Deep-branching acetogens in serpentinized subsurface fluids of Oman

Abstract

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Keywords: Acetothermia; acetogenesis; metagenomics; origin of life; serpentinization.

Fig. 1.

Phylogenomic analysis and ecological distribution of Acetothermia MAGs recovered from SO subsurface fracture waters. (A) ML phylogenetic reconstruction of MAGs recovered in this study in comparison with those recovered from previous studies, with type I MAGs from the SO highlighted in orange and type II MAGs highlighted in blue. The year of the sample is shown, followed by the well designation from which the MAGs were recovered. Biomass from fracture waters was collected from 50-m (NSHQ14B) and 85-m (NSHQ14C) depths from NSHQ14 in 2017. The three proposed orders for the Acetothermia/Bipolaricualaeota are shown on the far right. Branch length is scaled to that shown in the lower left for expected substitutions per site. Bootstrap values >90% (out of 1,000 bootstraps) are indicated by black circles. Thermotogae representatives were used as the outgroup including Petrotoga mobilis SJ95, Thermotoga petrophila RKU-1, and Mesotoga inferna. (B) The abundances of types I and II populations are shown as the percentage of total metagenome reads mapped to either MAG type for each 2017 metagenome. The 2015 and 2020 metagenomes were not used for abundance calculations, given their relatively low sequencing depth and limited sampling scope, respectively. Metagenomes are arranged by the fluid type designation of their well waters and then ordered by ascending pH values for well waters, as indicated in parentheses next to each metagenome.

Fig. 2.

Metabolic reconstructions for Acetothermia type I and II MAGs recovered from the SO subsurface fracture waters. A composite metabolic reconstruction map is shown for type I and II MAGs, with encoded proteins/pathways in orange denoting type I MAGs and those in blue denoting the type II MAGs. Energy conservation pathways highlighted in the text are grouped in gray boxes, with the rest of the pathways representing central carbon metabolism. Question marks show areas of uncertainty in reconstructing aspects of metabolism. Subunits or proteins highlighted in black were not observed in the corresponding MAGs. Abbreviations are as follows: anaerobic carbon monoxide dehydrogenase (Coo), pyruvate Fd oxidoreductase (Pfor), ADP-forming acetyl-CoA synthetase (ACD), AMP-forming acetyl-CoA synthase (ACS), citrate synthase (GltA), aconitate hydratase (Aco), isocitrate dehydrogenase (Idh), 2-oxoglutarate Fd oxidoreductase (Kor), succinyl-CoA synthetase (Suc), succinate dehydrogenase (Sdh), fumarate hydratase (FumC), malate dehydrogenase (Mdh), pyruvate orthophosphate dikanase (PpdK), pyruvate-water dikinase (PpsA), enolase (Eno), phosphoglycerate kinase (PgK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase (TPI), fructose-1,6-bisphosphatase I (FBP), glucose/mannose-6-phosphate isomerase (Pgi-Pmi), glucose-6-phosphate isomerase (Pgil), Fd, reduced Fd (Fd²⁻), nitrate reductase (Nar), cytochrome c nitrite reductase (Nrf), heme-Cu oxidase (HCO), methyl-viologen reducing [NiFe]-hydrogenase (Mvh), and heterodisulfide reductase (Hdr).

Fig. 3.

Putative autotrophs in subsurface fracture water communities from the SO. Each column shows the relative abundances of reconstructed MAGs (>5% relative abundance) within each well water community, with the corresponding taxonomic classification of MAGs given on the left. The circles represent individual MAGs and are scaled based on the relative abundance of that MAG among others within that community (based on the legend at the top left of the plot). The circles are colored according to the autotrophic carbon fixation pathway that is inferred to be encoded within the MAG, as based on the legend at the top right of the plot. Well names are followed by the pH of the sampled fracture waters in parentheses. Taxonomic classifications are given at the lowest characterized taxonomic designation, followed by either the specific uncultured group it belongs to (alphanumeric designations) or are otherwise followed by “unclassified” if the MAG is not related to previously characterized genomes within the Genome Toolkit Database. The type II Methanobacterium is the same as described in Fones et al. (27), and, while it encodes the WL pathway, it is dependent on formate as an electron donor and carbon source. The 2015 and 2020 metagenomes were not used for abundance calculations, given their relatively low sequencing depth and limited sampling scope, respectively.

Fig. 4.

Phylogenetic placement of key proteins involved in acetogenesis and Acetothermia lineages recovered from the SO subsurface waters among other bacterial lineages. (A) ML phylogeny of the oxidoreductase subunits of the carbon monoxide/acetyl-CoA synthase (CODH/ACS) complex (CdhABC) encoded by the type II Acetothermia in context of other archaeal-like CdhABC (alignment length of 1,908 amino acid positions). Each subunit was individually aligned, and a concatenation of the three was subjected to ML analysis. The type II Acetothermia CdhABC are highlighted in bolded blue text. CdhABC from MAGs recovered from serpentinite-influenced environments are indicated on the right. Black circles show >90% bootstrap support (out of 1,000 bootstraps). Branch length is scaled based on the expected number of substitutions per site legend at the bottom left. CdhABC clades are collapsed as triangles, with the taxonomic groups they correspond to indicated next to the triangles. The monophyletic bacterial CdhABC clade is shown by an arrow. The tree is shown with a midpoint-rooted visualization. (B) Unrooted ML phylogeny of concatenated protein alignments from 30 housekeeping single-copy genes within 722 bacterial genomes representative of 128 phyla in the Genome Taxonomy Database (filtered alignment length of 9,162 amino acid positions; see Materials and Methods for additional analytical details). Phylum-level groupings are provided next to the shaded regions, with multiple phyla delineated with slashes. Genome information for the 722 entries is provided in Dataset S1. Black circles show >90% bootstrap support (out of 1,000 bootstraps). Bootstraps are only shown for phylum or higher-level groupings for clarity in interpreting the tree. Branch length is scaled based on the expected number of substitutions per site, as indicated on the left. Type I and II Acetothermia lineages are indicated with orange and blue stars, respectively. The placement of recently hypothesized root positions for the bacterial domain is shown with black (44) or white (94) stars.