Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field

Abstract

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H₂), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H₂ and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H₂ and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H₂) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Keywords: acetogenesis; formate; hydrogenase; hydrothermal; metagenomics; methanogenesis; serpentinization; sulfate reduction.

FIG 1

The Lost City hydrothermal field is located at 30°N, west of the Mid-Atlantic Ridge, on the southern wall of the Atlantis Massif. Part A shows a three-dimensional view of the field (after Kelley et al., 2005 [6]) featuring the massive Poseidon structure, which is composed of several actively venting chimneys. Hypothetical flow paths are informed by the aqueous geochemistry results reported here, by Aquino et al. (in review [40]), and by prior studies referenced in the main text. Part B is a nonmetric multidimensional scaling (NMDS) ordination of 16S rRNA amplicon sequence data where each data point represents the microbial community composition of one hydrothermal fluid sample. Sizes of data points are scaled to the measured sulfate concentration of that sample (Table 1).

FIG 2

Relative abundances of selected ASVs in Lost City hydrothermal fluid samples. Amplicon libraries were generated from both DNA and RNA extractions; bubbles representing relative abundances in RNA libraries are highlighted with black borders. ASVs were selected to highlight the taxa that were the focus of this study, as well as additional taxa that are expected to be associated with hydrothermal environments and provide context for interpreting differences among fluid samples. A full table of ASV counts is provided in Table S2.

FIG 3

Abundance of refined MAGs in Lost City hydrothermal fluid samples. Total mapped read coverage was normalized to genome size and to the size of the metagenome or metatranscriptome library. The final normalized coverage is reported as a proportional unit (transcripts/fragments per million; TPM) suitable for cross-sample comparisons. Bubbles representing coverage in metatranscriptomes (MT), rather than metagenomes (MG), are highlighted with black borders. For clarity, not all MAGs are shown. A full coverage table is provided in Table S4.

FIG 4

Presence and absence of key genes in refined MAGs. Genes defined by KEGG Orthology (see Table S5) were selected to highlight potential metabolic capabilities to metabolize hydrogen gas, to reduce sulfate to sulfide (SRB), to fix nitrogen (N₂) gas, to fix carbon dioxide via the Wood-Ljungdahl pathway (CODH/ACS), and to utilize formate or glycine as carbon sources. Patescibacteria MAGs (including Paceibacteria and Gracilibacteria) are not shown here because they lack all of the gene shown here.

FIG 5

Phylogeny of the large catalytic subunit of membrane-bound hydrogenase (mbhL). Sequences identified in refined MAGs from this study are highlighted in bold font. The two archaeal sequences from Lost City (Methanosarcinaceae and ANME-1) form their own clade apart from all known mbhL sequences. The mbhL sequence from a Lost City Bipolaricaulota MAG clusters together with Bipolaricaulota MAGs from other hydrothermal environments. Bootstrap support values are shown for each node. An expanded version of this figure, including the gene order for the mbh gene cluster is provided as Fig. S8.

FIG 6

Phylogeny of divergent FDH-like sequences. Sequences identified in refined MAGs from this study are highlighted in bold font. Their closest matches in the NCBI nr database are from other serpentinite-hosted springs (Voltri Massif, Hakuba Happo, and The Cedars). The FDH-like sequences shown here include an iron-sulfur binding domain and a molybdopterin oxidoreductase domain, which are encoded as two separate coding regions in some species and as a fused coding region in others (see Fig. S9 an expanded version of this figure, including genomic context). The phylogeny was constructed from the conserved oxidoreductase domain. Bootstrap support values are shown for each node. The Lost City Bipolaricaulota sequence was identified in multiple BinSanity bins classified as Bipolaricaulota, but it was not included in the final, manually refined MAGs.

FIG 7

Abundance of key genes in Lost City hydrothermal fluid samples. Metagenomic coverage was normalized to predicted protein length and to the size of the metagenome or metatranscriptome library. The final normalized coverage is reported as a proportional unit (transcripts/fragments per million; TPM) suitable for cross-sample comparisons. Bubbles representing coverage in metatranscriptomes (MT), rather than metagenomes (MG), are highlighted with black borders. Genes are defined with KEGG Orthology; see Table S5.