William J. Brazelton

Methane- and sulfur-metabolizing microbial communities dominate the Lost City hydrothermal field ecosystem.

ABSTRACT Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.

Archaea and bacteria with surprising microdiversity show shifts in dominance over 1,000-year time scales in hydrothermal chimneys.

The Lost City Hydrothermal Field, an ultramafic-hosted system located 15 km west of the Mid-Atlantic Ridge, has experienced at least 30,000 years of hydrothermal activity. Previous studies have shown that its carbonate chimneys form by mixing of ∼90 °C, pH 9–11 hydrothermal fluids and cold seawater. Flow of methane and hydrogen-rich hydrothermal fluids in the porous interior chimney walls supports archaeal biofilm communities dominated by a single phylotype of Methanosarcinales. In this study, we have extensively sampled the carbonate-hosted archaeal and bacterial communities by obtaining sequences of >200,000 amplicons of the 16S rRNA V6 region and correlated the results with isotopic (230Th) ages of the chimneys over a 1,200-year period. Rare sequences in young chimneys were commonly more abundant in older chimneys, indicating that members of the rare biosphere can become dominant members of the ecosystem when environmental conditions change. These results suggest that a long history of selection over many cycles of chimney growth has resulted in numerous closely related species at Lost City, each of which is preadapted to a particular set of reoccurring environmental conditions. Because of the unique characteristics of the Lost City Hydrothermal Field, these data offer an unprecedented opportunity to study the dynamics of a microbial ecosystem’s rare biosphere over a thousand-year time scale.

Metagenomic Evidence for H2 Oxidation and H2 Production by Serpentinite-Hosted Subsurface Microbial Communities

Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H2). In order to assess the potential for microbial H2 utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H2-oxidizers. Both sites also yielded metagenomic evidence for microbial H2 production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood–Ljungdahl pathway. In general, our results point to H2-oxidizing Betaproteobacteria thriving in shallow, oxic–anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H2-powered primary production in serpentinite-hosted subsurface habitats.

Using CRISPRs as a metagenomic tool to identify microbial hosts of a diffuse flow hydrothermal vent viral assemblage

Metagenomic analyses of viruses have revealed widespread diversity in the viriosphere, but it remains a challenge to identify specific hosts for a viral assemblage. To address this problem, we analyze the viral metagenome of a northeast Pacific hydrothermal vent with a comprehensive database of spacers derived from the clustered regularly interspaced short palindromic repeat (CRISPR) putative immune system. CRISPR spacer matches to the marine vent virome suggest that viruses infecting hosts from diverse taxonomic groups are present in this vent environment. Comparative virome analyses show that CRISPR spacers from vent isolates and from thermophiles in general have a higher percentage of matches to the vent virome than to other marine or terrestrial hot spring viromes. However, a high percentage of hits to spacers from mesophilic hosts, combined with a moderately high modeled alpha diversity, suggest that the marine vent virome is comprised of viruses that have the potential to infect diverse taxonomic groups of multiple thermal regimes in both the bacterial and the archaeal domains.

Bacterial Communities Associated with Subsurface Geochemical Processes in Continental Serpentinite Springs

ABSTRACT Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.

Physiological Differentiation within a Single-Species Biofilm Fueled by Serpentinization

ABSTRACT Carbonate chimneys at the Lost City hydrothermal field are coated in biofilms dominated by a single phylotype of archaea known as Lost City Methanosarcinales. In this study, we have detected surprising physiological complexity in single-species biofilms, which is typically indicative of multispecies biofilm communities. Multiple cell morphologies were visible within the biofilms by transmission electron microscopy, and some cells contained intracellular membranes that may facilitate methane oxidation. Both methane production and oxidation were detected at 70 to 80°C and pH 9 to 10 in samples containing the single-species biofilms. Both processes were stimulated by the presence of hydrogen (H2), indicating that methane production and oxidation are part of a syntrophic interaction. Metagenomic data included a sequence encoding AMP-forming acetyl coenzyme A synthetase, indicating that acetate may play a role in the methane-cycling syntrophy. A wide range of nitrogen fixation genes were also identified, many of which were likely acquired via lateral gene transfer (LGT). Our results indicate that cells within these single-species biofilms may have differentiated into multiple physiological roles to form multicellular communities linked by metabolic interactions and LGT. Communities similar to these Lost City biofilms are likely to have existed early in the evolution of life, and we discuss how the multicellular characteristics of ancient hydrogen-fueled biofilm communities could have stimulated ecological diversification, as well as unity of biochemistry, during the earliest stages of cellular evolution. IMPORTANCE Our previous work at the Lost City hydrothermal field has shown that its carbonate chimneys host microbial biofilms dominated by a single uncultivated “species” of archaea. In this paper, we integrate evidence from these previous studies with new data on the metabolic activity and cellular morphology of these archaeal biofilms. We conclude that the archaeal biofilm must contain cells that are physiologically and possibly genetically differentiated with respect to each other. These results are especially interesting considering the possibility that the first cells originated and evolved in hydrothermal systems similar to Lost City. Our previous work at the Lost City hydrothermal field has shown that its carbonate chimneys host microbial biofilms dominated by a single uncultivated “species” of archaea. In this paper, we integrate evidence from these previous studies with new data on the metabolic activity and cellular morphology of these archaeal biofilms. We conclude that the archaeal biofilm must contain cells that are physiologically and possibly genetically differentiated with respect to each other. These results are especially interesting considering the possibility that the first cells originated and evolved in hydrothermal systems similar to Lost City.

Did life originate from a global chemical reactor

Many decades of experimental and theoretical research on the origin of life have yielded important discoveries regarding the chemical and physical conditions under which organic compounds can be synthesized and polymerized. However, such conditions often seem mutually exclusive, because they are rarely encountered in a single environmental setting. As such, no convincing models explain how living cells formed from abiotic constituents. Here, we propose a new approach that considers the origin of life within the global context of the Hadean Earth. We review previous ideas and synthesize them in four central hypotheses: (i) Multiple microenvironments contributed to the building blocks of life, and these niches were not necessarily inhabitable by the first organisms; (ii) Mineral catalysts were the backbone of prebiotic reaction networks that led to modern metabolism; (iii) Multiple local and global transport processes were essential for linking reactions occurring in separate locations; (iv) Global diversity and local selection of reactants and products provided mechanisms for the generation of most of the diverse building blocks necessary for life. We conclude that no single environmental setting can offer enough chemical and physical diversity for life to originate. Instead, any plausible model for the origin of life must acknowledge the geological complexity and diversity of the Hadean Earth. Future research may therefore benefit from identifying further linkages between organic precursors, minerals, and fluids in various environmental contexts.

Metagenomic Comparison of Two Thiomicrospira Lineages Inhabiting Contrasting Deep-Sea Hydrothermal Environments

Background The most widespread bacteria in oxic zones of carbonate chimneys at the serpentinite-hosted Lost City hydrothermal field, Mid-Atlantic Ridge, belong to the Thiomicrospira group of sulfur-oxidizing chemolithoautotrophs. It is unclear why Thiomicrospira-like organisms thrive in these chimneys considering that Lost City hydrothermal fluids are notably lacking in hydrogen sulfide and carbon dioxide. Methodology/Principal Findings Here we describe metagenomic sequences obtained from a Lost City carbonate chimney that are highly similar to the genome of Thiomicrospira crunogena XCL-2, an isolate from a basalt-hosted hydrothermal vent in the Pacific Ocean. Even though T. crunogena and Lost City Thiomicrospira inhabit different types of hydrothermal systems in different oceans, their genomic contents are highly similar. For example, sequences encoding the sulfur oxidation and carbon fixation pathways (including a carbon concentration mechanism) of T. crunogena are also present in the Lost City metagenome. Comparative genomic analyses also revealed substantial genomic changes that must have occurred since the divergence of the two lineages, including large genomic rearrangements, gene fusion events, a prophage insertion, and transposase activity. Conclusions/Significance Our results show significant genomic similarity between Thiomicrospira organisms inhabiting different kinds of hydrothermal systems in different oceans, suggesting that these organisms are widespread and highly adaptable. These data also indicate genomic processes potentially associated with the adaptation of these lineages into strikingly different habitats.

Is the Genetic Landscape of the Deep Subsurface Biosphere Affected by Viruses

Viruses are powerful manipulators of microbial diversity, biogeochemistry, and evolution in the marine environment. Viruses can directly influence the genetic capabilities and the fitness of their hosts through the use of fitness factors and through horizontal gene transfer. However, the impact of viruses on microbial ecology and evolution is often overlooked in studies of the deep subsurface biosphere. Subsurface habitats connected to hydrothermal vent systems are characterized by constant fluid flux, dynamic environmental variability, and high microbial diversity. In such conditions, high adaptability would be an evolutionary asset, and the potential for frequent host–virus interactions would be high, increasing the likelihood that cellular hosts could acquire novel functions. Here, we review evidence supporting this hypothesis, including data indicating that microbial communities in subsurface hydrothermal fluids are exposed to a high rate of viral infection, as well as viral metagenomic data suggesting that the vent viral assemblage is particularly enriched in genes that facilitate horizontal gene transfer and host adaptability. Therefore, viruses are likely to play a crucial role in facilitating adaptability to the extreme conditions of these regions of the deep subsurface biosphere. We also discuss how these results might apply to other regions of the deep subsurface, where the nature of virus–host interactions would be altered, but possibly no less important, compared to more energetic hydrothermal systems.

Multiple scales of diversification within natural populations of archaea in hydrothermal chimney biofilms

Corroborative data collected from 16S rRNA clone libraries, intergenic transcribed spacer (ITS) region clone libraries, and 16S rRNA hypervariable region tag pyrosequencing demonstrate microdiversity within single-species archaeal biofilms of the Lost City Hydrothermal Field. Both 16S rRNA clone libraries and pyrosequencing of the V6 hypervariable region show that Lost City Methanosarcinales (LCMS) biofilms are dominated by a single sequence, but the pyrosequencing data set also reveals the presence of an additional 1654 rare sequences. Clone libraries constructed with DNA spanning the V6 hypervariable region and ITS show that multiple ITS sequences are associated with the same dominant V6 sequence. Furthermore, ITS variability differed among three chimney samples, and the sample with the highest ITS diversity also contained the highest V6 diversity as measured by clone libraries as well as tag pyrosequencing. These results indicate that the extensive microdiversity detected in V6 tag sequences is an underestimate of genetic diversity within the archaeal biofilms.