A non-methanogenic archaeon within the order Methanocellales

Abstract

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO₂-reducing, electron-fueled acetogen without electron bifurcation.

Fig. 1. The Cedars serpentinization site and in situ filtration.

a Barnes Spring complex in The Cedars area. Yellow arrowhead shows the location of Barnes Spring 5 (BS5). b Bird’s eye view of the BS5 pool. c The source water of BS5 flow (BS5sc) at the bottom of BS5 pool (yellow rectangle in b). d Grotto Pool Spring1 (GPS1) located near the Grotto pool near camp site. e Microbes in the GPS1 source spring water (sample GPS1) was collected by using in-line filtration system (green rectangle in panel d).

Fig. 2. Phylogenomic position and key genes for methanogenesis encoded in the Methanocellales archaeon Met12.

a Maximum likelihood phylogenetic tree of publicly available genomes belonging to a phylum Halobacteriota with newly identified MAG, Met12 (Left). Orders Methanocellales (purple) and Methanosarcinales (red) were highlighted respectively. The existence of key genes for methanogenesis in the genomes is shown in the square and triangle blocks (right). A filled block indicates full gene set of the module coded in the genomes, whereas filled block with lighter color indicates only partial gene set of the module coded in the genomes. The orthologous clusters and the related genes are shown in Supplementary Data 1. Types of energy metabolism determined by physiological analysis of cultivated organisms are shown in red filled circles, whereas those predicted from genomic constitutions are shown in open circles. Genome size is shown by blue bar chart. b Energy and carbon metabolisms of the Methanocellales archaeon Met12 predicted from the genomic constitution. Proteins, complexes, cofactors and electron carriers are colored based on the functional categories. Cdh acetyl-CoA decarbonylase/synthase complex Fwd/Fmd formylmethanofuran dehydrogenase, Ftr formylmethanofuran:tetrahydromethanopterin N-formyltransferase, Mch methenyltetrahydromethanopterin cyclohydrolase, Mtd methenyltetrahydromethanopterin dehydrogenase, Mer methenyltetrahydromethanopterin reduce,ase, Mtr tetrahydromethanopterin S-methyltransferase, Mcr methyl-coenzyme M reductase, Acs acetyl-CoA synthetase, YaaH succinate-acetate transporter, SbtA Na⁺-dependent bicarbonate transporter, Frh coenzyme F420-reducing hydrogenase, Ech/Eha/Ehb energy-converting hydrogenase, Mvh F420-non-reducing hydrogenase, Hdr heterodisulfide reductase, Mrp Multiple resistance and pH antiporter, Ntp archaeal type A1AO-ATPase, Rnf proton/sodium-translocating ferredoxin-NAD:oxidoreductase complex, MHcytC multi-heme c-type cytochrome, Fdx ferredoxin, MP methanopterin, MFR methanofuran, H₄MPT tetrahydromethanopterin, CoA coenzyme A, F₄₂₀ coenzyme F₄₂₀. The missing components for the methanogenic/methanotrophic pathway are translucent.

Fig. 3. Scatter plots of in situ gene expression for Met12 among BS5sc in 2014, GPS1 in 2012, BS5 pool in 2011, and BS5 pool in 2012.

The scatter plots were generated based on in situ gene expression profiles of the whole CDSs of Met12 (Supplementary Data 2). Proximate line was shown as the linear model of the expression correlation (dashed line), and R-squared value was calculated. Key genes for methanogenesis (Supplementary Data 1) were highlighted as blue circles, while multi-heme c-type cytochromes (MHcytCs) were highlighted as red circles. Highly expressed archaeal pilin and 4-heme MHcytC, mmcX, were indicated.

Fig. 4. Phylogeny and 3D structure of 4-heme c-type cytochrome MmcX of the Met12.

a Protein sequences of MmcX of the Met12 and the close relatives were aligned using MUSCLE and organized into a phylogenetic tree using maximum likelihood (ML). b The 3D protein structure model of MmcX of the Met12. The protein backbone trace was cyan, and the four heme molecules were shown in red ball and stick. c, A close-up view of the MHcytC to show the heme array in MmcX, with the distances between iron atoms of hemes. Heme molecules are labeled from N-terminal with numbers in circles. d, The heme array in MmcX of the Met12 and the histidine coordination for the hemes.

Fig. 5. Characterization of 4-heme c-type cytochrome MmcX and reconstruction of metabolic pathway of Met12.

a Electrotrophic current consumption from an electrode poised at −400 mV (vs. SHE) for wild-type MR-1, ΔomcAΔmtrC mutant and mmcX-ΔomcAΔmtrC. Data are presented as mean values ± SD (n = 3 biologically independent experiments). b Electrogenic current generation to an electrode poised at +200 mV (vs. SHE) for wild-type MR-1, ΔomcAΔomcA mutant and mmcX-ΔomcAΔmtrC. Data are presented as mean values ± SD (n = 3 biologically independent experiments). c Estimated electron-fueled acetogenic pathway of the Met12 in highly alkaline ultra-reducing serpentinized subsurface groundwater.

Fig. 6. Phylogenetic trees of carbonate alkaline transporter (SbtA) genes.

Protein sequences were aligned using MUSCLE and organized into a phylogenetic tree using maximum likelihood (ML). ORFs from the metagenomes of serpentinization settings are highlighted with different colors. SbtA function was described elsewhere.

Fig. 7. Global distribution of Met12-like MAGs in other serpentinization sites.

a BLAST Ring Image Generator shows the blastn analysis of the Met12 genome against three genomes of close relative strains (Methanocella paludicola, Ca. Methanoperedens nitroreducens, Methanosarcina acetivorans, and Methermicoccus shengliensis), MAG-838 from Lost City, the Old City metagenome, and the Prony Bay metagenome. The lower and upper identity threshold in the blastn analysis is shown in the circles. b Presence and absence of Met12-like MAGs in various serpentinization sites. The analyses were conducted for The Cedars serpentinizing groundwater (PRJDB2971), Lost City hydrothermal vents (PRJNA779602), Old City hydrothermal vents (PRJNA556392), Voltari Massif travertine, BR2 (PRJNA265986), Cabeco de Vide travertine, AC3 (PRJNA265986), Prony Hydrothermal Field, ST09 (PRJNA265986), and Santa Elena Ophiolite alkaline spring, Spring9 (PRJNA340462). The global map was provided from NOAA National Centers for Environmental Information. 2022: ETOPO 2022 15 Arc-Second Global Relief Model (10.25921/fd45-gt74. Accessed 2024/03/08)/CC0-1.0.

Fig. 8. Gain and loss of orthologous genes and methanogenesis-related genes.

Numbers of orthologous genes for gain, loss, hold, and missing were shown by pie charts at the branch point of unrooted phylogenomic tree of orders Methanocellales, Methanosarcinales, Syntropharchaeales, and Methanotrichales genomes. From the 110 methanogenesis-related genes (inner pie chart), specific key gene sets of the methanogenic process described in Fig. 1 were depicted in the colored boxes with + as gained gene set (red line) or − as lost gene set (blue line). Multi-heme c-type cytochromes that is not assigned as the key methanogenesis genes were also shown in yellow boxes.