Geochemical features of hydrothermal fluids
The temperature of the hydrothermal fluid discharged from the EPR-L chimney was 231 °C, and the concentrations of sulfide and hydrogen were 1,970 and 268 μM, respectively. For EPR-M, the temperature was measured at 35 °C with low concentrations of sulfide (0.42 μM) and hydrogen (below detection). Methane was also detected in both the EPR-L and -M fluid at a concentration of 25.8 and 21.7 μM, respectively. Despite the stark difference in temperature, dissolved Mg2+, K+, Ca2+, Na+ and Cl- concentrations and pH of the two fluid samples were largely identical (Table 1).
Microbial taxonomic diversity based on full-length 16S rRNA genes
After removing low-quality metagenomic reads, a total of 319,138,012 and 352,707,814 reads were obtained from EPR-L and -M, respectively. Subsequently, these reads were de novo assembled to 644,620 and 545,008 contigs for EPR-L and -M, respectively (Additional file 2: Table S1). In particular, 162 and 372 full-length 16S rRNA genes were retrieved from the EPR-L and -M metagenomes, respectively. The phylogenetic analyses of the 16S rRNA gene showed that the active EPR-L 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 sharp contrast, the bacterial community of the inactive EPR-M 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 striking 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 significant higher abundance (P-value < 0.05) in the active EPR-L sample compared to EPR-M (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 EPR-M chimney, and the majority (42.6% of rbcL; 86.2% of rbcS and 74.9% 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 EPR-M 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 EPR-L community (Fig.2a).
Nitrogen metabolism: Genes encoding the periplasmic nitrate reductase (napA/B) and membrane-bound nitrate reductase (narG/H) were identified in both EPR-L and EPR-M samples, but with distinctly different abundances (Fig. 2b). In the EPR-M chimney, narG/H were significantly enriched, with 42.8% of narG assigned to Alphaproteobacteria (Additional file 1: Figure S1b), while napA/B were more enriched in the active EPR-L chimney, with 97.8% 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 EPR-M chimney, with 73.9% 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 EPR-L chimney compared to EPR-M (Fig.2b), with the majority of them assigned to Campylobacteria and Aquificae (Additional file 1: Figure S1a; 80.1% of norB and 75.4% of nosZ). On the other hand, the EPR-M community was more enriched in genes encoding for subunits of the nitrogenase (nifD/K/H), which is involved in N2-fixation, compared to EPR-L (Fig.2b), with 43.3% 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 EPR-M sample (Fig.2c). Particularly, most aprA/B were taxonomically assigned to Gamma- and Deltaproteobacteria (Additional file 1: Figure S1b; 59.6% of aprA and 68.4% 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 EPR-L were of the reductive type, including Deltaproteobacteria, Archaeoglobus and Acidobacteria, while 36 of 72 dsrA genes from EPR-M 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 EPR-L and EPR-M, however the majority of soxB from EPR-L were assigned to Aquificae and Campylobacteria, while those from EPR-M 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 EPR-L 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 EPR-L 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 high-quality MAGs with a completeness ≥ 70% and potential contamination ≤ 10 % were obtained for further analysis from EPR-L and -M metagenomes, respectively. A total of 66.4% of these MAGs have a completeness ≥ 80% and a contamination ≤ 5% (Additional file 2: Table S2). For EPR-L and -M, 42.3% and 48.3% reads were retrieved to their respective MAGs. Among these MAGs, 20 and 34 genomes of the top 50 most abundant microbial species were identified from ERP-L and -M, respectively, including the top three taxa of the EPR-L community and the most and third most abundant taxa of EPR-M (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). Particularly, for the 71 MAGs from the EPR-L 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 EPR-L metagenome (Fig.4). Thermodesulfobacteria was the second most abundant bacterial MAG (8.0%), and 1 in 4 recovered MAGs belonging to this phylum had the highest relative abundance among all MAGs from EPR-L sample (L-MaxBin-1, 6.6%, Additional file 2: Table S9). 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), Chlorobi (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 Nanohalarchaeota phyla (1). L-Maxbin-88 was not close to any other taxa in the phylogenetic tree.
For the EPR-M 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) [31]. 11 Deltaproteobacterial MAGs were recovered with a total relative abundance of 4.9%. In addition, Chlorobi (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 EPR-M microbial community. Phylogenetic analysis showed that the 6 Nitrospirae MAGs were assigned into two distinct clades (Fig.3): one is named “sulfide mineral” clade comprised of 4 Nitrospirae MAGs from EPR-M and other 5 MAGs either from inactive sulfide chimneys or subseafloor massive sulfides (SMS) [31, 65], the other 2 Nitrospirae MAGs are away from the “sulfide mineral” clade (Additional file 1: Figure S6). Besides, 3 MAGs assigned to novel taxa in the candidate phyla radiation (CPR), including candidate TM6, SR1 and Campbellbacteria, were retrieved from the EPR-M chimney. 2 MAGs were assigned to the phylum Micrachaeota in DPANN group, and 1 MAG (M-MaxBin-034) was not close to any other microbial taxa.
Index of replication value (iRep) of bacterial MAGs
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 EPR-L and -M samples, respectively. The average iRep value of bacterial MAGs from the recently inactive EPR-M is 1.51, which is higher than that from the active EPR-L (1.42) (Additional file 2: Table S6). In the EPR-L, 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 EPR-M, Calditrichaeota had the highest iRep value (1.8), followed by Chlorobi (1.74), Chloroflexi (1.59), Nitrospirae (1.49), Alpha-/Deltaproteobacteria (1.48 and 1.42, respectively) and Gammaproteobacteria (1.4). iRep value for each MAGs 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 metabolic potential of the main microbial groups from both chimneys were revealed by respective MAG analysis (Fig.4):
The active EPR-L chimney
The dominating Campylobacteria (17 MAGs) and Aquificae (11 MAGs) are potential sulfur/hydrogen-oxidizing bacteria with capabilities of denitrification and carbon fixation through the rTCA cycle. 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 S5) 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 EPR-L 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 S7). Moreover, genes encoding for the thiosulfate reductase (phsA/B) and tetrathionate reductase (ttrA) were also identified in 2 of them. Thermodesulfobacteria MAGs from EPR-L chimney share highly similar metabolic potential with their sulfur-disproportioning isolates [66].
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 Chlorobi, are potential organotrophic bacteria, either using fermentation or respiration, as indicated by the absence of genes indicating autotrophic pathways and having considerable number of genes related to carbohydrate degradation (Fig.4).
The recently extinct EPR-M chimney
The dominating Gammaproteobacteria (16 MAGs) in the EPR-M 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 Gammaproteobacterial 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 Gammaproteobacterial 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 EPR-M 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 S8). 3 of them encode the key enzyme of cbb3-type cytochrome c oxidase (Additional file 2: Table S9). Furthermore, 5 of all 9 Nitrospirae from the “sulfide mineral” clade (including the 2 most abundant recovered from the EPR-M and the other 3 derived from recently extinct sulfide chimneys and SMS, respectively [31, 65]) encode the cyc2 gene (Additional file 2: Table S8). 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) [67] (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 S8). Besides the 4 MAGs assigned to the “sulfide mineral” clade, the other two Nitrospirae MAGs recovered from the EPR-M 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 [31] (Additional file 2: Table S8).
Based on the prevalence of genes encoding for narGH, napAB, and the subsequent DNRA pathway (nrfA/H, nirB and nirD; Additional file 2: Table S5) in their genomes, other microorganisms in the EPR-M chimney including the Chlorobi, PVC, 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 identified in their MAGs, especially for the Calditrichaeota, Chlorobi, and PVC (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 EPR-M suggests that they are largely assigned to the putative FormII/BMS clade (Additional file 1: Figure S5). The Euryarchaeota MAGs recovered from EPR-M 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).