Diverse archaeal lineages were detected in the deep sediment of soda-saline lakes
From the five artificially-separated ponds in a soda-saline lake named Habor Lake (DK), four deep sediment (40–50 cm depth) samples of each pond were collected (20 samples in total). DK and Sed indicated Habor Lake and the sediment samples. The numbers after DK and Sed showed the salinity of brine and that of pore water in the sediment. The porosities were approximately 20–26%. The total salinities of pore waters in the deep sediment ranged from 7.0% up to 33.0%, while pH values were 9.72–9.96 (Table 1). To have a glance at the archaeal microbiome of deep sediment, the variable regions V3-V4 of 16S rRNA gene were amplified using archaea-specific primers and the high-throughput sequencing was performed. A total of 3,566,000 reads were obtained across all samples (Table S1). After clustering sequences at 99% similarity level and resampling to a minimum of 131,986 reads per sample, a total of 8598 OTUs were obtained, in which 1920 archaeal OTUs were assigned (Table S2). The rarefaction curve showed that all twenty curves tend to be flat, so it suggested that the sequencing depth was enough to cover most of the local species (Fig. S1a). The Good’s coverage (99.17–100%) also supported this conclusion (Fig. S1b). Both Chao1 and Shannon indexes indicated that the alpha diversity index tended to decrease along with elevated salinities (Fig. S1c, d). The influence of deep sediment salinity on biodiversity tendency was similar as that of brine and surface sediment [12]. The principal coordinate analysis (PCoA) (Fig. S2a) and non-metric multidimensional scaling (NMDS) (Fig. S2b) showed that deep sediment salinity influenced the archaeal composition profiles.
Table 1 Geochemical characteristic of the deep sediments
Sediment name*
|
DK1Sed7
|
DK3Sed8
|
DK15Sed18
|
DK24Sed24
|
DK33Sed33
|
Porosity (%)
|
0.26 ± 0.02
|
0.22 ± 0.01
|
0.25 ± 0.01
|
0.23 ± 0.01
|
0.20 ± 0.01
|
Salinity# (%)
|
7
|
8.6
|
18
|
24
|
33
|
pH#
|
9.92 ± 0.07
|
9.92 ± 0.07
|
9.93 ± 0.03
|
9.96 ± 0.01
|
9.72 ± 0.12
|
Cl- (g/kg)
|
0.10 ± 0.02
|
0.13 ± 0.02
|
0.38 ± 0.02
|
0.85 ± 0.36
|
0.80 ± 0.27
|
CO32- (g/kg)
|
0.0035 ± 0.00
|
0.01 ± 0.00
|
0.01 ± 0.00
|
0.06 ± 0.00
|
0.15 ± 0.01
|
HCO3- (g/kg)
|
0.13 ± 0.03
|
0.25 ± 0.00
|
0.50 ± 0.15
|
0.30 ± 0.03
|
0.53 ± 0.01
|
SO42- (mmol/g)
|
3.29 ± 0.06
|
4.06 ± 0.04
|
1.26 ± 0.00
|
0.88 ± 0.07
|
2.30 ± 0.03
|
SO32- (mmol/g)
|
0.88 ± 0.04
|
0.64 ± 0.03
|
0.58 ± 0.03
|
0.85 ± 0.08
|
0.85 ± 0.03
|
NH4+ (μmol/g)
|
2.76 ± 0.47
|
3.29 ± 0.30
|
7.80 ± 0.37
|
3.72 ± 0.51
|
4.96 ± 0.95
|
NO3- (μmol/g)
|
740.74 ± 824.18
|
179.46 ± 7.77
|
207.70 ± 6.81
|
176.17 ± 12.49
|
199.36 ± 14.27
|
NO2- (μmol/g)
|
222.54 ± 143.89
|
62.10 ± 2.78
|
118.63 ± 9.27
|
39.34 ± 7.95
|
45.04 ± 21.26
|
# Salinity, pH and concentration of inorganic ions of pore water were measured.
* DK and Sed indicated Habor Lake and the sediment samples. The numbers after DK and Sed showed the salinity of brine and that of pore water in the sediment.
The archaeal composition profiles at the phylum level were presented in Fig. S3. A large number of archaeal phyla were detected, including Euryarchaeota, Nanoarchaeota, Thaumarchaeota, Diapherotrites, Crenarchaeota, Hadesarchaeota, Altiarchaeota, Hydrothermarchaeota, Asgardaeota and unclassified lineages. Euryarchaeota was the most abundant archaeal phylum, and Nanoarchaeota, Diapherotrites and Crenarchaeota accounted for a considerable proportion in the archaeome of deep sediments. At class-level, a surprising result showed that Thermoplasmata and Halobacteria in Euryarchaeota were the most abundant archaeal classes, and the abundance of Thermoplasmata was even higher than that of Halobacteria in the deep sediments with total salinities greater than 18% (Fig. 1). Besides, we also found diverse methanogens, including Methanomicrobia (mainly consisted of Methanomicrobiales, Methanosarcinales and uncultured), Methanobacteriales (in Methanobacteria), Methanofastidiosales (in Thermococci) and Methanomassiliicoccales (in Thermoplasmata) (Table S2).
Genomes recovery of novel archaeal lineage from metagenomes
To unbiasedly evaluate the archaeal community in the polyextreme deep sediment, high-throughput metagenomic sequencing of the five deep sediments (mixing four samples from the same pond) was performed. A total of 88.35 Gb clean data was obtained in the five metagenomes (Table S3). Through extracting draft genomes (bins) from metagenomes, 69 archaeal genomes and 518 bacterial genomes (taxonomic classifications were assigned based on the Genome Database Taxonomy (GTDB)) was recovered, and the archaeal communities were further quantified in the deep sediments (Table S4). Archaea accounted for 2.2% to 35.73% of the microbial community in the deep sediments (Fig. 2a), and the most abundance was in the sample DK33Sed33 with the highest salinity. The relative abundance of Thermoplasmatota was almost equal to or much higher than Halobacteriota (Fig. 2a). In the archaeal microbiome composed of 69 MAGs, there were 31 Thermoplasmatota MAGs, 28 Halobacteriota MAGs, and 10 other archaeal MAGs (from phyla Hadarchaeota, Iainarchaeota, Nanoarchaeota, Thermoproteota, and PWEA01, detailed shown in Fig. 2b). Interestingly, Thermoplasmatota and Halobacteriota MAGs were in the majority in the relative abundance and the MAGs number (Fig.1 and Fig. 2).
Considering the high abundance of Thermoplasmatota in deep sediments and most of which were classified as uncultured, the detailed phylogenetic position of these 31 MAGs was further analyzed by constructing an evolutionary tree with 770 Thermoplasmatota genomes (classified based on GTDB) as reference. Until now, Thermoplasmatota was classified into 21 order level taxonomic units using 122 single-copy conserved proteins. Thirty of the 31 Thermoplasmatota MAGs were assigned to the order PWKY01 by GTDB, while the other one belonged to the order Methanomassiliicoccales (Fig. 3). The order PWKY01 accounted for the most diversity of Thermoplasmatota and was also the most abundant archaeal order in our deep sediment samples (Fig. S4a).
There were 12 released PWKY01 MAGs in GTDB, and all of them were assembled from the sediment metagenomes of soda lake (Table S5). It was obvious that order PWKY01 could be regarded as the haloalkaliphilic and anaerobic environment-specific lineage. All 30 PWKY01 MAGs were classified into four genera (SKVC01, PWKY01, B1SED10-34 and PWHR01), and the relative abundance of each MAGs was estimated in the archaeome. The 30 PWKY01 MAGs was renamed HAT1 to HAT30 for short (Table S4). Three SKVC01 MAGs (HAT24, HAT25 and HAT26) exhibited the high relative abundance in DK1Sed7 and DK3Sed8 (relative low salinity within our samples, 7-8%), while 4 PWKY01 MAGs (HAT27, HAT28, HAT29 and HAT30) in DK3Sed8 and DK15Sed18 (higher salinity, 8-18%). B1SED10-34 and PWHR01 tended to inhabit in DK15Sed18 and DK24Sed24 (18-24%), even some PWHR01 MAGs exhibited the highest relative abundance in DK33Sed33 (33%) with the highest salinity, such as HAT12 and HAT13 (Fig. 3). Therefore, these four genera (SKVC01, PWKY01, B1SED10-34 and PWHR01) tended to distribute in different salinities in the soda lake deep sediments (Fig. 3 and Fig. S4b).
To identify the relationship between the Thermoplasmata OTUs (assigned based on SILVA) and the Thermoplasmatota MAGs (assigned based on GTDB), the 16S rRNA gene sequences was extracted from MAGs and alignment with the 16S amplicon sequence data was performed. The phylogenetic tree of class Thermoplasmata OTUs based on the 16S rRNA amplicon sequences was constructed and the unclassified taxa was clustered into five clades (Fig. S5a). The relative abundances of unclassified lineages were much higher, and Clade IV and V were the dominant lineages (Fig. S5b). The otu7254, otu8240 and otu2742 in clade IV showed 100% identity with order PWKY01 MAGs HAT6, HAT24 and HAT30, respectively (Fig. S5a & Table S6). Evidently, the order PWKY01 was identical to clade IV detected by 16S rRNA amplicon, and both high-throughput sequencing approaches revealed that this novel order was the abundant lineage in the deep sediment. Based on the results above, we proposed that this order was named Candidatus Natranaeroarchaeales (Natr.an.ae.ro.ar.chae.a’les. N.Gr. n. natron derived from Arabic natrun soda (sodium carbonate); Gr. pref. an not; Gr. n. aer air; N.L. neut. n. archaeum [from Gr. adj. archaios, -e, -on] ancient archaeon; L. fem. pl. suff. -ales, ending to denote an order; N.L. fem. pl. n. Natranaeroarchaeales, the order denoting the soda-requiring anaerobic archaea, affiliated to Thermoplasmatota).
Environmental adaptation mechanism of Ca. Natranaeroarchaeales
To further understand the environmental adaptation mechanism of Ca. Natranaeroarchaeales, the high- (≥ 90% complete, < 5% contamination) and medium-quality (90% > complete ≥ 50%, < 10% contamination) MAGs of the four genera were selected to perform the isoelectric point calculation of predicted proteomes and functional annotation.
The isoelectric point profiles of predicted proteomes of 13 high- and medium-quality Ca. Natranaeroarchaeales MAGs obtained in this article were different from Halobacteria and nonhalophiles, but similar to that of halophilic bacteria (Fig. S6). Based on the isoelectric point profiles and average electric points, they were separated into four classes (Fig. 4). Like the reference species Sipribacter salinus and Halomonas elongate in class I (Fig. 4a), HAT24 may use dissolved solutes rather than inorganic salts (KCl) to resist the high osmotic pressure; however, no gene involved in the biosynthesis or transporting of frequently-used ectoine, trehalose and glycine betaine (Table S8). Maybe other dissolved solutes were employed by HAT24. The average pI (6.03) was most close to neutral pH (Table S7). In class II, III, and IV, the peaks of isoelectric point profiles move gradually to acidified pH, and the average pI keeps decreased (Fig. 4bcd & Table S7). Similar to the reference species Natranaerobiusthermophilus in class II and Salinibacter ruber in class IV, the species represented by MAGs in class II, III, and IV seemed to live in high saline environments by absorbing inorganic salts (“salt-in” strategy) rather than dissolved solutes.
To explore the molecular basis for halophilic and alkaliphilic adaptation, the related functional genes were analyzed. The marker genes for biosynthesis of ectoine, glycine betaine, and trehalose (most used solutes by halophilic bacteria) were not complete (Table S8), while genes for betaine and choline transport were less found in Ca. Natranaeroarchaeales. Therefore, the “salt-in” strategy may be mainly adopted by most Ca. Natranaeroarchaeales members to maintain osmotic pressure balance, and the dissolved organic solutes might be also used by some taxa (for example HAT24). Most high- and medium-quality MAGs contained trkAHG genes (detailed in Supplementary Results, Fig. S7), whose products functioned in potassium uptake. As for the adaptation to alkalinity, the most important mechanism is to keep intracellular neutral pH. Multicomponent Na+:H+ antiporter (MnhABCDEFG) was found in all of high- and medium-quality MAGs (Fig. S7), and they could maintain intracellular pH homeostasis.
A mixotrophic lifestyle of Ca. Natranaeroarchaeales
To decipher the metabolic potentials and putative ecological functions of Ca.Natranaeroarchaeales, the encoding genes of 11 high-quality MAGs (including 4 MAGs recovered in this article and 7 MAGs downloaded from GTDB) and 14 medium-quality MAGs (9 in this article and 5 from GTDB) were further investigated (Fig. 5 and S8). These 25 MAGs were classified into four genera named SKVC01, PUNK01, PWKY01 and PWHR01 by GTDB-tk pipeline according GTDB database and manual modification from phylogenomic tree (Fig. 3).
The most remarkable feature was that almost all of high-quality MAGs (nine out of eleven) from four genera contained anaerobic carbon-monoxide dehydrogenase catalytic subunit (AcsA) which was the key enzyme in the Wood-Ljungdahl pathway (WL pathway) (Fig. 5). AcsA usually constitutes Cdh/Acs complex and performs CO utilization and CO2 fixation. Seven out of nine AcsA containing MAGs also had formate--tetrahydrofolate ligase (Fhs), methylenetetrahydrofolate dehydrogenase (NADP+) / methenyltetrahydrofolatecyclohydrolase (FolD) and methylenetetrahydrofolate reductase (MTHFR), while the other two MAGs had two of Fhs, FolD and MTHFR catalyzing the one carbon metabolism in WL pathway (Fig. 5). Of the nine high-quality MAGs, CSSed10_214 and T1Sed10_119m (belonging to PWKY01 and PWHR01, respectively) had formate dehydrogenase (NADP+) (FdhAB) which catalyzed the transformation of CO2 into formate (Fig. 5). One medium-quality MAGs B1Sed10_107R1 belonging to genus PWKY01 also had all six above-mentioned genes in WL pathway (Table S8). Notably, like Thermoplasmatales, MG-II, MG-III, and Thermoprofundales (MBG-D), the genes encoding Cdh/Acs complex were defective even absent in the available genomes. It was widely accepted that some subunits of Cdh/Acs complexes from Thermoplasmata might share low similarity with those from other archaea [26] . Therefore, it was derived that some members (at least these three MAGs CSSed10_214, T1Sed10_119m and B1Sed10_107R1) had the potential of assimilating inorganic carbons via WL pathway.
Part of product acetyl-CoA could flow into gluconeogenesis, considering the compete pathway in almost all high-quality MAGs (Fig. 5 & S9). Carbonate could be utilized because of the presence of phosphate pentose pathway, although Embden-Meyerhof pathway was blocked (lacking phosphofructose kinase) while Entner-Doudoroff were incomplete (detailed in Supplementary Results, Fig. S9 & S10). In almost all high-quality MAGs, acetyl-CoA could enter into incomplete reduced citrate cycle and was convert to succinate and 2-oxoglutarate (Fig. 5), and could further turn into glutamate and glutamine (detailed in Supplementary Results, Fig. S11). Acetyl-CoA was also the important precursor for the biosynthesis of membrane lipid via mevalonate pathway (detailed in Supplementary Results, Fig. S12).
In summary, the Ca. Natranaeroarchaeales members had the potential of inorganic carbon fixation, and all taxa may utilize the extracellular organic acids. Thus, this lineage may have a mixotrophic lifestyle.
Energy metabolism coupling with sulfur respiration
The genes involved in sulfur metabolism were rich in Ca. Natranaeroarchaeales. Sulfate could be reduced to sulfite via the assimilatory sulfate reduction pathway by 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS), sulfate adenylyltransferase (Sat), adenylylsulfate kinase (CysC) and phosphoadenosine phosphosulfate reductase (CysH) in most high-quality MAGs of the four genera, although the assimilatory (CysJI and Sir) or dissimilatory (DsrAB and AsrABC) sulfite reduction was less found. Sir was found in only one high-quality MAG HAT24, and anaerobic sulfite reductase subunit B (AsrB) was found in three high-quality MAGs HAT25, CSSed162cmB_145, and CSSed10_245. No CysJI or DsrAB were found in both high- and medium- quality MAGs. Some Ca. Natranaeroarchaeales had the potential of reduction of S0 and thiosulfate and disproportionation of thiosulfate (Fig. 5). All of the high-quality MAGs had at least one subunit of sulfhydrogenase HydGBAD (functioning in the reduction of S0 and elemental sulfur), thiosulfate reductase (PhsA) or thiosulfate/3-mercaptopyruvate sulfurtransferase (TST). The hydGBAD genes clustered in one operon in high-quality MAGs HAT25, CSSed162cmB_145, B1Sed10_191R1, and medium-quality MAGs CSSed165cm_322 and T1Sed10_113R1 (containing two clusters). Almost all (ten of eleven) high-quality MAGs contained Phs and TST, and more than half (nine of fourteen) medium-quality MAGs also had Phs. Phs transforms thiosulfate into sulfite and sulfide with quinol (CoQ) as the cofactor, while TST transfers the sulfur atom onto one thiol group to form a disulfide group and also produces sulfite and sulfide (Table S8). The CoQ could be reduced by NADH dehydrogenase, and this process coupled with the formation of transmembrane proton gradient for ATP regeneration (Fig. 5). All high-quality Ca. Natranaeroarchaeales MAGs contained some subunits of NADH dehydrogenase (NuoEFIKLMN) and almost complete V/A type ATPases (AtpABCDEFIK) which took part in ATP regeneration via electromotive force (Fig. 5 & Table S8) coupled with thiosulfate reduction.
As we know, sulfide could be assimilated into amino acids (cysteine and methionine) biosynthesis, or released to the environment. No more than one-third of all 25 high- and medium- quality MAGs contained cysteine synthase (CysK) (Fig. S11), so we proposed the above-mentioned MAGs or closely related species may generate energy by sulfur respiration and may involve in the sulfur cycle at the same time, e.g. sulfidogenesis in the anaerobic conditions of soda-saline lake sediment. Only one doubt was the reduction of sulfite. Maybe sulfite would be exported similar as sulfide.
Hydrogenotrophic acidogenesis coupled with energy metabolism by CoM and CoB
There were diverse hydrogenase and related proteins annotated in Ca. Natranaeroarchaeales, including energy-converting hydrogenase (EhbAEHI), coenzyme F420 hydrogenase (FrhABG), NADP-reducing hydrogenase (HndAC), Bidirectional [NiFe] hydrogenase (HoxEHY), Membrane-bound hydrogenase (MbhJKL) and F420-non-reducing hydrogenase (MvhADG) (Table S8). Besides, almost all high-quality MAGs (except HAT15) had complete or nearly complete (lacking one subunit) hydrogenase maturation proteins (HypABCDEF). It was suggested that hydrogen may be the main source of electron donor. Notably, while some subunits were missing in several hydrogenases in these MAGs, the F420-non-reducing hydrogenase (MvhADG) containing three subunits was present in almost all high-quality MAGs (except HAT15) and most medium-quality MAGs of genera PWKY01 and PWHR01 (Fig. 5 & Table S8).
Interestingly, genes mvhADG formed a gene operon with heterodisulfide reductase genes (hdrABC) in at least nine MAGs (seven shown in Fig. 6 and HAT29 and HAT17 in Fig. S13, contigs listed in Table S9), and these six proteins constituted a complex catalyzing the reduction of CoM-S-S-CoB and ferredoxin by molecular hydrogen. Complex HdrABC-MvhADG coupled with methane biosynthesis in some methanogens, for example Methanomassiliicoccales ([27-29], discussed below). However, none of Ca. Natranaeroarchaeales contained a methyl-coenzyme M reductase (Table S8). Interestingly, the oxidation of HS-CoM and HS-CoB was replaced by a fumarate reductase (TfrAB) (Fig. 5). It was supported that trfAB genes are also located in one operon with hdrABC and mvhADG and/or at the downstream of hdrB in thirteen MAGs (seven shown in Fig. 6, while HAT25, CSSen162cmB_145, B1Sed10_107R1, T1Sed10_92, HAT28 and HAT29 in Fig. S13). Same as the incomplete reductive citrate cycle, tfrA and tfrB (or tfrB-homolog) genes distributed in almost all high-quality MAGs and most medium-quality MAGs (Fig. 6 & Fig. S14). More details on trfAB were in the Supplementary Results.
The clustered genes in one operon usually were involved in the same life process. Following the incomplete reductive citrate cycle, succinate was transferred to succinyl-CoA, and then was converted into 2-oxoglutarate with reductive ferredoxin as cofactor. The reductive ferredoxin may be produced from an electron bifurcation catalyzed by HdrABC-MvhADG complex (Fig. 5). Thus, the production of 2-oxoglutarate was coupled with hydrogen utilization by HdrABC-MvhADG complex in gene distribution and metabolic functions. Alternatively, succinate was produced meanwhile reductive ferredoxin was oxidized by Na+-translocating ferredoxin:NAD+ oxidoreductase which was also annotated in almost high-quality MAGs and some medium-quality MAGs (Fig. 5 & Table S8). In this process, transmembrane Na+ gradient could be formed for ATP regeneration and NAD+ could be reduced for NADH regeneration in sulfur respiration (Fig. 5). So Ca. Natranaeroarchaeales could perform hydrogenotrophic acidogenesis and sulfur respiration (described above). In addition, most of hdrABC-mvhADG-tfrAB operon containing MAGS had four subunits of 2-oxoglutarate/2-oxoacid ferredoxin oxidoreductase (KorDABC) (Fig. 5), and this enzyme could catalyze the reversable transformation between succinyl-CoA and 2-oxoglutarate [30]. Genes korDABC located at the downstream of hdrABC-mvhADG-tfrAB operon and shared the same promoter (Fig. 6, S13 & S14). This result suggested that 2-oxoglutarate may be the one of the final products. Besides, one or both subunits of fumAB involving in the formation of fumarate from malate were located at the upstream of hdrABC-mvhADG-tfrAB operon in HAT24, HAT26, CSSed10_245 and CSSed10_214 (Fig. 6). Genes fumAB was separated by seven genes from hdrABC-mvhADG-tfrAB operon in MAG B1Sed10_191R1 (Fig. S13). It also supported the relationship between hydrogen metabolism and incomplete reductive TCA. The evidence of reversed TCA flow also included that none MAGs had the potential of phosphorylation (lacking cytochrome c reductase and cytochrome c oxidase) and photophosphorylation (lacking all of photosystem I, II, bacteriorhodopsin (K04641) and halorhodopsin (K04642)). This is reasonable considering the sediment is an anaerobic and dark environment.