Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.

Processing of matched and mismatched rNMPs in DNA by archaeal ribonucleotide excision repair.

Ribonucleoside monophosphates (rNMPs) are the main non-canonical nucleotides in genomic DNA, and their incorporation can occur as mismatches or matches in vivo. To counteract the mutagenic potential of rNMPs in DNA, all organisms evolved ribonucleotide excision repair (RER), a mechanism initiated by type 2 RNase H. Here, we describe the in vitro reconstitution of matched and mismatched rNMP repair using archaeal RER enzymes. Our data suggest two types of RER pathways, including the classical flap RER and a backup RER with the order of reactions changed for Fen1 and Pols. The genomic rNMP level in RER-deficient or PolB-deficient archaeal cells along with in vitro reconstitution of RER suggests an in vivo role of PolD in RER. Our results provide insights into how matched and mismatched rNMPs may be processed by RER.

The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator.

Global transcriptional regulators are crucial for supporting rapid adaptive responses in changing environments. In Thermococcales, the TrmB sugar-sensing regulator family is well represented but knowledge of the functional role/s of each of its members is limited. In this study, we examined the link between TrmBL4 and the degree of protein secretion in different sugar environments in the hyperthermophilic Archaeon Thermococcus barophilus. Although the absence of TrmBL4 did not induce any growth defects, proteomics analysis revealed different secretomes depending on the sugar and/or genetic contexts. Notably, 33 secreted proteins present in the supernatant were differentially detected. Some of these proteins are involved in sugar assimilation and transport, such as the protein encoded by TERMP_01455 (cyclomaltodextrin glucanotransferase), whereas others have intracellular functions, such as the protein encoded by TERMP_01556 (pyruvate: ferredoxin oxidoreductase Δsubunit). Then, using reverse transcription quantitative polymerase chain reaction experiments, we observed effective transcription regulation by TrmBL4 of the genes encoding at least two ABC-type transporters according to sugar availability.

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.

The Piezo-Hyperthermophilic Archaeon Thermococcus piezophilus Regulates Its Energy Efficiency System to Cope With Large Hydrostatic Pressure Variations.

Deep-sea ecosystems share a common physical parameter, namely high hydrostatic pressure (HHP). Some of the microorganisms isolated at great depths have a high physiological plasticity to face pressure variations. The adaptive strategies by which deep-sea microorganisms cope with HHP variations remain to be elucidated, especially considering the extent of their biotopes on Earth. Herein, we investigated the gene expression patterns of Thermococcus piezophilus, a piezohyperthermophilic archaeon isolated from the deepest hydrothermal vent known to date, under sub-optimal, optimal and supra-optimal pressures (0.1, 50, and 90 MPa, respectively). At stressful pressures [sub-optimal (0.1 MPa) and supra-optimal (90 MPa) conditions], no classical stress response was observed. Instead, we observed an unexpected transcriptional modulation of more than a hundred gene clusters, under the putative control of the master transcriptional regulator SurR, some of which are described as being involved in energy metabolism. This suggests a fine-tuning effect of HHP on the SurR regulon. Pressure could act on gene regulation, in addition to modulating their expression.

Physiological and Genomic Characterization of a Hyperthermophilic Archaeon Archaeoglobus neptunius sp. nov. Isolated From a Deep-Sea Hydrothermal Vent Warrants the Reclassification of the Genus Archaeoglobus.

Hyperthermophilic archaea of the genus Archaeoglobus are the subject of many fundamental and biotechnological researches. Despite their significance, the class Archaeoglobi is currently represented by only eight species obtained as axenic cultures and taxonomically characterized. Here, we report the isolation and characterization of a new species of Archaeoglobus from a deep-sea hydrothermal vent (Mid-Atlantic Ridge, TAG) for which the name Archaeoglobus neptunius sp. nov. is proposed. The type strain is SE56T (=DSM 110954T = VKM B-3474T). The cells of the novel isolate are motile irregular cocci growing at 50-85°C, pH 5.5-7.5, and NaCl concentrations of 1.5-4.5% (w/v). Strain SE56T grows lithoautotrophically with H2 as an electron donor, sulfite or thiosulfate as an electron acceptor, and CO2/HCO3 - as a carbon source. It is also capable of chemoorganotrophic growth by reduction of sulfate, sulfite, or thiosulfate. The genome of the new isolate consists of a 2,115,826 bp chromosome with an overall G + C content of 46.0 mol%. The whole-genome annotation confirms the key metabolic features of the novel isolate demonstrated experimentally. Genome contains a complete set of genes involved in CO2 fixation via reductive acetyl-CoA pathway, gluconeogenesis, hydrogen and fatty acids oxidation, sulfate reduction, and flagellar motility. The phylogenomic reconstruction based on 122 conserved single-copy archaeal proteins supported by average nucleotide identity (ANI), average amino acid identity (AAI), and alignment fraction (AF) values, indicates a polyphyletic origin of the species currently included into the genus Archaeoglobus, warranting its reclassification.

Thermococcus henrietii sp. nov., a novel extreme thermophilic and piezophilic sulfur-reducing archaeon isolated from a deep-sea hydrothermal chimney.

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12cT, was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12cT grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12cT falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1T. Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12cT is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12cT (=UBOCC M-2417T=DSM 111004T).

Genetic Potential of Dissulfurimicrobium hydrothermale, an Obligate Sulfur-Disproportionating Thermophilic Microorganism.

The biochemical pathways of anaerobic sulfur disproportionation are only partially deciphered, and the mechanisms involved in the first step of S0-disproportionation remain unknown. Here, we present the results of sequencing and analysis of the complete genome of Dissulfurimicrobium hydrothermale strain Sh68T, one of two strains isolated to date known to grow exclusively by anaerobic disproportionation of inorganic sulfur compounds. Dissulfurimicrobium hydrothermale Sh68T is a motile, thermophilic, anaerobic, chemolithoautotrophic microorganism isolated from a hydrothermal pond at Uzon caldera, Kamchatka, Russia. It is able to produce energy and grow by disproportionation of elemental sulfur, sulfite and thiosulfate. Its genome consists of a circular chromosome of 2,025,450 base pairs, has a G + C content of 49.66% and a completion of 97.6%. Genomic data suggest that CO2 assimilation is carried out by the Wood-Ljungdhal pathway and that central anabolism involves the gluconeogenesis pathway. The genome of strain Sh68T encodes the complete gene set of the dissimilatory sulfate reduction pathway, some of which are likely to be involved in sulfur disproportionation. A short sequence protein of unknown function present in the genome of strain Sh68T is conserved in the genomes of a large panel of other S0-disproportionating bacteria and was absent from the genomes of microorganisms incapable of elemental sulfur disproportionation. We propose that this protein may be involved in the first step of elemental sulfur disproportionation, as S0 is poorly soluble and unable to cross the cytoplasmic membrane in this form.

Complete genome sequence of Thermosulfurimonas marina SU872T, an anaerobic thermophilic chemolithoautotrophic bacterium isolated from a shallow marine hydrothermal vent.

Thermosulfurimonas marina strain SU872T is a thermophilic, anaerobic, chemolithoautotrophic bacterium, isolated from a shallow-sea hydrothermal vent in the Pacific Ocean near Kunashir Island, that is able to grow by disproportionation of inorganic sulfur compounds and dissimilatory nitrate reduction to ammonium. Here we report the complete genome sequence of strain SU872T, which presents one circular chromosome of 1,763,258 bp with a mean G + C content of 58.9 mol%. The complete genome harbors 1827 predicted protein-encoding genes, 47 tRNA genes and 3 rRNA genes. Genes involved in sulfur and nitrogen metabolism were identified. This study expands our knowledge of sulfur and nitrogen use in energy metabolism of high temperatures areas of shallow-sea hydrothermal environments. In order to highlight Thermosulfurimonas marina metabolic features, its genome was compared with that of Thermosulfurimonas dismutans, the only other species described within the Thermosulfurimonas genus.

Genome analysis of Thermosulfuriphilus ammonigenes ST65T, an anaerobic thermophilic chemolithoautotrophic bacterium isolated from a deep-sea hydrothermal vent.

Thermosulfuriphilus ammonigenes ST65T is an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent chimney. T. ammonigenes is an obligate chemolithoautotroph utilizing elemental sulfur as an electron donor and nitrate as an electron acceptor with sulfate and ammonium formation. It also is able to grow by disproportionation of elemental sulfur, thiosulfate and sulfite. Here, we present the complete genome sequence of strain ST65T. The genome consists of a single chromosome of 2,287,345 base pairs in size and has a G + C content of 51.9 mol%. The genome encodes 2172 proteins, 48 tRNA genes, and 3 rRNA genes. Genome analysis revealed a complete set of genes essential to CO2 fixation and gluconeogenesis. Homologs of genes encoding known enzyme systems for nitrate ammonification are absent in the genome of T. ammonigenes assuming unique mechanism for this pathway. The genome of strain ST65T encodes a complete set of genes necessary for dissimilatory sulfate reduction, which are probably involved in sulfur disproportionation and anaerobic oxidation. This is the first reported genome of a bacterium from the genus Thermosulfuriphilus, providing insights into the microbial contribution into carbon, sulfur and nitrogen cycles in the deep-sea hydrothermal vent environment.

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin.

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYROT was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYROT were motile cocci. The strain SLHTYROT grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L-1 NaCl (optimum 135 g L-1). Strain SLHTYROT was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYROT was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYROT was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYROT should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYROT (=DSM 108854 = JCM 32768 = UBOCC-M-3308).

Genomic Characterization and Environmental Distribution of a Thermophilic Anaerobe Dissulfurirhabdus thermomarina SH388T Involved in Disproportionation of Sulfur Compounds in Shallow Sea Hydrothermal Vents.

Marine hydrothermal systems are characterized by a pronounced biogeochemical sulfur cycle with the participation of sulfur-oxidizing, sulfate-reducing and sulfur-disproportionating microorganisms. The diversity and metabolism of sulfur disproportionators are studied to a much lesser extent compared with other microbial groups. Dissulfurirhabdus thermomarina SH388T is an anaerobic thermophilic bacterium isolated from a shallow sea hydrothermal vent. D. thermomarina is an obligate chemolithoautotroph able to grow by the disproportionation of sulfite and elemental sulfur. Here, we present the results of the sequencing and analysis of the high-quality draft genome of strain SH388T. The genome consists of a one circular chromosome of 2,461,642 base pairs, has a G + C content of 71.1 mol% and 2267 protein-coding sequences. The genome analysis revealed a complete set of genes essential to CO2 fixation via the reductive acetyl-CoA (Wood-Ljungdahl) pathway and gluconeogenesis. The genome of D. thermomarina encodes a complete set of genes necessary for the dissimilatory reduction of sulfates, which are probably involved in the disproportionation of sulfur. Data on the occurrences of Dissulfurirhabdus 16S rRNA gene sequences in gene libraries and metagenome datasets showed the worldwide distribution of the members of this genus. This study expands our knowledge of the microbial contribution into carbon and sulfur cycles in the marine hydrothermal environments.

Complete genome sequence of Methanofervidicoccus sp. A16, a thermophilic methanogen isolated from Mid Cayman Rise hydrothermal vent.

Methanofervidicoccus sp. A16 is a novel thermophilic and obligate hydrogenotrophic methanogen isolated from a deep-sea hydrothermal vent chimney sample at the Mid Cayman spreading center, Caribbean Sea. Here we report the complete genome of strain A16, which has one circular chromosome of 1,485,358 bp with a mean G+C content of 35.01 mol%. The complete genome harbors 1442 predicted protein-encoding genes. Genes involved in hydrogenotrophic methane production and N2 fixation were identified in this genome. This study expands our knowledge of methanogenesis at high temperatures and the involvement of these microorganisms in the carbon and nitrogen cycles of deep-sea hydrothermal environments.

Experimental and Simulation Efforts in the Astrobiological Exploration of Exooceans.

The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus' plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.

RNA processing machineries in Archaea: the 5'-3' exoribonuclease aRNase J of the ß-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3'-5' exoribonucleolytic RNA exosome machinery.

A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.

Genomic Insights into the Carbon and Energy Metabolism of a Thermophilic Deep-Sea Bacterium Deferribacter autotrophicus Revealed New Metabolic Traits in the Phylum Deferribacteres.

Information on the biochemical pathways of carbon and energy metabolism in representatives of the deep lineage bacterial phylum Deferribacteres are scarce. Here, we report the results of the sequencing and analysis of the high-quality draft genome of the thermophilic chemolithoautotrophic anaerobe Deferribacter autotrophicus. Genomic data suggest that CO2 assimilation is carried out by recently proposed reversible tricarboxylic acid cycle ("roTCA cycle"). The predicted genomic ability of D. autotrophicus to grow due to the oxidation of carbon monoxide was experimentally proven. CO oxidation was coupled with the reduction of nitrate to ammonium. Utilization of CO most likely involves anaerobic [Ni, Fe]-containing CO dehydrogenase. This is the first evidence of CO oxidation in the phylum Deferribacteres. The genome of D. autotrophicus encodes a Nap-type complex of nitrate reduction. However, the conversion of produced nitrite to ammonium proceeds via a non-canonical pathway with the participation of hydroxylamine oxidoreductase (Hao) and hydroxylamine reductase. The genome contains 17 genes of putative multiheme c-type cytochromes and "e-pilin" genes, some of which are probably involved in Fe(III) reduction. Genomic analysis indicates that the roTCA cycle of CO2 fixation and putative Hao-enabled ammonification may occur in several members of the phylum Deferribacteres.

Chemosynthetic ectosymbionts associated with a shallow-water marine nematode.

Prokaryotes and free-living nematodes are both very abundant and co-occur in marine environments, but little is known about their possible association. Our objective was to characterize the microbiome of a neglected but ecologically important group of free-living benthic nematodes of the Oncholaimidae family. We used a multi-approach study based on microscopic observations (Scanning Electron Microscopy and Fluorescence In Situ Hybridization) coupled with an assessment of molecular diversity using metabarcoding based on the 16S rRNA gene. All investigated free-living marine nematode specimens harboured distinct microbial communities (from the surrounding water and sediment and through the seasons) with ectosymbiosis seemed more abundant during summer. Microscopic observations distinguished two main morphotypes of bacteria (rod-shaped and filamentous) on the cuticle of these nematodes, which seemed to be affiliated to Campylobacterota and Gammaproteobacteria, respectively. Both ectosymbionts belonged to clades of bacteria usually associated with invertebrates from deep-sea hydrothermal vents. The presence of the AprA gene involved in sulfur metabolism suggested a potential for chemosynthesis in the nematode microbial community. The discovery of potential symbiotic associations of a shallow-water organism with taxa usually associated with deep-sea hydrothermal vents, is new for Nematoda, opening new avenues for the study of ecology and bacterial relationships with meiofauna.

Electroactive Bacteria Associated With Stainless Steel Ennoblement in Seawater.

Microorganisms can increase the open-circuit potential of stainless steel immersed in seawater of several hundred millivolts in a phenomenon called ennoblement. It raises the chance of corrosion as the open-circuit potential may go over the pitting corrosion potential. Despite the large impact of the ennoblement, no unifying mechanisms have been described as responsible for the phenomenon. Here we show that the strict electrotroph bacterium "Candidatus Tenderia electrophaga" is detected as an ennoblement biomarker and is only present at temperatures at which we observe ennoblement. This bacterium was previously enriched in biocathode systems. Our results suggest that "Candidatus Tenderia electrophaga," and its previously described extracellular electron transfer metabolism coupled to oxygen reduction activity, could play a central role in modulating stainless steel open-circuit potential and consequently mediating ennoblement.

The Effect of Crowding on Protein Stability, Rigidity, and High Pressure Sensitivity in Whole Cells.

In live cells, high concentrations up to 300-400 mg/mL, as in Eschericia coli (Ellis, R. J. Curr. Opin. Struct. Biol. 2001, 11, 114) are achieved which have effects on their proper functioning. However, in many experiments only individual parts of the cells as proteins or membranes are studied in order to get insight into these specific components and to avoid the high complexity of whole cells, neglecting by the way the influence of crowding. In the present study, we investigated cells of the order of Thermococcales, which are known to live under extreme conditions, in their intact form and after cell lysis to extract the effect of crowding on the molecular dynamics of the proteome and of water molecules. We found that some parameters characterizing the dynamics within the cells seem to be intrinsic to the cell type, as flexibility typical for the proteome, others are more specific to the cellular environment, as bulk water's residence time and some fractions of particles participating to the different motions, which make the lysed cells' dynamics similar to the one of another Thermococcale adapted to live under high hydrostatic pressure. In contrast to studies on the impact of crowding on pure proteins we show here that the release of crowding constraints on proteins leads to an increase in the rigidity and a decrease in the high pressure sensitivity. In a way similar to high pressure adaptation in piezophiles, the hydration water layer is decreased for the lysed cells, demonstrating a first link between protein adaptation and the impact of crowding or osmolytes on proteins.