Text/Listen to Jun Chat
Edit/listen to Jun Chat
Many archaeal lineages inhabit marine surface sediments, although their distribution, metabolic capacity and interspecific interactions remain relatively blank in the field of scientific research.
We recovered the metagenomic assembled genomes (MAGs) of archaea from CR margins and compared them with close relatives from shallower environments.
This experiment studied 31 mags from 6 different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales and Hadesarchaea) and performed a comprehensive analysis of the representative mags of Lokiarchaeota and Bathyarchaeota phylum.
We combine genomics and thermodynamic models to highlight the ecological and physiological conditions of symbiotic interactions between different archaeal lineages and use phylogenetic inference to explain the development of atypical symbiotic interactions of atypical symbiotic effects of abyssal archaea in CR deep sediments.
If Lokiarchaeota binds to organisms that use nitrates, nitrites, and sulfites as electron acceptors, then it is genetically possible for Lokiarchaeota to degrade benzoates.
These metabolic features suggest that this abyssal archaea lineage may be a transition between methanogenic and acetone-producing abyssal archaeal lineages.
I. Research progress on archaeal distribution, ecological role and adaptation strategies in deep-sea sediments
Marine subsurface sediments are rich in different archaeal lineages, although their distribution, ecological roles, and adaptation strategies have not been fully studied.
Metagenomic sequencing and single-cell genomics enable the discovery of nascent organisms, the elucidation of new metabolisms, the expansion of known lineages, and the redefinition of parts of the gene tree.
Genomes decomposed on the deep seafloor are still scarce, meaning that the adaptation of specific ecological niches in the deep-sea biosphere is not well understood.
Genomic analysis of Asgardian archaea shows that its eukaryotic homologs are abundant and indicate a wide variety of metabolic functions.
From autotrophic lifestyles that rely primarily on carbon-fixed production via the Wood-Ljungdahl pathway and acetone, to heteroorganic trophic lifestyles that consume protein and aliphatic hydrocarbons using methylcom reductase-like enzymes.
Abyssal Archaeota, a member of another phylum of archaea characterized by a broad metabolic range, is able to heterotrophic to remove proteins, carbohydrates, short-chain lipids and other reducing compounds as substrates, as well as their methane metabolism potential.
The deep-sea paleontological genome also indicates the potential for carbon fixation and acetone production, and the evolutionary pathways of methanogenesis and acetone production pathways of deep-sea archaeota remain unanswered.
We studied in detail archaeal genomes recovered from metagenomics at CR margins and compared them with close relatives recovered from shallower sites.
This experiment studied 31 archaeal metagenomic assembly genomes (MAGs) belonging to 6 different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales and Hadesarchaea).
We performed representative MAGs analysis of two new archaeal lineages, Lokiarchaeota and Bathyarchaeota, and the analysis showed that the Lokiarchaeota genome encodes genes for anaerobic treatment and degradation of aliphatic and aromatic hydrocarbons.
Genome classification for DNA extraction and sequencing and metagenomic assembly
The maximum gene tree was calculated using IQ-Tree based on the tandem of 16 ribosomal proteins (L2, L3, L4, L5, L6, L14, L15, L16, L18, L22, L24, S3, S8, S10, S17, and S19).
Evolutionary distances are calculated based on best-fit surrogate models (VT+F+R10) and single branching locations are tested using 1000 ultra-fast bootstrap and approximate Bayesian calculations.
Use the HMMsearch tool to screen predictive proteins from all MAGs against a custom HMM database representing key genes for specific metabolic pathways.
Assay completion of pathways was assessed by querying predictive proteins in the KEGG database by using BlastKoal, carbohydrate-active enzymes were identified, and cell localization identification was performed.
USENOBLAST was used to identify proteases, peptidases, and peptidase inhibitors, transporter identification, and detect the phylogenetic distribution of predicted proteins in each bin using USEARCH-ublast.
Use the corresponding KEGG entry as a search keyword, manually organize aligned sequences, calculate the phylogenetic tree, and describe evolutionary relationships using the best-fit model.
Perform initial Gibbs free energy (ΔrG') calculations for redox reactions proposed in CR Lokiarchaeota and its potential partners, using an equilibrizer, apply a pH of 8 and a concentration of 1 mm per reactant.
By calculating the Gibbs free energy of the five redox reactions at the CR sites U1378 and U1379 in situ, the feasibility of the coupled redox reaction proposed for the new CR Lokiarchaeota genome was further confirmed.
Gibbs free energy is calculated as:
In this study, ΔG0r was calculated using standard Gibbs generated free energy thermodynamic data for each substance and corrected to near-in-situ pressure and temperature (4°C) using R-wrapped CHNOSZ.
Qr is the reaction quotient and can be calculated by a relation
Third, the advantages of archaeal lineage diversity based on 16S rRNA gene analysis
In sediment samples at 5 depths at the CR margin, archaea were abundant.
A total of 126 16S rRNA gene sequences were recovered from metagenomics by collecting metagenomic data and examining small subunit ribosomal genes.
Sequences associated with archaea (31 sequences account for 25% of the total) represent a wide range of lineage diversity, including deep-sea archaeota, pyrogens, and Lokiarchaeota.
In the 5 depths analyzed, 31 different archaeal MAG sketches were recovered and only 11 MAGs with high integrity and low contamination (greater than 60% integrity and less than 5% contamination) were genomically analyzed.
Genome quality was further assessed by comparing their predicted proteins to an NCBI (nr) database to assess the degree of phylogenetic consistency within the classified genome.
The taxonomic attribution (60-75%) of most of the predicted proteins in each MAG was consistent with the respective phylogenetic taxa, but only 15% of the coding proteins in the CR_12 belonged to Heimdallarchaeota and were excluded from further analysis.
Using 16 ribosomal proteins to determine the phylogenetic location of the draft genome, the MAGs found experimentally belong to 6 different phylogenetic lineages, namely Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales and Hadesarchaea.
All archaeal MAGs recovered from CR deposits contain eukaryotic characteristic proteins (ESPs), which include protein homologs specifically for information processing, transport mechanisms, ubiquitin system, cell division, and cytoskeletal formation.
Asgardian archaea genomes recovered from CRs, such as Heimdallarchaeota and Lokiarchaeota MAGs (CR_06, CR_11), show a large number of eukaryotic homologues and cover a wide range of ESPs classes.
ESP was also detected in deep-sea archaeota, Hadesarchaeota and Thermoplasmata MAGs recovered in CR, and only the Asgardian genome has homologs of cell division and cytoskeleton.
The MCR complex gene (mcrABCDG) is absent in both the microgenome and the entire metagenomics, indicating that short-chain alkanes in CR sediments are not oxidized by the MCR complex.
Screen all metagenomic readouts and mags for possible alternative hydrocarbon degradation pathways using customized HMM searches specifically targeting key metabolic genes for aliphatic and aromatic hydrocarbon degradation pathways.
Multiple pathways have been successfully discovered, including the coupling of glycyl radicalase-associated genes to n-alkansuccinate synthase (AssA) and benzylsuccinate synthetase (BssA), which activate n-ane and monoaromatic compounds by forming C-C bonds between n-ane and monoaromatic compounds and fumarate to form hydrocarbon adducts, respectively.
Since Lokiarchaeota CR_06 had the lowest levels of contamination, phylogenetic analysis of BCR subunit B recovered from Lokiarchaeota CR_06 revealed that they belonged to the Bzd class and consisted of 4 subunits (BzdONPQ).
This BCR type was originally discovered in β genus Proteus, Azoarchaea genus Evancyi, and BCRs detected in CR_06 were closely related to BCRs detected in different β, δ Proteus genus, and other archaeal lineages such as Lokiarchaeota, Bathyarchaeota and Archaeoglobus.
Subunits P and Q have ATP-binding/ATPase functional domains and are linked together by [4Fe-4S] clusters.
The reduced ferro-returning toxin transfers electrons to the coenzyme A ester-binding domain (O and N subunits), catalyzing the cleavage of the benzoyl-CoA aromatic ring to generate diene-CoA products.
In CR_06, 4 subunits of the BCR complex are located on a continuous operon (CR_06-contig-100_3495), but no reliable phylogenetic marker genes have been found in adjacent genomic neighborhoods due to the high fragmentation of the genome.
For high fragmentation levels of CR_06 MAG (number of scaffolds = 1110), although the genome exhibits a high coverage level of > 200x, a plausible explanation is that there is a high level of intralineage strain heterogeneity.
CR_ Lokiarchaeota (CR_06) and other Asgard archaea located at the edge of CR may feed on aromatic hydrocarbons and grow heterotroph
The deletion of genes encoding different types of cytochrome oxidase and anaerobic respiration in the genomic content of Asgard MAGs (CR_06, 07, 08, 11 and 12) indicates that they are unable to fully mineralize these hydrocarbons to CO2 and H2O, which would allow for high energy yields.
They encode the genes that mediate the fermentation of these organic macromolecules into acetate esters and other reducing products, which are thermodynamically unfavorable under CR conditions with a positive ΔrG' value (ΔrG' = 196.3 [kJ/mol]), because it means that these genes are selectively maintained.
Due to the incomplete genome (86% intact), we cannot rule out the possibility of the presence of intact aromatic hydrocarbon mineralization pathways, using one or more oxidized substrates as electron absorption.
It is more likely that Lokiarchaetoa derives its energy through symbiotic interactions with partners capable of oxidizing biodegradable intermediates.
Inference of potential symbiotic partner identities under ocean subsurface conditions by comparing all possible metabolic and thermodynamic scenarios, and each scenario is measured in terms of the presence of metabolic pathways in metagenomic datasets and thermodynamic feasibility under each condition.
We calculated the Gibbs free energy of the coupling reaction under various substrate concentration conditions, and the optimal conditions showed that degradation of parabens, a central metabolic intermediate in the aromatic hydrocarbon degradation pathway, may occur under the following metabolic conditions:
(a) benzoate mineralization to CO2 and H2O, reduction of nitrite to ammonia (ΔrG ' = −1206.3 [kJ/mol]);
(b) benzoate mineralization to produce CO2 and H2O, sulfite reduction to hydrogen sulfide (ΔrG ' = −373.6 [kJ/mol]);
(c) Paraben mineralization to CO2 and H2O, nitrate reduction to nitrite (ΔrG ' = - 119.9 [kJ/mol]).
The type and complexity of the exchange substrate is another key factor affecting Lokiarchaeota trophies and identity as foster partners.
The presence of the 3b and 3d groups of genes encoding membrane-binding electron bifurcation [NiFe] hydrogenases, which combine the oxidation of NADH+ and NADH+ with the evolution of H2, also suggests a co-exchange of hydrogen between Asgard and their partners.
Effective substrate and electron exchange between symbiotic partners requires the presence of a biological catheter (e.g., type IV hairs or flagella) or some sort of electron shuttle that allows extracellular electron transfer (e.g., polyheme cytochrome), we screened these mechanisms for CR_06 and identified two candidate mechanisms for interspecific substrate and electron exchange.
Metabolic analysis showed that CR_14 contained genes encoding the methanogenic pathway of incomplete methyl nutrients, but lacked key genes encoding the MCR complex (mcrABCDG).
CR_14 shows the potential ability to utilize group 4 hydrogenases (formate hydrolases) to reduce formic acid to trimethylamine as a donor of electrons as electrons and hydrogen donors by trimethylamine oxide reductase or anaerobic dimethyl sulfoxide reductase (TMAO/DMSO reductase).
The gene encoding trimethylamine-specific corcoroid protein and the presence of multiple methyltransferases CoB-CoM isodisulfide reductase/F420 non-reducing hydrogenase (hdrABCD and mvhADG) suggest that CR_14 has the ability to recover coenzyme M (CoM) and Coenzyme B (CoB) and transfer methyl groups from trimethylamine to CoM-sh.
We found a gene encoding tetrahydromethanepterin s-methyltransferase (mtrA-h), indicating that the mtrA protein transfers methyl groups from CoM to 5,6,7,8-tetrahydromethaneterin (H4MPT) and uptakes methyl groups to acetyl-CoA via the β subunit of the CODH/ACS complex, replacing the function of the Wood-Ljungdahl pathway methyl branch.
CR_14 is the only deep-sea archaeal MAG with the transfer of methyl groups from monomethylated, dimethylated, and trimethylated compounds.
These collective metabolic signatures of the deep-sea archaeota CR_14 suggest that the genome may represent a lineage that creates a bridge between methanogenic and acetate-producing deep-sea archaeota by adopting an atypical methanogenic lifestyle.
The phylogenetic tree of the acetyl-CoA synthetase β subunit (AcsB) shows that the CR_14 AcsB gene is clustered with genes recovered from other abyssal archaea lines, the Asgard phylum, Chloroflexi, and Altiarchaeales.
The uniqueness of the clade stems from previous assumptions that Altiarchaeales members possess an ancient version of AcsB.
Members of Chloroflexi encode this archaeal version of the ACSβ subunit, similar to the ACSβ subunit in the deep-sea archaeota, Asgard phylum, and Altiarchaeales.
This ACS type supports the hydrogen-dependent autotrophic lifestyle of Asgardian archaea, and we hypothesize that the deep-sea archaeota has an ACS type capable of utilizing different carrier proteins (CoM, H4MPT, and possibly other unknown carrier proteins) to bind methyl groups into acetyl-CoA, allowing deep-sea archaeota to absorb methyl groups from various methylated compounds.
All CR archaeal MAGs are rich in ESP-coding genes, and the widespread distribution of these eukaryotic homologs suggests that ESPs are more prevalent in anaerobic archaea than previously recognized.
The sediments used in the experiment were collected from deeper depths, so the analysis focused on mags belonging to deep-sea archaeota and Lokiarchaeota to understand their ecological potential under deep-sea sediment conditions.
Deep-sea sediments are rich in aliphatic and aromatic hydrocarbons, which provide a significant portion of the energy and carbon needs of the microorganisms involved.
Degradation of hydrocarbons is limited to a limited number of bacterial and archaeal phylums (such as Aminicenantes, TA06, Aerophobetes, Atribacteria, Helarchaeota and Bathyarchaeota).
Given the high energy requirements for aromatic decomposition under conditions of limited energy in seafloor sediments, the discovery of atp-dependent BCR complexes (Bzd classes) in the genome of CR_Asgard/CR_Lokiarchaeota has a fermented and/or acetone-producing lifestyle.
Lokiarchaeota's potential partners belong to different metabolomes (e.g., nitrate reducing agents, nitrite reducing agents, sulfate reducing agents, sulfite reducing agents, and thiosulfate reducing agents), but under subsurface conditions, only sulfite, nitrate, and nitrite reducing agents are thermodynamically preferred.
In this experiment, we used a metagenomics-based genomics approach to successfully recover 31 mags from the metagenomic dataset of 5 different samples, belonging to the phylum Archaea (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales and Hadesarchaea).
Only 11 MAGs met our completion and contamination thresholds (> 60% completion and <10% contamination) and were incorporated into detailed genomic analysis and metabolic reconstruction.
More than 90% of the high-quality genomes belong to the deep-sea archaeota and Asgard phylums, and the phylogenetic relationships of the proteins predicted in each MAG confirm the appropriate quality of the MAG considered in this study.
Under the same environmental conditions, different species of homotrophs may need to share different qualities of substrates (e.g., acetate, propionate, and other short-chain fatty acids).
This suggests that in addition to the previously proposed hydrogen nutrition partners, there are Lokiarchaeota cotrophic partners capable of acetic acid oxidation and short-chain fatty acid oxidation.
We hypothesize that thermodynamic advantages and potential diversity of shared metabolites will drive Lokiarchaeota to perform syncytial rather than just individual metabolite exchange.
Abyssal archaeota has been shown to have the ability to produce methane and acetone, and in general, the abyssal archaea lineage is widely distributed in different marine benthic habitats compared to conventional methanogens, which may benefit from their acetone-producing ability to degrade a variety of organic compounds under thermodynamically favorable conditions.
The redox potential and availability of oxidized substrates in CR sediments may be detrimental to the occurrence of complete methanogenesis and associated methanogenic archaea.
Experiments found archaea magnans in samples collected from deep underground sediments, some of which were of high quality and integrity.
Many CR archaeal MAGs, particularly those belonging to the Asgardian host eukaryotic characteristic, are able to degrade fats and aromatic hydrocarbons and establish hypothetical partnerships with metabolically diverse symbionts.
These environmental conditions include the presence of high levels of methylated compounds, such as methylated amines, in the deep-sea environment, and the lack of specialized pathways needed to recover these methylated compounds and transport the extracted methyl groups directly to the Wood-Ljungdahl pathway.
In these CR sediments, different environmental conditions may capture an intermediate transition between acetone-producing and methane-producing lifestyles.
 He Tianliang, "Study on the Anti-tumor Metastasis Activity of Microorganism-Virus Interactions and Their Secondary Metabolites" in the Deep Sea Hydrothermal Vent
 Gao Zhiyuan, "Study on the Microbial Diversity of the Deep Sea Environment and the Metabolic Potential of Intestinal Microorganisms of Ultra-abyssal Sea Cucumbers"
 Yali Huang, "Deep-sea metagenomic library screening and new functional genes"
 Xie Wei, "Environmental Adaptability and Genetic Resources of Microbial Community in Deep-sea Hydrothermal Vents"
 Li Hui, "Application of metagenomic technology in the development of uncultured environmental microbial gene resources"
 Zeng Runying, "Progress in Research and Development Technology of Deep-sea Microbial Resources"
 Muhammad Zohaib Nawaz, Detection of small RNA in deep-sea microorganisms based on multiple omics approaches