Genome analysis of a new sulphur disproportionating species Thermosulfurimonas strain F29 and comparative genomics of sulfur-disproportionating bacteria from marine hydrothermal vents.

This paper reports on the genome analysis of strain F29 representing a new species of the genus Thermosulfurimonas. This strain, isolated from the Lucky Strike hydrothermal vent field on the Mid-Atlantic Ridge, is able to grow by disproportionation of S0 with CO2 as a carbon source. Strain F29 possesses a genome of 2,345,565 bp, with a G+C content of 58.09%, and at least one plasmid. The genome analysis revealed complete sets of genes for CO2 fixation via the Wood-Ljungdahl pathway, for sulphate-reduction and for hydrogen oxidation, suggesting the involvement of the strain into carbon, sulphur, and hydrogen cycles of deep-sea hydrothermal vents. Strain F29 genome encodes also several CRISPR sequences, suggesting that the strain may be subjected to viral attacks. Comparative genomics was carried out to decipher sulphur disproportionation pathways. Genomes of sulphur-disproportionating bacteria from marine hydrothermal vents were compared to the genomes of non-sulphur-disproportionating bacteria. This analysis revealed the ubiquitous presence in these genomes of a molybdopterin protein consisting of a large and a small subunit, and an associated chaperone. We hypothesize that these proteins may be involved in the process of elemental sulphur disproportionation.

Evolution of Thermophilic Microbial Communities from a Deep-Sea Hydrothermal Chimney under Electrolithoautotrophic Conditions with Nitrate.

Recent studies have shown the presence of an abiotic electrical current across the walls of deep-sea hydrothermal chimneys, allowing the growth of electroautotrophic microbial communities. To understand the role of the different phylogenetic groups and metabolisms involved, this study focused on electrotrophic enrichment with nitrate as electron acceptor. The biofilm density, community composition, production of organic compounds, and electrical consumption were monitored by FISH confocal microscopy, qPCR, metabarcoding, NMR, and potentiostat measurements. A statistical analysis by PCA showed the correlation between the different parameters (qPCR, organic compounds, and electron acceptors) in three distinct temporal phases. In our conditions, the Archaeoglobales have been shown to play a key role in the development of the community as the first colonizers on the cathode and the first producers of organic compounds, which are then used as an organic source by heterotrophs. Finally, through subcultures of the community, we showed the development of a greater biodiversity over time. This observed phenomenon could explain the biodiversity development in hydrothermal contexts, where energy sources are transient and unstable.

Hydrothermal plumes as hotspots for deep-ocean heterotrophic microbial biomass production.

Carbon budgets of hydrothermal plumes result from the balance between carbon sinks through plume chemoautotrophic processes and carbon release via microbial respiration. However, the lack of comprehensive analysis of the metabolic processes and biomass production rates hinders an accurate estimate of their contribution to the deep ocean carbon cycle. Here, we use a biogeochemical model to estimate the autotrophic and heterotrophic production rates of microbial communities in hydrothermal plumes and validate it with in situ data. We show how substrate limitation might prevent net chemolithoautotrophic production in hydrothermal plumes. Elevated prokaryotic heterotrophic production rates (up to 0.9 gCm-2y-1) compared to the surrounding seawater could lead to 0.05 GtCy-1 of C-biomass produced through chemoorganotrophy within hydrothermal plumes, similar to the Particulate Organic Carbon (POC) export fluxes reported in the deep ocean. We conclude that hydrothermal plumes must be accounted for as significant deep sources of POC in ocean carbon budgets.

Identification of enriched hyperthermophilic microbial communities from a deep-sea hydrothermal vent chimney under electrolithoautotrophic culture conditions.

Deep-sea hydrothermal vents are extreme and complex ecosystems based on a trophic chain. We are still unsure of the identities of the first colonizers of these environments and their metabolism, but they are thought to be (hyper)thermophilic autotrophs. Here we investigate whether the electric potential observed across hydrothermal chimneys could serve as an energy source for these first colonizers. Experiments were performed in a two-chamber microbial electrochemical system inoculated with deep-sea hydrothermal chimney samples, with a cathode as sole electron donor, CO2 as sole carbon source, and nitrate, sulfate, or oxygen as electron acceptors. After a few days of culturing, all three experiments showed growth of electrotrophic biofilms consuming the electrons (directly or indirectly) and producing organic compounds including acetate, glycerol, and pyruvate. Within the biofilms, the only known autotroph species retrieved were members of Archaeoglobales. Various heterotrophic phyla also grew through trophic interactions, with Thermococcales growing in all three experiments as well as other bacterial groups specific to each electron acceptor. This electrotrophic metabolism as energy source driving initial microbial colonization of conductive hydrothermal chimneys is discussed.

H2-dependent formate production by hyperthermophilic Thermococcales: an alternative to sulfur reduction for reducing-equivalents disposal.

Removal of reducing equivalents is an essential catabolic process for all microorganisms to maintain their internal redox balance. The electron disposal by chemoorganotrophic Thermococcales generates H2 by proton reduction or H2S in presence of S0. Although in the absence of S0 growth of these (hyper)thermopiles was previously described to be H2-limited, it remains unclear how Thermococcales could be present in H2-rich S0-depleted habitats. Here, we report that 12 of the 47 strains tested, distributed among all three orders of Thermococcales, could grow without S0 at 0.8 mM dissolved H2 and that tolerance to H2 was always associated with formate production. Two conserved gene clusters coding for a formate hydrogenlyase (FHL) and a putative formate dehydrogenase-NAD(P)H-oxidoreductase were only present in H2-dependent formate producers, and were both systematically associated with a formate dehydrogenase and a formate transporter. As the reaction involved in this alternative pathway for disposal of reducing equivalents was close to thermodynamic equilibrium, it was strongly controlled by the substrates-products concentration ratio even in the presence of S0. Moreover, experimental data and thermodynamic modelling also demonstrated that H2-dependent CO2 reduction to formate could occur within a large temperature range in contrasted hydrothermal systems, suggesting it could also provide an adaptive advantage.

Persephonella atlantica sp. nov.: How to adapt to physico-chemical gradients in high temperature hydrothermal habitats.

A novel thermophilic, microaerophilic and anaerobic, hydrogen- sulphur- and thiosulphate-oxidising bacterium, designated MO1340T, was isolated from a deep-sea hydrothermal chimney collected from the Lucky Strike hydrothermal vent field on the Mid-Atlantic Ridge. Cells were short, motile rods of 1.4-2.2µm length and 0.5-0.8µm width. Optimal growth was observed for a NaCl concentration of 2.5 % (w/v) at pH 6.5. As for other members of the genus Persephonella, strain MO1340T was strictly chemolithoautotrophic and could oxidise hydrogen, elemental sulphur or thiosulphate using oxygen as electron acceptor. Anaerobic nitrate reduction using hydrogen could also be performed. Each catabolic reaction had a different optimal growth temperature (65 to 75°C) and an optimal dissolved oxygen concentration (11.4 to 119.7 µM at 70°C for aerobic reactions) that varied according to the electron donors utilised. These experimental results are consistent with the distribution of these catabolic substrates along the temperature gradient observed in active hydrothermal systems. They strongly suggest that this adaptive strategy could confer a selective advantage for strain MO1340T in the dynamic part of the ecosystem where hot, reduced hydrothermal fluid mixes with cold, oxygenated seawater. Phylogenetic analysis indicated that strain MO1340T was a member of the genus Persephonella within the order Hydrogenothermales as it shared a 16S rRNA gene sequence similarity <95.5 % and ANI respectively 75.66 % with closest described Persephonella (P. hydrogeniphila 29WT). On the basis of the physiological and genomic properties of the new isolate, the name Persephonella atlantica sp. nov. is proposed. The type strain is MO1340T (=UBOCC-M-3359T =JCM 34026T).

The first head-tailed virus, MFTV1, infecting hyperthermophilic methanogenic deep-sea archaea.

Deep-sea hydrothermal vents are inhabited by complex communities of microbes and their viruses. Despite the importance of viruses in controlling the diversity, adaptation and evolution of their microbial hosts, to date, only eight bacterial and two archaeal viruses isolated from abyssal ecosystems have been described. Thus, our efforts focused on gaining new insights into viruses associated with deep-sea autotrophic archaea. Here, we provide the first evidence of an infection of hyperthermophilic methanogenic archaea by a head-tailed virus, Methanocaldococcus fervens tailed virus 1 (MFTV1). MFTV1 has an isometric head of 50 nm in diameter and a 150 nm-long non-contractile tail. Virions are released continuously without causing a sudden drop in host growth. MFTV1 infects Methanocaldococcus species and is the first hyperthermophilic head-tailed virus described thus far. The viral genome is a double-stranded linear DNA of 31 kb. Interestingly, our results suggest potential strategies adopted by the plasmid pMEFER01, carried by M. fervens, to spread horizontally in hyperthermophilic methanogens. The data presented here open a new window of understanding on how the abyssal mobilome interacts with hyperthermophilic marine archaea.

Production of Current by Syntrophy Between Exoelectrogenic and Fermentative Hyperthermophilic Microorganisms in Heterotrophic Biofilm from a Deep-Sea Hydrothermal Chimney.

To study the role of exoelectrogens within the trophic network of deep-sea hydrothermal vents, we performed successive subcultures of a hyperthermophilic community from a hydrothermal chimney sample on a mix of electron donors in a microbial fuel cell system. Electrode (the electron acceptor) was swapped every week to enable fresh development from spent media as inoculum. The MFC at 80 °C yielded maximum current production increasing from 159 to 247 mA m-2 over the subcultures. The experiments demonstrated direct production of electric current from acetate, pyruvate, and H2 and indirect production from yeast extract and peptone through the production of H2 and acetate from fermentation. The microorganisms found in on-electrode communities were mainly affiliated to exoelectrogenic Archaeoglobales and Thermococcales species, whereas in liquid media, the communities were mainly affiliated to fermentative Bacillales and Thermococcales species. The work shows interactions between fermentative microorganisms degrading complex organic matter into fermentation products that are then used by exoelectrogenic microorganisms oxidizing these reduced compounds while respiring on a conductive support. The results confirmed that with carbon cycling, the syntrophic relations between fermentative microorganisms and exoelectrogens could enable some microbes to survive as biofilm in extremely unstable conditions. Graphical Abstract Schematic representation of cross-feeding between fermentative and exoelectrogenic microbes on the surface of the conductive support. B, Bacillus/Geobacillus spp.; Tc, Thermococcales; Gg, Geoglobus spp.; Py, pyruvate; Ac, acetate.

The interplay at the replisome mitigates the impact of oxidative damage on the genetic integrity of hyperthermophilic Archaea.

8-oxodeoxyguanosine (8-oxodG), a major oxidised base modification, has been investigated to study its impact on DNA replication in hyperthermophilic Archaea. Here we show that 8-oxodG is formed in the genome of growing cells, with elevated levels following exposure to oxidative stress. Functional characterisation of cell-free extracts and the DNA polymerisation enzymes, PolB, PolD, and the p41/p46 complex, alone or in the presence of accessory factors (PCNA and RPA) indicates that translesion synthesis occurs under replicative conditions. One of the major polymerisation effects was stalling, but each of the individual proteins could insert and extend past 8-oxodG with differing efficiencies. The introduction of RPA and PCNA influenced PolB and PolD in similar ways, yet provided a cumulative enhancement to the polymerisation performance of p41/p46. Overall, 8-oxodG translesion synthesis was seen to be potentially mutagenic leading to errors that are reminiscent of dA:8-oxodG base pairing.

Specific enrichment of hyperthermophilic electroactive Archaea from deep-sea hydrothermal vent on electrically conductive support.

While more and more investigations are done to study hyperthermophilic exoelectrogenic communities from environments, none have been performed yet on deep-sea hydrothermal vent. Samples of black smoker chimney from Rainbow site on the Atlantic mid-oceanic ridge have been harvested for enriching exoelectrogens in microbial electrolysis cells under hyperthermophilic (80 °C) condition. Two enrichments were performed in a BioElectrochemical System specially designed: one from direct inoculation of crushed chimney and the other one from inoculation of a pre-cultivation on iron (III) oxide. In both experiments, a current production was observed from 2.4 A/m2 to 5.8 A/m2 with a set anode potential of -0.110 V vs Ag/AgCl. Taxonomic affiliation of the exoelectrogen communities obtained on the electrode exhibited a specific enrichment of Archaea belonging to Thermococcales and Archeoglobales orders, even when both inocula were dominated by Bacteria.

Relative Abundance and Diversity of Bacterial Methanotrophs at the Oxic-Anoxic Interface of the Congo Deep-Sea Fan.

Sitting at ∼5,000 m water depth on the Congo-Angola margin and ∼760 km offshore of the West African coast, the recent lobe complex of the Congo deep-sea fan receives large amounts of fluvial sediments (3-5% organic carbon). This organic-rich sedimentation area harbors habitats with chemosynthetic communities similar to those of cold seeps. In this study, we investigated relative abundance, diversity and distribution of aerobic methane-oxidizing bacteria (MOB) communities at the oxic-anoxic interface of sedimentary habitats by using fluorescence in situ hybridization and comparative sequence analysis of particulate mono-oxygenase (pmoA) genes. Our findings revealed that sedimentary habitats of the recent lobe complex hosted type I and type II MOB cells and comparisons of pmoA community compositions showed variations among the different organic-rich habitats. Furthermore, the pmoA lineages were taxonomically more diverse compared to methane seep environments and were related to those found at cold seeps. Surprisingly, MOB phylogenetic lineages typical of terrestrial environments were observed at such water depth. In contrast, MOB cells or pmoA sequences were not detected at the previous lobe complex that is disconnected from the Congo River inputs.

Complete Genome Sequence of the Hyperthermophilic Piezophilic Archaeon Pyrococcus kukulkanii NCB100 Isolated from the Rebecca's Roost Hydrothermal Vent in the Guaymas Basin.

Members of the order Thermococcales are common inhabitants of high-temperature hydrothermal vent systems (black smokers) that are represented in clone libraries mostly by isolates from the Thermococcus genus. We report the complete sequence of a novel species from the Pyrococcus genus, P. kukulkanii strain NCB100, which has been isolated from a flange fragment of the Rebecca's Roost hydrothermal vent system in the Guaymas Basin.

Endemicity of the cosmopolitan mesophilic chemolithoautotroph Sulfurimonas at deep-sea hydrothermal vents.

Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with ⩾98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

Draft Genome Sequences of Two Marinitoga camini Isolates Producing Bacterioviruses.

Here, we present the draft genome sequences of two thermophilic Marinitoga strain members of the Thermotogales order, Marinitoga camini DV1155 and Marinitoga camini DV1197. These strains were isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge.

Pyrococcus kukulkanii sp. nov., a hyperthermophilic, piezophilic archaeon isolated from a deep-sea hydrothermal vent.

A novel hyperthermophilic, piezophilic, anaerobic archaeon, designated NCB100T, was isolated from a hydrothermal vent flange fragment collected in the Guaymas basin at the hydrothermal vent site named 'Rebecca's Roost' at a depth of 1997 m. Enrichment and isolation were performed at 100 °C under atmospheric pressure. Cells of strain NCB100T were highly motile, irregular cocci with a diameter of ~1 µm. Growth was recorded at temperatures between 70 and 112 °C (optimum 105 °C) and hydrostatic pressures of 0.1-80 MPa (optimum 40-50 MPa). Growth was observed at pH 3.5-8.5 (optimum pH 7) and with 1.5-7 % NaCl (optimum at 2.5-3 %). Strain NCB100T was a strictly anaerobic chemo-organoheterotroph and grew on complex proteinaceous substrates such as yeast extract, peptone and tryptone, as well as on glycogen and starch. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The fermentation products from complex proteinaceous substrates were CO2 and H2. The G+C content of the genomic DNA was 41.3 %. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NCB100T belongs to the genus Pyrococcus, showing 99 % similarity with the other described species of the genus Pyrococcus. On the basis of physiological characteristics, DNA G+C content, similarity level between ribosomal proteins and an average nucleotide identity value of 79 %, strain NCB100T represents a novel species for which the name Pyrococcus kukulkanii sp. nov. is proposed. The type strain is NCB100T (=DSM 101590T=Souchothèque de Bretagne BG1337T).

Crassaminicella profunda gen. nov., sp. nov., an anaerobic marine bacterium isolated from deep-sea sediments.

A novel, anaerobic, chemo-organotrophic bacterium, designated strain Ra1766H(T), was isolated from sediments of the Guaymas basin (Gulf of California, Mexico) taken from a depth of 2002  m. Cells were thin, motile, Gram-stain-positive, flexible rods forming terminal endospores. Strain Ra1766H(T) grew at temperatures of 25-45 °C (optimum 30 °C), pH 6.7-8.1 (optimum 7.5) and in a salinity of 5-60 g l(-1) NaCl (optimum 30 g l(-1)). It was an obligate heterotrophic bacterium fermenting carbohydrates (glucose and mannose) and organic acids (pyruvate and succinate). Casamino acids and amino acids (glutamate, aspartate and glycine) were also fermented. The main end products from glucose fermentation were acetate, butyrate, ethanol, H2 and CO2. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, nitrate, nitrite and Fe(III) were not used as terminal electron acceptors. The predominant cellular fatty acids were C14  : 0, C16 : 1ω7, C16 : 1ω7 DMA and C16 : 0. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipids. The G+C content of the genomic DNA was 33.7 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Ra1766H(T) was affiliated to cluster XI of the order Clostridiales, phylum Firmicutes. The closest phylogenetic relative of Ra1766H(T) was Geosporobacter subterraneus (94.2% 16S rRNA gene sequence similarity). On the basis of phylogenetic inference and phenotypic properties, strain Ra1766H(T) ( = DSM 27501(T) = JCM 19377(T)) is proposed to be the type strain of a novel species of a novel genus, named Crassaminicella profunda.

Biogeochemical insights into microbe-mineral-fluid interactions in hydrothermal chimneys using enrichment culture.

Active hydrothermal chimneys host diverse microbial communities exhibiting various metabolisms including those involved in various biogeochemical cycles. To investigate microbe-mineral-fluid interactions in hydrothermal chimney and the driver of microbial diversity, a cultural approach using a gas-lift bioreactor was chosen. An enrichment culture was performed using crushed active chimney sample as inoculum and diluted hydrothermal fluid from the same vent as culture medium. Daily sampling provided time-series access to active microbial diversity and medium composition. Active archaeal and bacterial communities consisted mainly of sulfur, sulfate and iron reducers and hydrogen oxidizers with the detection of Thermococcus, Archaeoglobus, Geoglobus, Sulfurimonas and Thermotoga sequences. The simultaneous presence of active Geoglobus sp. and Archaeoglobus sp. argues against competition for available carbon sources and electron donors between sulfate and iron reducers at high temperature. This approach allowed the cultivation of microbial populations that were under-represented in the initial environmental sample. The microbial communities are heterogeneously distributed within the gas-lift bioreactor; it is unlikely that bulk mineralogy or fluid chemistry is the drivers of microbial community structure. Instead, we propose that micro-environmental niche characteristics, created by the interaction between the mineral grains and the fluid chemistry, are the main drivers of microbial diversity in natural systems.

Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

Structural Iron (II) of Basaltic Glass as an Energy Source for Zetaproteobacteria in an Abyssal Plain Environment, Off the Mid Atlantic Ridge.

To explore the capability of basaltic glass to support the growth of chemosynthetic microorganisms, complementary in situ and in vitro colonization experiments were performed. Microbial colonizers containing synthetic tholeitic basaltic glasses, either enriched in reduced or oxidized iron, were deployed off-axis from the Mid Atlantic Ridge on surface sediments of the abyssal plain (35°N; 29°W). In situ microbial colonization was assessed by sequencing of the 16S rRNA gene and basaltic glass alteration was characterized using Scanning Electron Microscopy, micro-X-ray Absorption Near Edge Structure at the Fe-K-edge and Raman microspectroscopy. The colonized surface of the reduced basaltic glass was covered by a rind of alteration made of iron-oxides trapped in a palagonite-like structure with thicknesses up to 150 µm. The relative abundance of the associated microbial community was dominated (39% of all reads) by a single operational taxonomic unit (OTU) that shared 92% identity with the iron-oxidizer Mariprofundus ferrooxydans PV-1. Conversely, the oxidized basaltic glass showed the absence of iron-oxides enriched surface deposits and correspondingly there was a lack of known iron-oxidizing bacteria in the inventoried diversity. In vitro, a similar reduced basaltic glass was incubated in artificial seawater with a pure culture of the iron-oxidizing M. ferrooxydans DIS-1 for 2 weeks, without any additional nutrients or minerals. Confocal Laser Scanning Microscopy revealed that the glass surface was covered by twisted stalks characteristic of this iron-oxidizing Zetaproteobacteria. This result supported findings of the in situ experiments indicating that the Fe(II) present in the basalt was the energy source for the growth of representatives of Zetaproteobacteria in both the abyssal plain and the in vitro experiment. In accordance, the surface alteration rind observed on the reduced basaltic glass incubated in situ could at least partly result from their activity.

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.

TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.