite the stark difference in temperature, the measured pH (25°C) showed similar values of 4.8 and 5.2, respectively (Table 1).
Microbial taxonomic diversity based on full-length 16S rRNA genes
After removing low-quality reads and assembly (general metagenomic information is shown in Additional file 2: Table S2), 162 and 372 full-length 16S rRNA genes were retrieved from the L- and M-vent metagenomes, respectively. The phylogenetic analyses of the 16S rRNA gene showed that the active L-vent chimney was dominated by Campylobacteria (phylum Campylobacterota) (55.4%), including the genera Sulfurovum (20.2%), Nitratifractor (8.6%), Sulfurimonas (8.6%) and Caminibacter (6.3%) (Additional file 2: Table S3). Bacteria belonging to the phylum Aquificae had the second highest relative abundance (14.7%), followed by members of the phylum Chlorobi (4.7%), Thermodesulfobacteria (3.2%) and Deinococcus-Thermus (2.4%) (Fig.1a). In contrast, the bacterial community of the inactive M-vent chimney was mainly composed of Gammaproteobacteria (22.9%) and Nitrospirae (17.3%), as well as Alpha- and Deltaproteobacteria (7.4% and 5.6%, respectively) (Fig.1b). The detailed taxonomic information and relative abundance of all reconstructed 16S rRNA genes are listed in Additional file 2: Table S3 and S4.
Distribution of key metabolic genes
Key genes for microbial carbon, nitrogen and sulfur metabolisms were searched in the metagenomes of the two chimneys, and differences were revealed regarding gene inventories and the pathways utilized by these two communities (Fig. 2).
Carbon fixation: Genes encoding for the ATP-citrate lyase (aclA/B), the key enzyme of reductive tricarboxylic acid (rTCA) cycle, were identified in significantly higher abundance (P-value < 0.05) in the active L-vent sample compared to M-vent (Fig.2a), and more than 99% of them share high similarities with those from Campylobacteria and Aquificae (Additional file 1: Figure S1a). In contrast, genes encoding enzymes of the Calvin-Benson-Bassham (CBB) cycle ribulose-bisphosphate carboxylase and phosphoribulokinase (rbcL/S and PRK) are significantly enriched in the inactive M-vent chimney, and the majority (43% of rbcL; 86% of rbcS and 75% of PRK) are assigned with Gammaproteobacteria (Additional file 1: Figure S1b). For the Wood–Ljungdahl (WL) pathway, genes encoding for the delta subunit of the archaeal acetyl-CoA decarbonylase/synthase complex (cdhD) and for the bacterial acetyl-CoA synthase (acsB) were more prevalent in the M-vent community, while the genes encoding for the alpha, beta, and epsilon subunits of the archaeal acetyl-CoA decarbonylase/synthase complex (chdA, cdhC and cdhB, respectively) were present in higher abundances in the L-vent community (Fig.2a).
Nitrogen metabolism: Genes encoding the periplasmic nitrate reductase (napA/B) and membrane-bound nitrate reductase (narG/H) were identified in both L- and M-vent samples, but with distinctly different abundances (Fig. 2b). In the M-vent chimney, narG/H were significantly enriched, with 43% of narG assigned to Alphaproteobacteria (Additional file 1: Figure S1b), while napA/B were more enriched in the active L-vent chimney, with 98% of napA assigned to Campylobacteria and Aquificae (Additional file 1: Figure S1a). Genes of the dissimilatory nitrate reduction to ammonia (DNRA) pathway were more abundant in the inactive M-vent chimney, with 74% of nitrite reductase large subunit (nirB) assigned to the Gammaproteobacteria (Additional file 1: Figure S1b). For the denitrification pathway, the gene encoding for the beta subunit of the nitric oxide reductase (norB) and for the nitrous-oxide reductase (nosZ) were identified in significantly higher abundance in the active L-vent chimney compared to M-vent (Fig.2b), with the majority of them assigned to Campylobacteria and Aquificae (Additional file 1: Figure S1a; 80% of norB and 75% of nosZ). On the other hand, the M-vent community was more enriched in genes encoding for subunits of the nitrogenase (nifD/K/H), which is involved in N2-fixation, compared to L-vent (Fig.2b), with 43% of nifH being assigned to Nitrospirae (Additional file 1: Figure S1b).
Sulfur metabolism: A significantly higher abundance of genes encoding for adenylylsulfate reductase (aprA/B) and sulfite reductase (dsrA/B) were identified in the M-vent sample (Fig.2c). Particularly, most aprA/B were taxonomically assigned to Gamma- and Deltaproteobacteria (Additional file 1: Figure S1b; 60% of aprA and 68% of aprB). Since the majority of dsrA/B were assigned to unclassified species, we inferred the taxonomy and catalytic type of dsrA based on their phylogenies. The results suggest that 13 of 14 dsrA presented in the L-vent were of the reductive type, including Deltaproteobacteria, Archaeoglobus and Acidobacteria, while 36 of 72 dsrA genes from M-vent were of the oxidative type belonging to sulfur-oxidizing Alpha- and Gammaproteobacteria, with the remainder being of the reductive type belonging to Deltaproteobacteria (10), Nitrospirae (12) and Acidobacteria (14) (Additional file 1: Figure S2). For the Sox sulfur oxidation system, similar abundances were found for soxB from L- and M-vent, however the majority of soxB from L-vent were assigned to Aquificae and Campylobacteria, while those from M-vent were largely assigned to Gamma- and Alphaproteobacteria (Additional file 1: Figure S3). On the other hand, soxA/C/Y/Z were found highly enriched in the active L-vent chimney, most of which (95%) were assigned to the Aquificae and Campylobacteria (Additional file 1: Figure S1a). Additionally, genes encoding for the sulfide-quinone oxidoreductase (sqr) were present in higher abundance in the L-vent community, with a similar taxonomic profile as the sox genes (Additional file 1: Figure S1a).
Phylogeny of metagenome-assembled genomes (MAGs)
After filtration of low-quality MAGs, 71 and 102 MAGs with a completeness ≥ 70% and potential contamination ≤ 10 %, which is higher than the MAG medium quality standard proposed by Genomic Standard Consortium (https://gensc.org/), were obtained for further analysis from L- and M-vent metagenomes, respectively (Additional file 2: Table S5). For L- and M-vent, 42.3% and 48.3% of reads were retrieved to their respective MAGs. Based on the sequencing depth, 20 and 34 of the top 50 most abundant RpS3 genes were identified in the MAGs recovered from L- and M-vent, respectively, including the top three of the L-vent community and the most and third most abundant taxa of M-vent (Additional file 1: Figure S4). Therefore, the retrieved MAGs are representative of the majority of microbial taxa of both communities.
Overall, the 173 retrieved MAGs could be taxonomically assigned to more than 20 phyla, including several novel candidate bacterial phyla without cultivated representatives (Fig.3). Relative abundance and major groups (>1%) and individual MAG were shown in Additional file 2: Table S6 and S10. Particularly, for the 71 MAGs from the L-vent chimney, 11 MAGs were taxonomically assigned to the phylum Aquificae, which was identified as the dominant taxon (14.5%) based on their reads mapped to the whole L-vent metagenome. 4 MAGs of Thermodesulfobacteria were the second most abundant bacterial group (8.0%). 17 MAGs belonged to Camplyobacteria representing only 3.6% of the whole microbial community, this discrepancy to the 16S rRNA gene-based results probably due to their high interspecies diversity and similar genomic features making it difficult to retrieve more MAGs. Chloroflexi (5 MAGs), FCB group (7 MAGs), Gammaproteobacteria (4 MAGs) and Thermotogae (2 MAGs) accounted for 2% ,1.7%, 1.5%, and 1.1%, respectively. For Archaea, 4 and 8 MAGs were assigned to the phyla Euryarchaeota and Crenarchaeota, representing 4.6% and 2.8%, respectively (Additional file 2: Table S6). Phylogenetic analysis indicated that 3 out of 4 Euryarchaeotal MAGs belonged to the methanogenic classes Methanococci and Methanopyri and most of the Crenarchaeota were distantly related to Ignicoccus (Additional file 1: Figure S8). Moreover, 4 MAGs were classified as DPANN groups, including Micrachaeota (2), Diapherotrites (1) and Nanohaloarchaeota phyla (1).
For the M-vent sample, 16 Gammaproteobacteria MAGs accounted for 11.4% of the whole community, most of which were assigned to Ca. Tenderia electrophaga and also closely related to those recovered from previously analyzed inactive chimneys (Additional file 1: Figure S7) . 11 Deltaproteobacterial MAGs were recovered with a total relative abundance of 4.9%. In addition, FCB group (11 MAGs; 4.99%), Calditrichaeota (6 MAGs; 3.72%), Alphaproteobacteria (7 MAGs; 2.77%), Nitrospirae (6 MAGs; 2.94%), PVC group (9 MAGs; 4.35%) and Acidobacteria (4 MAGs; 2.57%) were represented as major microbial groups among the MAGs of the M-vent microbial community. Phylogenetic analysis showed that the 4 out of the 6 Nitrospirae MAGs could be assigned to a newly identified “sulfide-mineral” clade, along with 5 additional MAGs either from inactive sulfide chimneys or subseafloor massive sulfides (SMS) [37, 72] (Fig.3), while the other 2 Nitrospirae MAGs were part of a separate clade (Additional file 1: Figure S6). The genome tree further showed that Nitrospirae are split into two distinct lineages with long phylogenetic distance (Fig.2), in line with the polyphyletic feature of Nitrospirae reported before , highlighting the need to reclassify this phylum. In addition, 3 MAGs were assigned to novel taxa in the candidate phyla radiation (CPR) and 2 MAGs were assigned to the phylum Micrachaeota in DPANN group.
Index of replication value (iRep) of bacterial MAGs
The iRep value provides information about the replication activity of specific MAGs at the time of sampling (see Method and Materials). Theoretically, a iRep value of 1.5 means that half of the cells in a population are replicating, but in reality there are several ways to achieve a given iRep value since the population is heterogeneous, i.e., some cells may not replicate and others are replicating at a faster rate with more than one replication fork . In this study, most of the retrieved bacterial MAGs in the chimneys represent active replicating bacterial taxa as indicated by iRep values calculated from 52 and 91 high-quality bacterial MAGs from the L- and M-vent samples, respectively. The average iRep value of bacterial MAGs from the inactive M-vent is 1.51, which is higher than that from the active L-vent (1.42) (Additional file 2: Table S6). In the L-vent, Camplyobacteria had the highest average iRep value (1.52), followed by Chloroflexi (1.5), Gammaproteobacteria (1.47), Thermodesulfobacteria (1.43) and Aquificae (1.40). In the M-vent, Calditrichaeota had the highest iRep value (1.8), followed by the FCB group (1.74), Chloroflexi (1.59), Nitrospirae (1.49), Alpha-/Deltaproteobacteria (1.48 and 1.42, respectively) and Gammaproteobacteria (1.4). iRep values for each MAG and the average iRep value of other major microbial groups (>1% in each sample) are shown in the Fig.4 and Additional file 2: Table S6, respectively.
Metabolic reconstruction of MAGs
The active L-vent chimney
Based on the analysis of MAGs, the Campylobacteria (17 MAGs) and Aquificae (11 MAGs) dominating the active L-vent are potential sulfur/hydrogen-oxidizing bacteria with capabilities of denitrification and carbon fixation through the rTCA cycle (Fig.4). All 28 MAGs encode at least one sqr gene, 45% and 24% of them also encode complete or near complete Sox system (Fig.4; Additional file 2: Table S6). The rTCA cycle is the sole carbon fixation pathway and was prevalently identified in the Aquificae and Campylobacteria MAGs (55% and 47%, respectively). In addition, the majority of Aquificae and Campylobacteria MAGs encode hydrogenase group 1 and 2 for hydrogen uptake. Further, napA/B genes were identified in every MAG assigned to Campylobacteria and 45% of Aquificae MAGs. Genes encoding for the enzymes catalyzing the subsequent steps of denitrification (nirS/K, norB/C, nosZ; Additional file 2: Table S7) were identified in 73% and 47% of the MAGs belonging to the Aquificae and Campylobacteria, respectively (Fig.4; Additional file 2: Table S6).
Besides Aquificae and Campylobacteria, 4 MAGs (8.2%) belonging to the Thermodesulfobacteria were identified in the L-vent chimney that have the capacity of reducing multiple sulfur species. They contained not only the key genes encoding the complete sulfate reducing pathway (i.e., sat, aprA/B and dsrA/B), but also other essential marker genes like dsrD, the sulfite reductase-associated electron transfer complex (dsrM/K/J/O/P), and the electron transfer complex (QmoA/B/C) (Additional file 2: Table S8). Moreover, genes encoding for the thiosulfate reductase (phsA/B) and tetrathionate reductase (ttrA) were also identified in 2 of them. Thermodesulfobacteria MAGs from L-vent chimney share highly similar metabolic potential with their sulfur-disproportioning isolates .
We also identified 2 MAGs belonging to the phylum Euryarchaeota that contained the complete gene cluster encoding for the methyl coenzyme M reductase mcrABG and also genes encoding for the Group 3/4 hydrogenase, indicating a methanogenic metabolism (Fig.4; Additional file 2: Table S6). The other major microbial groups, such as Chloroflexi and the FCB group, have organotrophic potential, either using fermentation or respiration, which is supported by the considerable number of genes related to carbohydrate degradation and nitrate reduction (Fig.4).
The recently extinct M-vent chimney
Based on the analysis of MAGs, the Gammaproteobacteria (16 MAGs) dominating the M-vent chimney are potential chemoautotrophic sulfur-oxidizing bacteria, using the CBB cycle for carbon fixation and reducing nitrate via the DNRA pathway (Fig.4). For sulfur oxidation, most of the MAGs contained the genes encoding for the Sox system (69%) and sqr gene (63%) (Fig.4; Additional file 2: Table S6). In addition, 50% of the MAGs also contain the gene encoding for the reverse DSR as evidenced by the phylogenetic assignment of the dsrA gene (Additional file 1: Figure S3), indicating the potential for the oxidative DSR pathway for sulfur oxidation. Moreover, 5 of these MAGs also contain the cyc2 gene encoding for an outer membrane c-type cytochrome, which is closely related with their expressed homologs in the electroautotrophic Ca. Tenderia electrophaga (Additional file 1: Figure S9).
Deltaproteobacteria (11 MAGs) were identified as one of the major microbial taxa in the M-vent chimney. Based on their gene content, they are putative sulfate-reducing bacteria (SRB) having the potential to oxidize organic matter through the WL pathway, with 64% of the MAGs encoding the reductive DSR pathway and WL pathway. Specifically, genes involved in carbohydrate degradation are significantly enriched in the Deltaproteobacteria (19.2 CAZyme genes per MAG on average; Additional file 2: Table S6). Based on the identification of genes encoding for napAB/narGH and the subsequent DNRA pathway in most of their MAGs, nitrate appears to be a potential alternative electron acceptor for Deltaproteobacteria.
The 4 Nitrospirae MAGs recovered belonging to the “sulfide-mineral” clade encode essential genes of DSR, WL pathway, nitrite reduction and nitrogen fixation, same as the other 5 MAGs in this clade (Fig.4; Additional file 2: Table S9). 3 of them encode the key enzyme of cbb3-type cytochrome c oxidase (Additional file 2: Table S10). Furthermore, 5 of all 9 Nitrospirae from the “sulfide mineral” clade (including the 2 most abundant recovered from the M-vent and the other 3 derived from recently extinct sulfide chimneys and SMS, respectively [37, 72]) encode the cyc2 gene (Additional file 2: Table S9). Their cyc2 genes are phylogenetically closely related to each other and distantly related to their counterparts identified in the genomes of Zeta-/Betaproteobacteria Fe-oxidizing bacteria (FeOB)  (Additional file 1: Figure S9). Moreover, these “sulfide mineral” Nitrospirae MAGs have relatively small genomes (<2Mb) and fewer genes involved in sulfur oxidation pathways (sqr and sox) compared with other Nitrospirae species (Additional file 2: Table S9). Besides the 4 MAGs assigned to the “sulfide mineral” clade, the other two Nitrospirae MAGs recovered from the M-vent have distinct metabolic features: one doesn’t have any genes involved in sulfate reduction, the other one encodes the rTCA pathway instead of the WL pathway and is closely related with another metabolically-similar Nitrospirae MAG recovered from a long-time inactive sulfide chimney  (Additional file 2: Table S9).
Based on the prevalence of genes encoding for narGH, napAB, and the subsequent DNRA pathway (nrfA/H, nirB and nirD; Additional file 2: Table S7) in their genomes, other microorganisms in the M-vent chimney including the FCB group, PVC group, Calditrichaeota, Alphaproteobacteria, Chloroflexi and Actinobacteria are likely to be nitrate-respiring heterotrophs (Fig.4). That’s also supported by the enrichment of genes for carbohydrate degradation (CAZyme) identified in their MAGs, especially for the Calditrichaeota, FCB and PVC group (Fig.4). Interestingly, some Calditrichaeota (1 in 6 MAGs), Alphaproteobacteria (2 in 7 MAGs) and Actinobacteria (2 in 3 MAGs) encode carbon monoxide dehydrogenase (coxM/L/S) which catalyzes CO oxidation. The phylogenetic analysis of coxL from M-vent suggests that they are largely assigned to the putative FormII/BMS clade (Additional file 1: Figure S5). The Euryarchaeota MAGs recovered from M-vent are potential sulfate reducing archaea that are phylogenetically closely related to the Archaeoglobi lineage, which is supported by the retrieved 16S rRNA gene of the genus Geoglobus (Additional file 2: Table S4). 3 of the 4 MAGs encode the complete reductive DSR pathway, the archaeal WL pathway as well as group 1 hydrogenase (Fig.4; Additional file 2: Table S6).