Dr Anne Postec
email: anne.postec@univ-amu.fr

Grégoire Galès

Anne-Hélène Prime

Manon Bartoli

Roy E Price

Céline Vandecasteele

Gaël Erauso

a novel thermophilic anaerobic heterotroph isolated from a shallow hydrothermal field of Panarea Island in the Aeolian archipelago, Italy

Keywords: Phylum Thermotogota (Class Thermotogae, Order Petrotogales, Family Petrotogaceae, Thermotogota, Marinitoga, thermophilic, anaerobic, shallow hydrothermal vents, Panarea Italy

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Members of the phylum Thermotogota are anaerobic rods, with a characteristic outer sheathlike structure called 'toga' [START_REF] Oren | Valid publication of the names of forty-two phyla of prokaryotes[END_REF][START_REF] Reysenbach | Phylum BII. Thermotogae phy. nov[END_REF][START_REF] Whitman | Proposal of the suffixota to denote phyla. Addendum to 'Proposal to include the rank of phylum in the International Code of Nomenclature of Prokaryotes[END_REF]. They mainly grow by fermentation of various organic compounds. Most of them are also able to grow by respiration notably by reduction of sulfur compounds. Within the phylum Thermotogota, the order Petrotogales is composed to date of a single family, the Petrotogaceae, including six genera: Defluviitoga, Geotoga, Marinitoga, Oceanotoga, Petrotoga, and Tepiditoga [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF][START_REF] Mori | Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov[END_REF]. The genus Marinitoga contains to date seven described species, here with their type strain: Marinitoga artica 2PyrY55-1 T , M. camini MV1075 T , M. hydrogenitolerans AT1271 T , M. lausensis LG1 T , M. litoralis MC3 T , M. okinawensis TFS10-5 T and M. piezophila KA3 T [START_REF] Postec | Marinitoga litoralis sp. nov., a thermophilic, heterotrophic bacterium isolated from a coastal thermal spring on Ile Saint-Paul, Southern Indian Ocean[END_REF][START_REF] Steinsbu | Marinitoga arctica sp. nov., a thermophilic, anaerobic heterotroph isolated from a Mid-Ocean Ridge vent field[END_REF][START_REF] L'haridon | Marinitoga lauensis sp. nov., a novel deepsea hydrothermal vent thermophilic anaerobic heterotroph with a prophage[END_REF][START_REF] Postec | Marinitoga hydrogenitolerans sp. nov., a novel member of the order Thermotogales isolated from a black smoker chimney on the Mid-Atlantic Ridge[END_REF][START_REF] Wery | Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent[END_REF][START_REF] Nunoura | Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough[END_REF][START_REF] Alain | Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent[END_REF]. Of the seven Marinitoga species isolated to date, most originated from deep-sea hydrothermal ecosystems except M. litoralis which was isolated from a coastal thermal spring. Thermotogota species have already been first isolated from the shallow hydrothermal system of the Aeolian archipelago, in particular, Thermotoga maritima isolated from Vulcano and known as the most thermophilic bacteria described to date [START_REF] Huber | Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C[END_REF]. Here we report the description of a novel thermophilic anaerobic Marinitoga strain isolated from a shallow hydrothermal vent of the Panarea island in the Aeolian archipelago in the Mediterranean Sea. It is proposed as a representative of a novel Marinitoga species.

Sampling site and chemical analyses

The Aeolian Islands archipelago is a volcanic arc generated by the subduction of the Ionian oceanic lithosphere under the Calabrian arc (Fig. S1). At Panarea island, located off the Sicilian coast, Italy, an underwater hydrothermal field in the caldera at approximately 20 mbsl (meters below sea level) was sampled in May 2017 [START_REF] Price | Subsurface hydrothermal processes and the bioenergetics of chemolithoautotrophy at the shallow-sea vents off Panarea Island (Italy)[END_REF][START_REF] Price | A review of the geochemistry and microbiology of shallow-sea hydrothermal vents[END_REF]. Hydrothermal sediments (coarse sand) bathed with hot acidic fluid were collected with a core sampler close to an iron-copper sulfide hydrothermal chimney at the Black Point site noted BP5 (38°38'23''N 15°06'28''E). The environmental parameters of the sampling station are described below. Less than 2 hours after sampling, back to the lab, these sediments and their pore fluid samples were transferred into 100 mL Schott bottles to fill them to avoid air and preserve anaerobic conditions. They were stored at 4°C until use for cultivation assays (Fig. S1e).

Physico-chemical characterization of the fluid was performed as detailed elsewhere by [START_REF] Price | Subsurface hydrothermal processes and the bioenergetics of chemolithoautotrophy at the shallow-sea vents off Panarea Island (Italy)[END_REF] [START_REF] Price | Subsurface hydrothermal processes and the bioenergetics of chemolithoautotrophy at the shallow-sea vents off Panarea Island (Italy)[END_REF]. Briefly, temperatures were measured in situ with a custom-built probe in an underwater housing (Max Planck Institute for Marine Microbiology, Bremen, Germany). A WTW pH meter 3210 and a Mic-D electrode with temperature compensation were used to measure the pH of each sample onshore. H2S concentration was measured after the addition of 1 mL of a 50 mM zinc acetate solution to 3 mL of sample, by analyzing decreased light intensity caused by the ZnS precipitate with a Merck photometer at a wavelength of 670 nm. Samples for anion (Br, Cl, SO4) or cation (Mg, Na, Ca, K, B, Sr, Si, Ba, Mn, and Fe) were filtered in the field at 0.2 μm and then frozen until measurement. Samples for cations analyses were acidified using ultrapure double distilled HNO3 to 1% by volume. Measurements were performed by Dionex IC system ion chromatography (anion) and inductively coupled plasma-optical emission spectrometry (cations, Perkin-Elmer Optima 3300 ICP-OES). The temperature measured in the sediment at the BP5 site varied from 50°C at the surface to around 70°C at 20 cm depth. The pH of the hydrothermal pore fluid of the BP5 sample was 5.0 to 5.5. The composition in H2S, major anions and major cations were as follows: H2S 233.1 µM; Cl -574 mM; Br -0,86 mM; SO4 2-30.3 mM; Na + 442 mM; Al 3+ 1.1 µM; Ca 2+ 11.6 mM; K + 9.8 mM; Mg 2+ 53.5 mM; Mn(tot) 30 µM; Si 4+ 0.67 mM; Sr 2+ 0.1 mM. Ba and Fe concentrations were below detection limits. These concentrations were close to those of seawater, except for the high H2S content.

Enrichment, isolation, and cultivation

The Panarea basal medium (PM) was composed of (per liter): 25 g NaCl, 6 g MgCl2•6H2O, 5 g MgSO4•7H2O, 0,7 g KCl, 85 mg NH4Cl, 50 mg K2HPO4, 50 mg KH2PO4, 1 g CaCl2•2H2O, 28 mg FeSO4•7H2O, 2 mg NaNO3, 88 mg NaBr, 84 mg NaHCO3, 200 mg MnCl2•4H2O, 1 mL Widdel traces elements solution [START_REF] Widdel | Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. III. Characterization of the filamentous gliding Desulfonema limicola gen. nov., and Desulfonema magnum sp. nov[END_REF], 1 mL Fe-Ni-Wo-Se solution (1.42 g FeSO4, 1.6 g NiSO4•6 H2O, 38 mg Na2WO4•2 H2O, and 3 mg Na2SeO3•5 H2O per liter of distilled water), and 0.2 mL of 4% resazurin solution. For some enrichment cultures, 1 g of yeast extract and 2 g of tryptone were added as substrates to the PM medium, then noted PMYT. The pH was adjusted to 5.5 with a 10 M KOH solution. The medium was boiled under a stream of O2-free N2 gas, cooled to room temperature, and dispensed in 50 mL serum bottles or Hungate tubes under a N2 gas stream before sterilization by autoclaving. The Hungate technique for anaerobic cultivation was used throughout this study [START_REF] Hungate | A roll tube method for the cultivation of strict anaerobes[END_REF]. About 1 g of sediment slurry sample BP5 was used to inoculate 20 mL of PM medium in 50 mL serum bottles, then incubated at 60°C. After 2 days of incubation, significant cell growth was observed in the culture BP5-C20 on PMYT medium.

Microbial diversity of the culture analysis by 16S rRNA gene metabarcoding (see [START_REF] Quéméneur | Hydrostatic pressure helps to cultivate an original anaerobic bacterium from the Atlantis Massif Subseafloor (IODP Expedition 357): Petrocella atlantisensis gen. nov. sp. nov[END_REF] for detailed procedure) showed that the genus Marinitoga was largely dominant, encompassing 95.93% of the OTUs detected in the culture, followed by the scarce genera (> 0.1%) Thermococcus (0.29%), Sulfurimonas (0.20%), Clostridium (0.19%), Thioprofundum (0.17%), Halospirulina (0.16%) and Kosmotoga (0.11%). Some of these genera are known to contain (hyper)thermophilic and anaerobic species able to use sulfur compounds in their energetic metabolism. Corynebacterium (0.12%) and Pseudomonas (0.11%) were also detected and are known to be potential contaminants.

The enrichment culture was subcultured several times under the same growth conditions before the isolation of colonies on roll tubes containing PMYT with 1.6% (w/v) agar.

Several colonies obtained after 6-day incubation at 60°C were creamy beige, round, and convex with smooth edges and a diameter of 0.5-1 mm. Some colonies were picked up and inoculated new series of roll tubes. The procedure was repeated three times before the culture was deemed pure. These colonies served as inoculum to obtain axenic culture. Among them, strain BP5-C20A T was selected for further characterization.

Phylogenetic analyses

For 16S rRNA gene analyses, genomic DNA isolation from a BP5-C20A T culture (50 mL) and amplification by PCR (using 27F/1492r primer set; [START_REF] Lane | Nucleic acid techniques in bacterial systematics[END_REF]) were performed as described previously [START_REF] Bes | Acetoanaerobium pronyense sp. nov., an anaerobic alkaliphilic bacterium isolated from a carbonate chimney of the Prony Hydrothermal Field (New Caledonia)[END_REF]. Sanger sequencing of the PCR product was performed at GATC Biotech AG (Germany). The 16S rRNA gene sequence was then compared with available sequences in the Genbank database using the BLASTN search [START_REF] Zhang | A Greedy Algorithm for Aligning DNA Sequences[END_REF]. All evolutionary analyses were performed using MEGAX [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF]. A multiple alignment was built using the MUSCLE program [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF].

Sequence positions with alignment uncertainty and gaps were omitted from the analysis. Phylogenetic trees were reconstructed using three methods: Neighbor-Joining, Minimum Evolution, and Maximum-Likelihood methods [START_REF] Felsentein | Evolutionary trees from DNA sequences: a maximum likelihood approach[END_REF][START_REF] Rzhetsky | Statistical properties of the ordinary least-squares, generalized leastsquares, and minimum-evolution methods of phylogenetic inference[END_REF][START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The evolutionary distances were computed using the Kimura 2-parameter method [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]. The analysis involved 30 nucleotide sequences having a total of 1599 positions in the final dataset. Branch robustness was estimated by the non-parametric bootstrap procedure [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF] (2000 replicates). The 16S rRNA gene sequences of strain BP5-C20A T was deposited in Genbank under accession number MZ571208.

Strain BP5-C20A T shared 97.5% 16S rRNA gene sequence similarity with the closest related species M. lauensis LG1 T , 96.6% with M. okinawensis TFS10-5 T and 96.3% with M. piezophila KA3 T as determined by EzBioCloud [START_REF] Yoon | Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies[END_REF] (Fig. 1). It belongs to Class Thermotogae, Order Petrotogales, Family Petrotogaceae, in the Phylum Thermotogota according to LPSN (https://www.bacterio.net/) [START_REF] Parte | List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ[END_REF].

Genome features

For whole-genome analyses, genomic DNA extraction of strain BP5-C20A T and sequencing at the GeT-PlaGe platform, (INRAE Toulouse, France) by combining long reads (Oxford Nanopore) and short-reads (Illumina MiSeq) technologies were performed as described previously [START_REF] Quéméneur | Hydrostatic pressure helps to cultivate an original anaerobic bacterium from the Atlantis Massif Subseafloor (IODP Expedition 357): Petrocella atlantisensis gen. nov. sp. nov[END_REF]. Read demultiplexing and adapter trimming were performed using qcat v1.1.0 (https://github.com/nanoporetech/qcat) and genome assembly with Unicycler v0.4.6 [START_REF] Wick | Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads[END_REF]. A complete genome sequence was obtained, deposited at DDBJ/ENA/GenBank, and annotated by using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). For further comparative analyses, the complete genome sequence was also annotated using MaGe (Microscope Genome Annotation) [START_REF] Vallenet | MicroScope: an integrated platform for the annotation and exploration of microbial gene functions through genomic, pangenomic and metabolic comparative analysis[END_REF]. A whole proteome-based taxonomic analysis of strain BP5-C20A T genome was performed using the bioinformatics platform Type Strain Genome Server (TYGS) (https://tygs.dsmz.de) [START_REF] Meier-Kolthoff | TYGS is an automated high-throughput platform for state-of-theart genome-based taxonomy[END_REF]. In brief, the TYGS analysis included a determination of the closest type strain genomes by (i) comparison against all type strain genomes available in the TYGS database via the MASH algorithm [START_REF] Ondov | Mash: fast genome and metagenome distance estimation using MinHash[END_REF], and (ii) the closely related type strains determined via the 16S rRNA gene sequences. For the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using Genome BLAST Distance Phylogeny (GBDP), and accurate intergenomic distances were inferred under the algorithm 'trimming' and distance formula d5. The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1 [START_REF] Lefort | FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program[END_REF]. Branch support was inferred from 100 replicates each. The trees were rooted at the midpoint [START_REF] Farris | Estimating Phylogenetic Trees from Distance Matrices[END_REF]. Values of average nucleotide identity (ANI) were calculated by using OrthoANI with USEARCH (http://www.ezbiocloud.net/tools/ani). Digital DNA-DNA hybridizations (dDDH) were calculated using the Genome-to-Genome Distance Calculator (GGDC) 2.1 (http://ggdc.dsmz.de/) [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF]. The percentage of conserved proteins (POCP) was calculated using the Protologger software (http://www.protologger.de/) [START_REF] Hitch | Automated analysis of genomic sequences facilitates high-throughput and comprehensive description of bacteria[END_REF].

The complete genome sequence of strain BP5-C20A T is a unique circular chromosome of 2.44 Mb long with a 27.3% G+C content, which is consistent with the genomes of other Marinitoga strains (size ranging from 2.23 Mb to 2.59 Mb and G+C content ranging from 27.2% to 29.2%) (Table S1). The dDDH values between the genomes of strain BP5-C20A T and other available Marinitoga genomes were found to be ≤ 31.0% (highest value with M. lauensis LG1 T ) and the ANI values were ≤ 85.70%, i.e. far below the species boundaries of 70% and 95~96% respectively, recommended by Chun et al. [START_REF] Chun | Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes[END_REF] (Table 1). Strain BP5-C20A T shared >80% POCP with other Marinitoga species, from 80.3% with M. piezophila to 83.8% with M.

hydrogenitolerans.

Strain BP5-C20A T genome comprises a total of 2311 genes among which 2243 protein-coding genes (CDS) (Table S1). There are 51 tRNA genes with 3 rRNA operons. The genome contains 4 CRISPR loci of CRISPR-associated cas genes. Strain BP5-C20A T genome has a complete pentose phosphate pathway and possesses a complete Embden-Meyerhoff-Parnas pathway to convert glucose into pyruvate. It has an impaired TCA cycle because it lacks genes coding for the fumarate reductase, fumarate hydratase, malate dehydrogenase, and aconite hydratase.

Strain BP5-C20A T genome possesses genes encoding flagella-related proteins, carbohydrates and amino acids transport and metabolism (92 ABC transporters), peptidases and proteases.

The distribution of genes into COG functional categories is shown in Table S2. The genome of strain BP5-C20A T also contains genes encoding hydrogenases, a Rnf complex and ATP synthases that explain hydrogen and energy production. The reducing power is transferred to a ferredoxin electron carrier (MARBP5_v1_0066) that may fuel a hydrogenase to produce H2.

Genes of a hydrogenase are found as an operon hndA, hndB, hndC and hndD (MARBP5_v1_2217-2220). Genes rnfA, rnfB, rnfC, rnfD, rnfE, rnfG were also found organized as an operon (MARBP5_v1_0453 to MARBP5_v1_0458). Rnf is a membrane-bound complex that couples electron transfer with translocation of ions (Na + ) across the membrane.

We hypothesize that this complex is associated to a F-type ATP synthase to build a simple twocomponent respiratory chain, as described in Thermotoga maritima [START_REF] Kuhns | The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima[END_REF]. Indeed, the genome encodes both a F-type ATP synthase generating ATP via proton translocation, encoded by atpA, atpB, atpC, atpD, atpE, atpF, atpG and atpH (MARBP5_v1_1427 to 1434) [START_REF] Perzov | Features of V-ATPases that distinguish them from F-ATPases[END_REF], and a V-type ATP synthase (MARBP5_v1_1639-1641 and MARBP5_v1_1643-1645-1646) known to use ATP to translocate proton across the membrane.

In a whole proteome-based analysis (Fig. S2), strain BP5-C20A T grouped with other Marinitoga, and clustered with other thermophilic members of the order Petrotogales.

Fatty acid analyses

The determination of cellular fatty acid composition was performed at the DSMZ identification service (Braunschweig, Germany). Cells were harvested in the late exponential growth phase.

After extraction using a modified version of the method of Miller [START_REF] Kuykendall | Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum[END_REF][START_REF] Miller | Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids[END_REF], fatty acids were separated by gas chromatography and detected using the Microbial Identification System (MIDI, Sherlock Version 6.1) [START_REF] Sasser | Identification of bacteria by gas chromatography of cellular fatty acids[END_REF].

The cells of strain BP5-C20A T contained C16:0 as major fatty acid (58.8% from total), similar to M. okinawensis [START_REF] Nunoura | Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough[END_REF]. Numerous minor fatty acids were also identified: C16:1ω9c (8.3%), C14:0 (6.1%), C16:1 ω7c (5.8%), C18:1 ω9c (4.5%), C16:0 iso (4.4%), C12:0 (3.2%), C18:0 (2.7%), C18:1 ω7c (1.8%), C10:0 (1.7%), C17:0 anteiso (1.6%) and C15:0 anteiso (1.1%).

Microscopic analyses

Cells were routinely observed under a Nikon ECLIPSE E600 phase-contrast microscope for culture monitoring. The cell structure of strain BP5-C20A T was analyzed using an FEI Tecnai G2 transmission electron microscope (TEM) operating at 200 kV. For observation of the cell wall, cells were first fixed 20 min with 10% (v/v) glutaraldehyde, stained overnight with 2% (w/v) osmium tetra oxide, and then embedded in EMbed-812 resin. Embedded specimens were sliced into ultrathin sections (90 nm in thickness), placed on a Cu TEM grid, and stained for 3 min with 2% (w/v) uranyl acetate and 1 min with 1% (w/v) lead before observation.

Cells of strain BP5-C20A T are rod-shaped, surrounded with a 'toga' which is the sheath-like outer structure characteristic of the members of the phylum Thermotogota (Fig. 2). They are motile and Gram-stain -negative. They have a mean length of 1.5-3.4 μm and a width of 0.5-0.7 μm in the exponential growth phase and appear singly or in short chains inside the toga.

Cells became elongated during the stationary growth phase. When cultured under unfavorable conditions, cells swell and exhibit a "balloon" shape before cell lysis occurs. No spore formation was observed, as is the case for each strain of the genus Marinitoga.

Physiological tests

The pH, temperature, and NaCl concentration ranges for growth were determined in Hungate tubes using Vulcano High Salinity (VHS) medium composed of (per liter of distilled water): 18 g NaCl, 1 g MgCl2•6 H2O, 1 g MgSO4•7 H2O, 0.3 g KCl, 0.3 g Na2SO4, 0.3 g (NH4)2SO4, 1 mL K2HPO4 5% solution, 1 mL KH2PO4 5% solution, 1 mL CaCl2•2 H2O 2% solution, 0.1 g Fe (III)-citrate, 0.1 g L-cysteine HCl, 0.1 g Yeast extract, 0.08 g Na acetate, 1.95 g MES, 1 mL Widdel's elements, 1 mL Fe-Ni-Wo-Se solution, and 0.2 mL of resazurine 4% solution. The VHS medium was routinely supplemented with 1 g yeast extract and 2 g tryptone as substrates, except for testing autotrophic capacities. pH was adjusted to 5.5 with a 10 M KOH solution and temperature for growth was 55°C except for determining optimal pH and temperature for growth.

Growth was determined by directly measuring the optical density at 600 nm in Hungate tubes using a spectrophotometer equipped with a tube holder (Cary 50, Varian). To determine pH range and optimum growth conditions, the VHS medium pH was adjusted from 3.7 to 8.7 with anaerobic sterile stock solutions of 0.1M HCl or 0.1M NaOH and was verified after inoculation.

Temperature tests were performed using water baths from 20 °C to 80 °C with an increment of 5°C. For NaCl requirement, NaCl was weighted directly in the tubes prior to adding the medium (0 to 70 g L -1 with an increment of 5 g L -1 from 0 to 40 g L -1 , then 10 g L -1 from 40 to 70 g L -1 ).

All cultures were performed in duplicate and reproduced twice. Growth rates were determined for subcultures under the same experimental conditions. Substrate utilization experiments were performed for strain BP5-C20A T in the VHS medium at pH 5.5 and 55°C. Sugars, organic acids, and alcohol were tested individually at 20 mM and proteinaceous compounds at 5 g L -1 . Autotrophic growth was tested by replacing N2 headspace with H2/CO2 (80/20, v/v) at 200 kPa with elemental sulfur as an electron acceptor. Electron acceptor utilization experiments were performed in the VHS medium added with 20 mM maltose as carbon and energy source. Each compound was tested in duplicate Hungate tubes and those for which growth was observed (OD significantly above that obtained on the inoculated duplicate controls on VHS medium only) were checked by two successive subcultures under the same conditions. Bacterial growth was monitored by measuring the increase in turbidity at 600 nm as mentioned above. H2S production was determined measuring the formation of colloidal CuS photometrically at 480 nm following the method described by Cord-Ruwisch [START_REF] Cord-Ruwisch | A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria[END_REF]. Metabolic end-products were measured by HPLC using an Aminex HPX-87H (Bio-Rad) column with 2.5 mM H2SO4 as mobile phase and gas-phase chromatography (Shimadzu GC 8A) after 5 days of incubation at 55 °C. Oxygen and hydrogen tolerances were tested at 55°C with shaking at 120 rpm. The gas phase was set from 20% to 0% O2 with a 2% increment, and from 100% H2 to 0% H2 with a 20% increment. The various gas phases were tested at atmospheric pressure. Hydrogen tolerance was tested with or without 0.5% elemental sulfur.

Susceptibility to antibiotics was investigated at 100 μg mL -1 , 50 μg mL -1 , 25 μg mL -1 , and 10 μg mL -1 . Ampicillin, bacitracin, chloramphenicol, cycloheximide, kanamycin, penicillin, streptomycin, and vancomycin were tested separately. Concentrated anaerobic solutions of antibiotics were added to the VHS medium just before inoculation. To measure antibiotics susceptibility, strain BP5-C20A T was cultured twice in VHS medium with the antibiotic, and growth was compared with an antibiotic-free control.

Growth occurs from 40°C to 70°C, with an optimum at 55°C, from 10 to 50 g L -1 (w/v) NaCl with an optimum at 25 g L -1 and from pH 4.7 to 6.5 with an optimum at pH 5.6 (Fig. S3). The µmax determined in optimal conditions (pH 5.6) was 0.18 h -1 (doubling time of 3 h 51 min).

From the substrates tested as carbon and energy sources for growth of strain BP5-C20A T , cellobiose, fructose, glucose, maltose, starch, sucrose, and xylan as carbohydrates, and yeast extract, peptone, and tryptone as complex compounds were used and improved growth. The following tested compounds were not used and did not improve growth: acetate, casamino acids, casein, ethanol, formate, galactose, glycerol, methanol, succinate, and xylose (Table 2).

Of the electron acceptors tested: elemental sulfur (5 g L -1 ), L-cystine (2 g L -1 ), sodium thiosulfate (20 mM), sodium sulfite (2 mM), sodium sulfate (20 mM), nitrate (20 mM), nitrite (2 mM), Fe III hydroxide (12 mM), and Fe IIII EDTA (12 mM), only elemental sulfur was used as an electron acceptor and reduced to H2S. During the growth of strain BP5-C20A T on glucose, acetate, carbon dioxide, and hydrogen were produced. Growth on yeast extract and tryptone was accompanied by acetate, H2, and CO2 production, with lactate, isovalerate, and isobutyrate detected as traces. H2S was only detected during growth on cultures containing elemental sulfur as an electron acceptor.

Autotrophic growth was examined in VHS medium in the absence of any organic compound.

Strain BP5-C20A T was not able to grow under autotrophic conditions using H2 as a sole energy source and elemental sulfur as electron acceptors, as all members of the genus Marinitoga.

Tolerance to oxygen was examined and strain BP5-C20A T was a strict anaerobe as M. lauensis [START_REF] L'haridon | Marinitoga lauensis sp. nov., a novel deepsea hydrothermal vent thermophilic anaerobic heterotroph with a prophage[END_REF] and M. okinawensis [START_REF] Nunoura | Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough[END_REF].

Growth did not occur under 100% H2 in the gas phase, but this growth inhibition was lifted in the presence of elemental sulfur as in M. camini [START_REF] Wery | Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent[END_REF]. Growth was observed without elemental sulfur in the medium under H2/CO2 (80/20, v/v) atmosphere. Strain BP5-C20A T growth was inhibited at 10 g mL -1 ampicillin, chloramphenicol, penicillin, and vancomycin; 50 g mL -1 bacitracin, cycloheximide and streptomycin; and 100 g mL -1 kanamycin.

Optimal growth conditions of strain BP5-C20A T , in terms of temperature, NaCl concentration, and pH are very similar to other species: the novel strain is thermophilic, slightly halophilic, and acidophilic. It differs from the closest Marinitoga type species in the use of substrates, especially in the inability to use acetate, casein, formate, and galactose contrary to M. lauensis

LG1 T (Table 2). Strain BP5-C20A T could use maltose and sucrose but not glycerol, at the difference of M. okinawensis TFS-10-5 T . They were all able to use elemental sulfur as an electron acceptor but differ in the use of L-cystine and thiosulfate. The phylogenetic and genome features, especially the dDDH and ANI values between the genomes of strains BP5-C20A T and its closest relatives, allow us to propose this strain as a new species of the genus Marinitoga.

Description of Marinitoga aeolica sp. nov.

Marinitoga aeolica (ae.o′li.ca. L. fem. adj. aeolica, from the Aeolian archipelago, referring to the Panarea Island near Sicily in Italy, the source of the first isolated strain). Cells are motile rods and occur singly, in pairs or in chains. They have a mean length of 1.5-3.4 μm and width 0.5-0.7 μm in exponential growth phase. Colonies are creamy, circular, and convex. Strictly anaerobic. The fatty acids identified were: C16:0, C16:1ω9c, C14:0, C16:1 ω7c, C18:1 ω9c, C16:0 iso C12:0, C18:0, C18:1 ω7c, C10:0, C17:0 anteiso and C15:0 anteiso. Obligate chemoorganotroph; the growth occurs preferentially on complex peptide organics such as yeast extract, peptone, tryptone. Able to use a range of carbohydrates: cellobiose, fructose, glucose, maltose, starch, sucrose, and xylan.

Elemental sulfur used as electron acceptor and reduced to sulfide. Optimal growth occurs at 55°C, pH 5.6 and 25 g L -1 (w/v) NaCl. The DNA G + C content of this strain is 27.3 %. The Genbank 16S rRNA gene sequence accession number of strain BP5-C20A T is MZ571208. The GenBank accession number of the complete genome sequence is CP069362. The type strain BP5-C20A T (=DSM 112332 T = JCM 39183 T ) was isolated from a shallow hydrothermal vent located at Panarea Island in the Aeolian archipelago near Sicily, Italy.

Conflict of Interest Statement

No Conflict of Interest to declare. values between strain BP5-C20A T genome and other related Marinitoga species for which genome data were available: M. lauensis LG1 T [START_REF] L'haridon | Marinitoga lauensis sp. nov., a novel deepsea hydrothermal vent thermophilic anaerobic heterotroph with a prophage[END_REF], M. camini str. 1197 [START_REF] Mercier | Draft Genome Sequences of Two Marinitoga camini Isolates Producing Bacterioviruses[END_REF], M. camini str.

1155 [START_REF] Mercier | Draft Genome Sequences of Two Marinitoga camini Isolates Producing Bacterioviruses[END_REF], M. piezophila KA3 T [START_REF] Lucas | Complete genome sequence of the thermophilic, piezophilic, heterotrophic bacterium Marinitoga piezophila KA3[END_REF], M. hydrogenitolerans AT1271 T , Marinitoga sp. 1137.

Accession numbers of the genomic sequences are given in brackets. dDDH were calculated using the Genome-to-Genome Distance Calculator (GGDC) 2.1 and formula d4 (http://ggdc.dsmz.de/) [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF]. ANI values were calculated using OrthoANI with usearch (http://www.ezbiocloud.net/tools/ani) [START_REF] Yoon | A large-scale evaluation of algorithms to calculate average nucleotide identity[END_REF].

BP5-C20A 21.0 76.82 * Formula d4 (a.k.a. GGDC formula 2): sum of all identities found in HSPs divided by overall HSP length (Formula d4 is independent of genome length and is thus robust against the use of incomplete draft genomes).

Table 2. Differential characteristics between strain BP5-C20A T and other members of the genus Marinitoga. Type strains of the Marinitoga species: 1, BP5-C20A T (this study); 2, M. lauensis

LG1 T [START_REF] L'haridon | Marinitoga lauensis sp. nov., a novel deepsea hydrothermal vent thermophilic anaerobic heterotroph with a prophage[END_REF]; 3, M. okinawensis TFS-10-5 T [START_REF] Nunoura | Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough[END_REF]; 4, M. piezophila KA3 T [START_REF] Alain | Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent[END_REF]; 5, M. camini MV1075 T [START_REF] Wery | Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent[END_REF]; 6, M. litoralis MC3 T [START_REF] Postec | Marinitoga litoralis sp. nov., a thermophilic, heterotrophic bacterium isolated from a coastal thermal spring on Ile Saint-Paul, Southern Indian Ocean[END_REF] ; 7, M. hydrogenitolerans AT1271 T [START_REF] Postec | Marinitoga hydrogenitolerans sp. nov., a novel member of the order Thermotogales isolated from a black smoker chimney on the Mid-Atlantic Ridge[END_REF]; 8, M. artica 2PyrY55-1 T [START_REF] Steinsbu | Marinitoga arctica sp. nov., a thermophilic, anaerobic heterotroph isolated from a Mid-Ocean Ridge vent field[END_REF]. +, Positive; (+), weakly positive; -, negative; ND, not determined.

Figure 1 .Figure 2 .

 12 Figure 1. Phylogenetic tree based on 16S rRNA gene sequence showing the position of strain BP5-C20A T , together with type species of the Petrogaceae family (Petrotogales order,

Table 1 .

 1 Digital DNA-DNA hybridization (dDDH) and Average Nucleotide Identity (ANI)

  a) means acetate was tested in the presence of CO2 in the gas phase.

			-50	10-55	10-50	10-45	8-46	10-65	15-55
	Optimum	25	30	30-35	30	20	26	30-40	25
	Carbon sources:
	Acetate	-	+ a	-	(+) a	-a	-	-	-
	Casein	-	+	-	+	-	+	+	-
	Formate	-	+	-	+	-	-	ND	-
	Galactose	-	+	-	+	-	+	-	-
	Glycerol	-	ND	+	ND	-	-	ND	ND
	Maltose	+	+	-	+	+	+	+	+
	Sucrose	+	ND	-	ND	+	ND	ND	+
	Tolerance of O2	No	No	No	ND	ND	<4	<4	ND
	Tolerance of 100% H2	No	No	Yes	No	No	Yes	Yes	Yes
	without S°
	G + C content (%)	27.3	27.9	28	29.2	29	26.2	27.2	27
	Electron acceptors	Elemental	Elemental	Elemental	Elemental	Elemental	Elemental	Elemental	Elemental
		sulfur	sulfur, L-	sulfur, L-	sulfur, L-	sulfur, L-	sulfur	sulfur, L-	sulfur, L-
			cystine,	cystine	cystine,	cystine		cystine,	cystine
			sodium		thiosulfate			thiosulfate
			thiosulfate

(

Acknowledgments

We thank the INGV Institute of Palermo (Italie) for their assistance in scuba diving sampling and Dr Valentina Esposito for giving us access to the ECCSEL-NatLab Italy laboratory in Panarea island. We thank the GeT core facility, Toulouse, France (http://get.genotoul.fr) for the genome sequencing of BP5-C20A T strain, supported by France génomique. The LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform.

Funding information

The project was financially supported by the Mission for inter Inter-disciplinary Initiatives (MITI) as part of the "X-life -Unknown branches of life" Challenge (CNRS, France).

maritima MSB-8 T (NR 029163.2) and Thermotoga neapolitana DSM 4359 T (NR 074959.1).

The scale bar represents 0.05 substitution per nucleotide.