The Ribofilm as a Concept for Life's Origins.

Recent phylogenetic data indicating that the first archaea were methane-producing galvanizes cross-disciplinary evidence supporting the hypothesis that life arose via thermodynamically directed events at hydrothermal vents. The new developments lead us to propose the concept of a ribofilm in which RNA's origin-of-life role is more akin to a slowly changing platform than a spontaneous self-replicator.

The origin of membrane bioenergetics.

Harnessing energy as ion gradients across membranes is as universal as the genetic code. We leverage new insights into anaerobe metabolism to propose geochemical origins that account for the ubiquity of chemiosmotic coupling, and Na(+)/H(+) transporters in particular. Natural proton gradients acting across thin FeS walls within alkaline hydrothermal vents could drive carbon assimilation, leading to the emergence of protocells within vent pores. Protocell membranes that were initially leaky would eventually become less permeable, forcing cells dependent on natural H(+) gradients to pump Na(+) ions. Our hypothesis accounts for the Na(+)/H(+) promiscuity of bioenergetic proteins, as well as the deep divergence between bacteria and archaea.

Life in the Dark: Phylogenetic and Physiological Diversity of Chemosynthetic Symbioses.

Possibly the last discovery of a previously unknown major ecosystem on Earth was made just over half a century ago, when researchers found teaming communities of animals flourishing two and a half kilometers below the ocean surface at hydrothermal vents. We now know that these highly productive ecosystems are based on nutritional symbioses between chemosynthetic bacteria and eukaryotes and that these chemosymbioses are ubiquitous in both deep-sea and shallow-water environments. The symbionts are primary producers that gain energy from the oxidation of reduced compounds, such as sulfide and methane, to fix carbon dioxide or methane into biomass to feed their hosts. This review outlines how the symbiotic partners have adapted to living together. We first focus on the phylogenetic and metabolic diversity of these symbioses and then highlight selected research directions that could advance our understanding of the processes that shaped the evolutionary and ecological success of these associations.

Discovery of active off-axis hydrothermal vents at 9° 54'N East Pacific Rise.

Comprehensive knowledge of the distribution of active hydrothermal vent fields along midocean ridges is essential to understanding global chemical and heat fluxes and endemic faunal distributions. However, current knowledge is biased by a historical preference for on-axis surveys. A scarcity of high-resolution bathymetric surveys in off-axis regions limits vent identification, which implies that the number of vents may be underestimated. Here, we present the discovery of an active, high-temperature, off-axis hydrothermal field on a fast-spreading ridge. The vent field is located 750 m east of the East Pacific Rise axis and ∼7 km north of on-axis vents at 9° 50'N, which are situated in a 50- to 100-m-wide trough. This site is currently the largest vent field known on the East Pacific Rise between 9 and 10° N. Its proximity to a normal fault suggests that hydrothermal fluid pathways are tectonically controlled. Geochemical evidence reveals deep fluid circulation to depths only 160 m above the axial magma lens. Relative to on-axis vents at 9° 50'N, these off-axis fluids attain higher temperatures and pressures. This tectonically controlled vent field may therefore exhibit greater stability in fluid composition, in contrast to more dynamic, dike-controlled, on-axis vents. The location of this site indicates that high-temperature convective circulation cells extend to greater distances off axis than previously realized. Thorough high-resolution mapping is necessary to understand the distribution, frequency, and physical controls on active off-axis vent fields so that their contribution to global heat and chemical fluxes and role in metacommunity dynamics can be determined.

Horizontal transmission enables flexible associations with locally adapted symbiont strains in deep-sea hydrothermal vent symbioses.

SignificanceIn marine ecosystems, transmission of microbial symbionts between host generations occurs predominantly through the environment. Yet, it remains largely unknown how host genetics, symbiont competition, environmental conditions, and geography shape the composition of symbionts acquired by individual hosts. To address this question, we applied population genomic approaches to four species of deep-sea hydrothermal vent snails that live in association with chemosynthetic bacteria. Our analyses show that environment is more important to strain-level symbiont composition than host genetics and that symbiont strains show genetic variation indicative of adaptation to the distinct geochemical conditions at each vent site. This corroborates a long-standing hypothesis that hydrothermal vent invertebrates affiliate with locally adapted symbiont strains to cope with the variable conditions characterizing their habitats.

Linking Zetaproteobacterial diversity and substratum type in iron-rich microbial mats from the Lucky Strike hydrothermal field (EMSO-Azores observatory).

Zetaproteobacteria have been reported in different marine and terrestrial environments all over the globe. They play an essential role in marine iron-rich microbial mats, as one of their autotrophic primary producers, oxidizing Fe(II) and producing Fe-oxyhydroxides with different morphologies. Here, we study and compare the Zetaproteobacterial communities of iron-rich microbial mats from six different sites of the Lucky Strike Hydrothermal Field through the use of the Zetaproteobacterial operational taxonomic unit (ZetaOTU) classification. We report for the first time the Zetaproteobacterial core microbiome of these iron-rich microbial mats, which is composed of four ZetaOTUs that are cosmopolitan and essential for the development of the mats. The study of the presence and abundance of different ZetaOTUs among sites reveals two clusters, which are related to the lithology and permeability of the substratum on which they develop. The Zetaproteobacterial communities of cluster 1 are characteristic of poorly permeable substrata, with little evidence of diffuse venting, while those of cluster 2 develop on hydrothermal slabs or deposits that allow the percolation and outflow of diffuse hydrothermal fluids. In addition, two NewZetaOTUs 1 and 2 were identified, which could be characteristic of anthropic iron and unsedimented basalt, respectively. We also report significant correlations between the abundance of certain ZetaOTUs and that of iron oxide morphologies, indicating that their formation could be taxonomically and/or environmentally driven. We identified a new morphology of Fe(III)-oxyhydroxides that we named "corals." Overall, our work contributes to the knowledge of the biogeography of this bacterial class by providing additional data from the Atlantic Ocean, a lesser-studied ocean in terms of Zetaproteobacterial diversity.IMPORTANCEUp until now, Zetaproteobacterial diversity studies have revealed possible links between Zetaproteobacteria taxa, habitats, and niches. Here, we report for the first time the Zetaproteobacterial core microbiome of iron-rich mats from the Lucky Strike Hydrothermal Field (LSHF), as well as two new Zetaproteobacterial operational taxonomic units (NewZetaOTUs) that could be substratum specific. We highlight that the substratum on which iron-rich microbial mats develop, especially because of its permeability to diffuse hydrothermal venting, has an influence on their Zetaproteobacterial communities. Moreover, our work adds to the knowledge of the biogeography of this bacterial class by providing additional data from the hydrothermal vent sites along the Mid-Atlantic Ridge. In addition to the already described iron oxide morphologies, we identify in our iron-rich mats a new morphology that we named corals. Finally, we argue for significant correlations between the relative abundance of certain ZetaOTUs and that of iron oxide morphologies, contributing to the understanding of the drivers of iron oxide production in iron-oxidizing bacteria.

Genetic divergence and migration patterns of a galatheoid squat lobster highlight the need for deep-sea conservation.

Information on genetic divergence and migration patterns of vent- and seep-endemic macrobenthos can help delimit biogeographical provinces and provide scientific guidelines for deep-sea conservation under the growing threats of anthropogenic disturbances. Nevertheless, related studies are still scarce, impeding the informed conservation of these hotspots of deep-sea biodiversity. To bridge this knowledge gap, we conducted a population connectivity study on the galatheoid squat lobster Shinkaia crosnieri - a deep-sea foundation species widely distributed in vent and seep ecosystems in the Northwest Pacific. With the application of an interdisciplinary methodology involving population genomics and oceanographic approaches, we unveiled two semi-isolated lineages of S. crosnieri with limited and asymmetrical gene flow potentially shaped by the geographic settings, habitat types, and ocean currents - one comprising vent populations in the Okinawa Trough, with those inhabiting the southern trough area likely serving as the source; the other being the Jiaolong (JR) seep population in the South China Sea. The latter might have recently experienced a pronounced demographic contraction and exhibited genetic introgression from the Okinawa Trough lineage, potentially mediated by the intrusion of the North Pacific Intermediate Water. We then compared the biogeographic patterns between S. crosnieri and two other representative and co-occurring vent- and seep-endemic species using published data. Based on their biogeographical subdivisions and source-sink dynamics, we highlighted the southern Okinawa Trough vents and the JR seep warrant imperative conservation efforts to sustain the deep-sea biodiversity in the Northwest Pacific.

Highly structured populations of copepods at risk to deep-sea mining: Integration of genomic data with demogenetic and biophysical modelling.

Copepoda is the most abundant taxon in deep-sea hydrothermal vents, where hard substrate is available. Despite the increasing interest in seafloor massive sulphides exploitation, there have been no population genomic studies conducted on vent meiofauna, which are known to contribute over 50% to metazoan biodiversity at vents. To bridge this knowledge gap, restriction-site-associated DNA sequencing, specifically 2b-RADseq, was used to retrieve thousands of genome-wide single-nucleotide polymorphisms (SNPs) from abundant populations of the vent-obligate copepod Stygiopontius lauensis from the Lau Basin. SNPs were used to investigate population structure, demographic histories and genotype-environment associations at a basin scale. Genetic analyses also helped to evaluate the suitability of tailored larval dispersal models and the parameterization of life-history traits that better fit the population patterns observed in the genomic dataset for the target organism. Highly structured populations were observed on both spatial and temporal scales, with divergence of populations between the north, mid, and south of the basin estimated to have occurred after the creation of the major transform fault dividing the Australian and the Niuafo'ou tectonic plate (350 kya), with relatively recent secondary contact events (<20 kya). Larval dispersal models were able to predict the high levels of structure and the highly asymmetric northward low-level gene flow observed in the genomic data. These results differ from most studies conducted on megafauna in the region, elucidating the need to incorporate smaller size when considering site prospecting for deep-sea exploitation of seafloor massive sulphides, and the creation of area-based management tools to protect areas at risk of local extinction, should mining occur.

Genetic sex determination in three closely related hydrothermal vent gastropods, including one species with intersex individuals.

Molluscs have undergone many transitions between separate sexes and hermaphroditism, which is of interest in studying the evolution of sex determination and differentiation. Here, we combined multi-locus genotypes obtained from restriction site-associated DNA (RAD) sequencing with anatomical observations of the gonads of three deep-sea hydrothermal vent gastropods of the genus Alviniconcha living in the southwest Pacific. We found that all three species (Alviniconcha boucheti, Alviniconcha strummeri, and Alviniconcha kojimai) share the same male-heterogametic XY sex-determination system but that the gonads of XX A. kojimai individuals are invaded by a variable proportion of male reproductive tissue. The identification of Y-specific RAD loci (found only in A. boucheti) and the phylogenetic analysis of three sex-linked loci shared by all species suggested that X-Y recombination has evolved differently within each species. This situation of three species showing variation in gonadal development around a common sex-determination system provides new insights into the reproductive mode of poorly known deep-sea species and opens up an opportunity to study the evolution of recombination suppression on sex chromosomes and its association with mixed or transitory sexual systems.

Bioenergetic characterization of hyperthermophilic archaean Methanocaldococcus sp. FS406-22.

Hyperthermophilic archaean Methanocaldococcus sp. FS406-22 (hereafter FS406) is a hydrogenotrophic methanogen isolated from a deep-sea hydrothermal vent. To better understand the energetic requirements of hydrogen oxidation under extreme conditions, the thermodynamic characterization of FS406 incubations is necessary and notably underexplored. In this work, we quantified the bioenergetics of FS406 incubations at a range of temperatures (65, 76, and 85 â„ƒ) and hydrogen concentrations (1.1, 1.4, and 2.1 mm). The biomass yields (C-mol of biomass per mol of H2 consumed) ranged from 0.02 to 0.19. Growth rates ranged from 0.4 to 1.5 h-1. Gibbs energies of incubation based on macrochemical equations of cell growth ranged from - 198 kJ/C-mol to - 1840 kJ/C-mol. Enthalpies of incubation determined from calorimetric measurements ranged from - 4150 kJ/C-mol to - 36333 kJ/C-mol. FS406 growth rates were most comparable to hyperthermophilic methanogen Methanocaldococcus jannaschii. Maintenance energy calculations from the thermodynamic parameters of FS406 and previously determined heterotrophic methanogen data revealed that temperature is a primary determinant rather than an electron donor. This work provides new insights into the thermodynamic underpinnings of a hyperthermophilic hydrothermal vent methanogen and helps to better constrain the energetic requirements of life in extreme environments.

Microplastic Contamination of a Benthic Ecosystem in a Hydrothermal Vent.

Plastic contamination is a global pervasive issue, extending from coastal areas and open oceans to polar regions and even the deep sea. Microplastic (MP) contamination in hydrothermal vents, which are known for their high biodiversity even under extreme conditions, has remained largely unexplored. Here, we present, for the first time, MP pollution in a deep-sea hydrothermal vent at one of the biodiversity hotspots─the Central Indian Ridge. Not only the environment (seawater: 2.08 ± 1.04 MPs/L, surface sediments: 0.57 ± 0.19 MP/g) but also all six major benthic species investigated were polluted by MPs. MPs mainly consisted of polypropylene, polyethylene terephthalate, and polystyrene fragments ≤100 µm and were characterized as being either transparent or white in color. Remarkably, bioaccumulation and even biomagnification of microplastics were observed in the top predators of the ecosystem, such as squat lobsters (14.25 ± 4.65 MPs/individual) and vent crabs (14.00 ± 2.16 MPs/individual), since they contained more MPs than animals at lower trophic levels (e.g., mussels and snails, 1.75-6.00 average MPs/individuals). These findings reveal MP contamination of an ecosystem in a hydrothermal vent, thereby suggesting that their accumulation and magnification can occur in top-level animals, even within remote and extreme environments.

Mesobacterium hydrothermale sp. nov., isolated from shallow-sea hydrothermal systems off Kueishantao Island.

A Gram-negative, rod-shaped, non-motile, aerobic bacterium, designated as strain TK19101T, was isolated from the intermediate seawater of yellow vent in the shallow-sea hydrothermal system located near Kueishantao Island. The strain was found to grow at 10-40 °C (optimum, 35 °C), at pH 6.0-8.0 (optimum, 7.0), and in 0-5% (w/v) NaCl (optimum, 1%). Strain TK19101T was catalase-positive and oxidase-positive. The predominant fatty acids (> 10%) in strain TK19101T cells were C16:0, summed feature 8 (C18:1 ω6c and/or C18:1 ω7c), and C18:0. The predominant isoprenoid quinone of strain TK19101T was ubiquinone-10. The polar lipids of strain TK19101T comprised phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phospholipid, and unknown polar lipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TK19101T belonged to the genus Mesobacterium. Strain TK19101T exhibited highest 16S rRNA gene sequence similarity value to Mesobacterium pallidum MCCC M24557T (97.48%). The estimated average nucleotide identity and digital DNA-DNA hybridization values between strain TK19101T and the closest related species Mesobacterium pallidum MCCC M24557T were 74.88% and 20.30%, respectively. The DNA G + C content was 63.49 mol%. On the basis of the analysis of 16S rRNA gene sequences, genotypic and phylogenetic data, strain TK19101T has a unique phylogenetic status and represents a novel species of genus Mesobacterium, for which the name Mesobacterium hydrothermale sp. nov. is proposed. The type strain is TK19101T (= MCCC 1K08936T = KCTC 8354T).

Helicovermis profundi gen. nov., sp. nov., a novel mesophilic, asporogenous bacterium within the Clostridia isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic bacterial strain, designated S502T, was isolated from a deep-sea hydrothermal vent at Suiyo Seamount, Japan. Cells were Gram-positive, asporogenous, motile, and curved rods, measuring 1.6-5.6 µm in length. The strain was an obligate anaerobe that grew fermentatively on complex substrates such as yeast extract and Bacto peptone. Elemental sulfur stimulated the growth of the strain, and was reduced to hydrogen sulfide. The strain grew within a temperature range of 10-23 °C (optimum at 20 °C), pH range of 4.8-8.3 (optimum at 7.4), and a NaCl concentration range of 1.0-4.0% (w/v) (optimum at 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the class Clostridia, with Fusibacter paucivorans strain SEBR 4211T (91.1% sequence identity) being its closest relative. The total size of the genome of the strain was 3.12 Mbp, and a G + C content was 28.2 mol%. The highest values for average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) value of strain S502T with relatives were 67.5% (with Marinisporobacter balticus strain 59.4MT), 51.5% (with M. balticus strain 59.4MT), and 40.9% (with Alkaliphilus serpentinus strain LacTT), respectively. Based on a combination of phylogenetic, genomic, and phenotypic characteristics, we propose strain S502T to represent a novel genus and species, Helicovermis profundi gen. nov., sp. nov., with the type strain S502T (= DSM 112048T = JCM 39167T).

Gene-centered metagenome analysis of Vulcano Island soil (Aeolian archipelago, Italy) reveals diverse microbial key players in methane, hydrogen and sulfur cycles.

The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H2, indicating that besides H2S, H2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H2S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems.

Protistan grazing impacts microbial communities and carbon cycling at deep-sea hydrothermal vents.

Microbial eukaryotes (or protists) in marine ecosystems are a link between primary producers and all higher trophic levels, and the rate at which heterotrophic protistan grazers consume microbial prey is a key mechanism for carbon transport and recycling in microbial food webs. At deep-sea hydrothermal vents, chemosynthetic bacteria and archaea form the base of a food web that functions in the absence of sunlight, but the role of protistan grazers in these highly productive ecosystems is largely unexplored. Here, we pair grazing experiments with a molecular survey to quantify protistan grazing and to characterize the composition of vent-associated protists in low-temperature diffuse venting fluids from Gorda Ridge in the northeast Pacific Ocean. Results reveal protists exert higher predation pressure at vents compared to the surrounding deep seawater environment and may account for consuming 28 to 62% of the daily stock of prokaryotic biomass within discharging hydrothermal vent fluids. The vent-associated protistan community was more species rich relative to the background deep sea, and patterns in the distribution and co-occurrence of vent microbes provide additional insights into potential predator-prey interactions. Ciliates, followed by dinoflagellates, Syndiniales, rhizaria, and stramenopiles, dominated the vent protistan community and included bacterivorous species, species known to host symbionts, and parasites. Our findings provide an estimate of protistan grazing pressure within hydrothermal vent food webs, highlighting the important role that diverse protistan communities play in deep-sea carbon cycling.

Parasite diversity at isolated, disturbed hydrothermal vents.

Habitat isolation and disturbance are important regulators of biodiversity, yet it remains unclear how these environmental features drive differences in parasite diversity between ecosystems. We test whether the biological communities in an isolated, frequently disturbed marine ecosystem (deep-sea hydrothermal vents) have reduced parasite richness and relatively fewer parasite species with indirect life cycles (ILCs) compared to ecosystems that are less isolated and less disturbed. We surveyed the parasite fauna of the biological community at the 9°50'N hydrothermal vent field on the East Pacific Rise and compared it to similar datasets from a well-connected and moderately disturbed ecosystem (kelp forest) and an isolated and undisturbed ecosystem (atoll sandflat). Parasite richness within host species did not differ significantly between ecosystems, yet total parasite richness in the vent community was much lower due to the low number of predatory fish species. Contrary to expectation, the proportion of ILC parasite species was not lower at vents due to a high richness of trematodes, while other ILC parasite taxa were scarce (nematodes) or absent (cestodes). These results demonstrate the success of diverse parasite taxa in an extreme environment and reinforce the importance of host diversity and food web complexity in governing parasite diversity.

Cellular mechanisms underlying extraordinary sulfide tolerance in a crustacean holobiont from hydrothermal vents.

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulfide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulfide-rich vent systems. Despite the toxicity of hydrogen sulfide, X. testudinatus occupies an ecological niche in a sulfide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulfide detoxification. Here sulfide is oxidized to thiosulfate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulfate and hypotaurine is mediated by the sodium-independent sulfate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulfide-oxidizing endo-symbiotic bacteria, creating an effective sulfide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet.

A community-supported metaproteomic pipeline for improving peptide identifications in hydrothermal vent microbiota.

Microorganisms in deep-sea hydrothermal vents provide valuable insights into life under extreme conditions. Mass spectrometry-based proteomics has been widely used to identify protein expression and function. However, the metaproteomic studies in deep-sea microbiota have been constrained largely by the low identification rates of protein or peptide. To improve the efficiency of metaproteomics for hydrothermal vent microbiota, we firstly constructed a microbial gene database (HVentDB) based on 117 public metagenomic samples from hydrothermal vents and proposed a metaproteomic analysis strategy, which takes the advantages of not only the sample-matched metagenome, but also the metagenomic information released publicly in the community of hydrothermal vents. A two-stage false discovery rate method was followed up to control the risk of false positive. By applying our community-supported strategy to a hydrothermal vent sediment sample, about twice as many peptides were identified when compared with the ways against the sample-matched metagenome or the public reference database. In addition, more enriched and explainable taxonomic and functional profiles were detected by the HVentDB-based approach exclusively, as well as many important proteins involved in methane, amino acid, sugar, glycan metabolism and DNA repair, etc. The new metaproteomic analysis strategy will enhance our understanding of microbiota, including their lifestyles and metabolic capabilities in extreme environments. The database HVentDB is freely accessible from http://lilab.life.sjtu.edu.cn:8080/HventDB/main.html.

Thermal Selection of Microbial Communities and Preservation of Microbial Function in Guaymas Basin Hydrothermal Sediments.

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, hydrothermal activity, and organic matter accumulation on the seafloor due to high sedimentation rates. In the hydrothermal sediments of Guaymas Basin, microbial community compositions and coexistence patterns change across steep gradients of temperature, potential carbon sources, and electron acceptors. Nonmetric multidimensional scaling and guanine-cytosine percentage analyses reveal that the bacterial and archaeal communities adjust compositionally to their local temperature regime. Functional inference using PICRUSt shows that microbial communities consistently maintain their predicted biogeochemical functions in different sediments. Phylogenetic profiling shows that microbial communities retain distinct sulfate-reducing, methane-oxidizing, or heterotrophic lineages within specific temperature windows. The preservation of similar biogeochemical functions across microbial lineages with different temperature adaptations stabilizes the hydrothermal microbial community in a highly dynamic environment. IMPORTANCE Hydrothermal vent sites have been widely studied to investigate novel bacteria and archaea that are adapted to these extreme environments. However, community-level analyses of hydrothermal microbial ecosystems look beyond the presence and activity of particular types of microbes and examine to what extent the entire community of bacteria and archaea is adapted to hydrothermal conditions; these include elevated temperatures, hydrothermally generated carbon sources, and inorganic electron donors and acceptors that are characteristic for hydrothermal environments. In our case study of bacterial and archaeal communities in hydrothermal sediments of Guaymas Basin, we found that sequence-inferred microbial function was maintained in differently structured bacterial and archaeal communities across different samples and thermal regimes. The resulting preservation of biogeochemical functions across thermal gradients is an important factor in explaining the consistency of the microbial core community in the dynamic sedimentary environment of Guaymas Basin.

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/ß-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal ß-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.