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# Thermospira aquatica gen. nov., sp. nov., a novel thermophilic spirochete isolated from a Tunisian hot spring, and description of the novel family Thermospiraceae

Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Anne Postec, Manon Bartoli-Joseph, Céline Vandecasteele, Gaël Erauso, Marc Labat

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Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Anne Postec, Manon Bartoli-Joseph, et al. Thermospira aquatica gen. nov., sp. nov., a novel thermophilic spirochete isolated from a Tunisian hot spring, and description of the novel family Thermospiraceae. International Journal of Systematic and Evolutionary Microbiology, 2023, 73 (1), 10.1099/ijsem.0.005690. hal-04417428

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https://hal.science/hal-04417428

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# Thermospira aquatica gen. nov., sp. nov., a novel thermophilic spirochete isolated from a Tunisian hot spring, and description of the novel family Thermospiraceae

Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Anne Postec, Manon Bartoli-Joseph, Céline Vandecasteele, Gaël Erauso, Marc Labat

► To cite this version:

Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Anne Postec, Manon Bartoli-Joseph, et al.. Thermospira aquatica gen. nov., sp. nov., a novel thermophilic spirochete isolated from a Tunisian hot spring, and description of the novel family Thermospiraceae. International Journal of Systematic and Evolutionary Microbiology, 2023, 73 (1), 10.1099/ijsem.0.005690. hal-04417428

HAL Id: hal-04417428

https://hal.science/hal-04417428v1

Submitted on 4 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1

*Thermospira aquatica* gen. nov., sp. nov., a novel thermophilic spirochete
isolated from a Tunisian hot spring, and description of the novel family

2

3

*Thermospiraceae*.

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5

Zouhaier Ben Ali Gam¹, Abdoulaye Thioye², Jean-Luc Cayol¹, Anne Postec¹, Manon

6

Bartoli-Joseph¹, Céline Vandecasteele³, Gaël Erauso¹ and Marc Labat¹*

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¹ Aix Marseille Université, Université de Toulon, CNRS, IRD, MIO, Mediterranean Institute
of Oceanography, 163 avenue de Luminy, F-13288, Marseille, France.

9

² Université Cheikh Anta Diop, Ecole Supérieure Polytechnique, Laboratoire de Microbiologie
Appliquée et de Génie Industriel, BP 5005, Dakar-Fann, Dakar, Sénégal.

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³ INRAe, US 1426, GeT-PlaGe, Genotoul, Castanet- Tolosan, France

*Correspondance to : Marc Labat, IRD, MIO UM110, Polytech-Marseille - Case 925, Aix-
Marseille Université, 163 avenue de Luminy, F-13288 Marseille cedex 9, France. Tel :
+33(0) 4 86 09 06 15 ; Fax : +33(0) 4 91 82 85 70 ;

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e-mail : marc.labat@univ-amu.fr.

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Running title: *Thermospira aquatica*

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New taxa – Spirochaetota.

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F1F22T is
KT767982 and the one for the whole genome sequence is CP073355.

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21

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The type strain is F1F22T deposit in DSMZ (=DSM 101182T) and in JCM (= JCM 31314T).

23
1

*Thermospira aquatica* gen. nov., sp. nov., a novel thermophilic spirochete
isolated from a Tunisian hot spring, and description of the novel family

2

3

*Thermospiraceae*.

4

5

Zouhaier Ben Ali Gam¹, Abdoulaye Thioye², Jean-Luc Cayol¹, Anne Postec¹, Manon

6

Bartoli-Joseph¹, Céline Vandecasteele³, Gaël Erauso¹ and Marc Labat¹*

7

8

¹ Aix Marseille Université, Université de Toulon, CNRS, IRD, MIO, Mediterranean Institute
of Oceanography, 163 avenue de Luminy, F-13288, Marseille, France.

9

² Université Cheikh Anta Diop, Ecole Supérieure Polytechnique, Laboratoire de Microbiologie

Appliquée et de Génie Industriel, BP 5005, Dakar-Fann, Dakar, Sénégal.

12

³ INRAe, US 1426, GeT-PlaGe, Genotoul, Castanet- Tolosan, France

*Correspondance to : Marc Labat, IRD, MIO UM110, Polytech-Marseille - Case 925, Aix-

Marseille Université, 163 avenue de Luminy, F-13288 Marseille cedex 9, France. Tel :
+33(0) 4 86 09 06 15 ; Fax : +33(0) 4 91 82 85 70 ;

e-mail : marc.labat@univ-amu.fr.

Running title: *Thermospira aquatica*

New taxa – Spirochaetota.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F1F22T is

KT767982 and the one for the whole genome sequence is CP073355.

21

The type strain is F1F22T deposit in DSMZ (=DSM 101182T) and in JCM (= JCM 31314T).

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24 Abstract

A novel thermophilic, anaerobic bacterium, strain F1F22<sup>T</sup>, was isolated from a hot spring water located north of Tunisia. The cells were non-motile, Gram-negative, and helical with hooked ends, 0.5 x 10-32 µm in size. Growth of the strain was observed at 45-70°C (optimum 55°C), in 0.0 - 1.0% (w/v) NaCl (optimum without NaCl) and at pH 6.5 - 8.5 (optimum pH 7.5). Yeast extract was required for growth, and the strain grew on glucose, sucrose, and maltose. The major fatty acids for the strain were C<sub>16:0</sub> (40.2%), iso-C<sub>16:0</sub> (30.2%) and C<sub>16:0</sub> DMA (14.5%). The genome consists of a circular chromosome (2.5 Mb) containing 2672 predicted protein-encoding genes with a G+C content of 43.15 mol%. Based on a comparative 16S rRNA gene sequence analysis, strain F1F22<sup>T</sup> formed a deeply branching lineage within the phylum *Spirochaetota*, class *Spirochaetia*, order *Brevinematales*, and had only low sequence similarity with other species of the phylum (lower than 83%). Genome-based analysis of average nucleotide identity and digital DNA-DNA hybridization of strain F1F22<sup>T</sup> with *Treponema caldarium*<sup>T</sup>, *Brevinema andersonii*<sup>T</sup>, and *Spirochaeta thermophila*<sup>T</sup> showed respectively values between 63.26% and 63.52%, and between 20% and 25%. Hence, we propose strain F1F22<sup>T</sup> as a novel family (*Thermospiraceae* fam. nov.), genus and species of *Brevinematales* : *Thermospira aquatica* gen. nov., sp. nov. (Type strain F1F22<sup>T</sup> =JCM 31314<sup>T</sup> =DSM 101182<sup>T</sup>).

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45
24 Abstract

A novel thermophilic, anaerobic bacterium, strain F1F22<sup>T</sup>, was isolated from a hot spring water located north of Tunisia. The cells were non-motile, Gram-negative, and helical with hooked ends, 0.5 x 10-32 µm in size. Growth of the strain was observed at 45-70°C (optimum 55°C), in 0.0 - 1.0% (w/v) NaCl (optimum without NaCl) and at pH 6.5 - 8.5 (optimum pH 7.5). Yeast extract was required for growth, and the strain grew on glucose, sucrose, and maltose. The major fatty acids for the strain were C<sub>16:0</sub> (40.2%), iso-C<sub>16:0</sub> (30.2%) and C<sub>16:0</sub> DMA (14.5%). The genome consists of a circular chromosome (2.5 Mb) containing 2672 predicted protein-encoding genes with a G+C content of 43.15 mol%. Based on a comparative 16S rRNA gene sequence analysis, strain F1F22<sup>T</sup> formed a deeply branching lineage within the phylum *Spirochaetota*, class *Spirochaetia*, order *Brevinematales*, and had only low sequence similarity with other species of the phylum (lower than 83%). Genome-based analysis of average nucleotide identity and digital DNA-DNA hybridization of strain F1F22<sup>T</sup> with *Treponema caldarium*<sup>T</sup>, *Brevinema andersonii*<sup>T</sup>, and *Spirochaeta thermophila*<sup>T</sup> showed respectively values between 63.26% and 63.52%, and between 20% and 25%. Hence, we propose strain F1F22<sup>T</sup> as a novel family (*Thermospiraceae* fam. nov.), genus and species of *Brevinematales* : *Thermospira aquatica* gen. nov., sp. nov. (Type strain F1F22<sup>T</sup> =JCM 31314<sup>T</sup> =DSM 101182<sup>T</sup>).

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44

45
46 Members of the phylum *Spirochaetota* are mainly helical, gram-negative bacteria, with free-living or host-associated lifestyles, some of which are highly prevalent disease-causing agents
47
48 [1,2]. They are widespread, either pathogenic in the human body or animals, or saprophytic in
49 diverse natural environments, including extreme ones like the deep-sea [3,4].

50 In 2013, a large taxonomic rearrangement of the phylum *Spirochaetota* was proposed. The
51 families *Brachyspiraceae*, *Brevinemataceae* and *Leptospiraceae* were transferred respectively
52 to the novel orders *Brachyspiriale*, *Brevinematales* and *Leptospiriales* all three belonging to
53 the class *Spirochaetia* and the genera *Borrelia* and *Cristispira* were transferred to the novel
54 family *Borreliaceae* within the order *Spirochaetales* [5]. The phylum *Spirochaetota* still
55 comprises a single class *Spirochaetia*, but currently contains four orders (*Brachyspiriale*,
56 *Brevinematales*, *Leptospiriales* and *Spirochaetales*) and seven families (*Brachyspiraceae*,
57 *Brevinemataceae*, *Borreliaceae*, *Leptospiraceae*, *Treponemataceae*, *Sphaerochaetaceae* and
58 *Spirochaetaceae*) [6].

59 Today, the phylum Spirochaetota counts 20 genera with various lifestyle and phenotypic or
60 genomic characteristics [2,7]. Previously, the presence of periplasmic endoflagella was
61 considered the only reliable phenotypic feature characteristic of this phylum [5]. However
62 today, even this feature is not a strict criterion as novel species have been described which do
63 not possess an endoflagellum such as the motile species *Rectinema subterraneum* [8] or partly
64 non-motile species within the genus *Sphaerochaeta* [6] or even the non-motile species
65 *Rectinema cohabitans* [9]. Therefore, the main characteristics of each family belonging to this
66 phylum have been detailed in [5, 6].

67 Only a few thermophilic species belonging to the phylum *Spirochaetota* have been isolated
68 from various aquatic springs, including not fully characterized strains from thermal springs
69 [10]. Within the order *Spirochaetales*, only two thermophiles have been previously
70 characterized: family *Spirochaetaceae*, with *Spirochaeta thermophila* isolated from a marine
46 Members of the phylum *Spirochaetota* are mainly helical, gram-negative bacteria, with free-living or host-associated lifestyles, some of which are highly prevalent disease-causing agents
47
48 [1,2]. They are widespread, either pathogenic in the human body or animals, or saprophytic in
49 diverse natural environments, including extreme ones like the deep-sea [3,4].

50 In 2013, a large taxonomic rearrangement of the phylum *Spirochaetota* was proposed. The
51 families *Brachyspiraceae*, *Brevinemataceae* and *Leptospiraceae* were transferred respectively
52 to the novel orders *Brachyspiriale*, *Brevinematales* and *Leptospiriales* all three belonging to
53 the class *Spirochaetia* and the genera *Borrelia* and *Cristispira* were transferred to the novel
54 family *Borreliaceae* within the order *Spirochaetales* [5]. The phylum *Spirochaetota* still
55 comprises a single class *Spirochaetia*, but currently contains four orders (*Brachyspiriale*,
56 *Brevinematales*, *Leptospiriales* and *Spirochaetales*) and seven families (*Brachyspiraceae*,
57 *Brevinemataceae*, *Borreliaceae*, *Leptospiraceae*, *Treponemataceae*, *Sphaerochaetaceae* and
58 *Spirochaetaceae*) [6].

59 Today, the phylum Spirochaetota counts 20 genera with various lifestyle and phenotypic or
60 genomic characteristics [2,7]. Previously, the presence of periplasmic endoflagella was
61 considered the only reliable phenotypic feature characteristic of this phylum [5]. However
62 today, even this feature is not a strict criterion as novel species have been described which do
63 not possess an endoflagellum such as the motile species *Rectinema subterraneum* [8] or partly
64 non-motile species within the genus *Sphaerochaeta* [6] or even the non-motile species
65 *Rectinema cohabitans* [9]. Therefore, the main characteristics of each family belonging to this
66 phylum have been detailed in [5, 6].

67 Only a few thermophilic species belonging to the phylum *Spirochaetota* have been isolated
68 from various aquatic springs, including not fully characterized strains from thermal springs
69 [10]. Within the order *Spirochaetales*, only two thermophiles have been previously
70 characterized: family *Spirochaetaceae*, with *Spirochaeta thermophila* isolated from a marine
71 hot spring [11], and family *Treponemataceae*, with *Treponema caldarium* initially proposed as
72 *Spirochaeta caldaria*, isolated from a freshwater hot spring [12]. A third thermophilic species
73 has been then described as a new genus but without an assigned family, *Exilispira thermophila*,
74 an anaerobic bacterium isolated from a deep-sea hydrothermal vent chimney [13]. This isolate
75 grew up to 60°C with an optimum of 50°C. Moreover, from the two known species of the genus
76 *Rectinema*, both described as mesophilic and isolated respectively in 2017 from a naphthalene-
77 degrading enrichment culture [9] and in 2020 from a deep terrestrial subsurface [8], only the
78 second ones can grow up to 50°C. Recently in 2020, two not sporulating thermophilic strains
79 (optimum 50-55°C), representative of a novel phylogenetically distinct group of thermophilic
80 spirochetes belonging to the order of *Brevinematales* was described with a proposed family
81 "*Longinemaceae*" and the species name "*Longinema margulisiae*" [14] but with unvalidly
82 published names of genus and family and with the whole genome sequence which remains
83 currently unavailable (accession number CP053071).

84 In this study, we have successfully isolated an anaerobic, thermophilic spirochete, designated
85 strain F1F22T, from the water of a Tunisian hot spring. We describe the detailed morphological,
86 physiological, and genomic characteristics of strain F1F22T and propose a novel species of a
87 new genus for this strain.

88 Thermal water has been collected in a hot spring (36° 48' 59" North, 10° 34' 07" East) in
89 Korbous city, north Tunisia. The temperature, pH, and salinity of
90 the fluid *in situ* was 60 °C, 7.8 and 8 g/l NaCl respectively. Strain F1F22T was isolated using
91 enrichment techniques described by Mechichi *et al.* [15]. A basal medium used for enrichment,
92 isolation, and cultivation was prepared using the anaerobic techniques [16-18]. The basal
93 medium contained (per liter of distilled water): KH2PO4, 0.20 g; NH4Cl, 0.30 g; NaCl, 1 g;
94 KCl, 0.50 g; MgCl2 · 6H2O, 0.40 g; cysteine · HCl, 0.50 g; CaCl2 · 2H2O, 0.15 g; yeast
95 extract, 0.50 g (Difco); trace-element solution [19], 1 ml; and 1 ml of 0.1% (w/v) resazurin. The
71 hot spring [11], and family *Treponemataceae*, with *Treponema caldarium* initially proposed as
72 *Spirochaeta caldaria*, isolated from a freshwater hot spring [12]. A third thermophilic species
73 has been then described as a new genus but without an assigned family, *Exilispira thermophila*,
74 an anaerobic bacterium isolated from a deep-sea hydrothermal vent chimney [13]. This isolate
75 grew up to 60°C with an optimum of 50°C. Moreover, from the two known species of the genus
76 *Rectinema*, both described as mesophilic and isolated respectively in 2017 from a naphthalene-
77 degrading enrichment culture [9] and in 2020 from a deep terrestrial subsurface [8], only the
78 second ones can grow up to 50°C. Recently in 2020, two not sporulating thermophilic strains
79 (optimum 50-55°C), representative of a novel phylogenetically distinct group of thermophilic
80 spirochetes belonging to the order of *Brevinematales* was described with a proposed family
81 "*Longinemaceae*" and the species name "*Longinema margulisiae*" [14] but with unvalidly
82 published names of genus and family and with the whole genome sequence which remains
83 currently unavailable (accession number CP053071).

84 In this study, we have successfully isolated an anaerobic, thermophilic spirochete, designated
85 strain F1F22T, from the water of a Tunisian hot spring. We describe the detailed morphological,
86 physiological, and genomic characteristics of strain F1F22T and propose a novel species of a
87 new genus for this strain.

88 Thermal water has been collected in a hot spring (36° 48' 59" North, 10° 34' 07" East) in
89 Korbous city, north Tunisia. The temperature, pH, and salinity of
90 the fluid *in situ* was 60 °C, 7.8 and 8 g/l NaCl respectively. Strain F1F22T was isolated using
91 enrichment techniques described by Mechichi *et al.* [15]. A basal medium used for enrichment,
92 isolation, and cultivation was prepared using the anaerobic techniques [16-18]. The basal
93 medium contained (per liter of distilled water): KH2PO4, 0.20 g; NH4Cl, 0.30 g; NaCl, 1 g;
94 KCl, 0.50 g; MgCl2 · 6H2O, 0.40 g; cysteine · HCl, 0.50 g; CaCl2 · 2H2O, 0.15 g; yeast
95 extract, 0.50 g (Difco); trace-element solution [19], 1 ml; and 1 ml of 0.1% (w/v) resazurin. The
pH was adjusted to 7.0 with 10 M KOH solution, then the medium was boiled under a stream of O₂-free N₂ gas and cooled at room temperature; 5 ml aliquots were dispensed into Hungate tubes under N₂/CO₂ (80/20, v/v) and subsequently sterilized by autoclaving at 121°C for 20 min. For enrichment purposes, prior to use, 0.2 ml of 10% (w/v) NaHCO₃, 0.2 ml of 2% (w/v) Na₂S · 9·H₂O and 0.1 ml glucose (1M) were injected from sterile stock solutions into pre-sterilized basal medium [15] and incubated at 50°C. For isolation purposes, positive enrichment cultures were serially diluted into Hungate tubes containing 5 ml of culture medium [16-19] and incubated at 50°C. The culture grown at the higher dilution was used as an inoculum for a novel dilution series. The process was repeated several times until isolates were deemed axenic. Purity was checked by microscopic examination of cultures grown in the basal medium. The last dilution culture showing only one cell morphology was considered for further studies.

Enrichment cultures incubated for three days showed substantial bacterial growth. Several isolates were obtained after three successive serially diluted sub-cultures, and single isolated colonies were selected from different enrichments obtained from water samples of the hot spring. Three were inventoried and finally named F1F22, F1F22p, and F1F22t. Strain F1F22 was chosen for further studies.

Physiological optimal growth conditions were determined in duplicate experiments conducted in basal medium where glucose (20 mM) was added, except when substrates utilization were determined. For testing pH on growth, the culture medium was adjusted to the desired pH using anaerobically prepared stock solutions of NaHCO₃ (10 %) or Na₂CO₃ (2 %). The temperature range for growth was determined using the culture medium adjusted to the optimum growth pH. For studies of NaCl requirements, NaCl was weighed directly into the tubes at concentrations ranging from 0 to 40 g l⁻¹ before dispensing basal medium without NaCl, followed by autoclaving. The tubes were incubated at 55 °C. Growth was measured by inserting
pH was adjusted to 7.0 with 10 M KOH solution, then the medium was boiled under a stream of O₂-free N₂ gas and cooled at room temperature; 5 ml aliquots were dispensed into Hungate tubes under N₂/CO₂ (80/20, v/v) and subsequently sterilized by autoclaving at 121°C for 20 min. For enrichment purposes, prior to use, 0.2 ml of 10% (w/v) NaHCO₃, 0.2 ml of 2% (w/v) Na₂S · 9·H₂O and 0.1 ml glucose (1M) were injected from sterile stock solutions into pre-sterilized basal medium [15] and incubated at 50°C. For isolation purposes, positive enrichment cultures were serially diluted into Hungate tubes containing 5 ml of culture medium [16-19] and incubated at 50°C. The culture grown at the higher dilution was used as an inoculum for a novel dilution series. The process was repeated several times until isolates were deemed axenic. Purity was checked by microscopic examination of cultures grown in the basal medium. The last dilution culture showing only one cell morphology was considered for further studies.

Enrichment cultures incubated for three days showed substantial bacterial growth. Several isolates were obtained after three successive serially diluted sub-cultures, and single isolated colonies were selected from different enrichments obtained from water samples of the hot spring. Three were inventoried and finally named F1F22, F1F22p, and F1F22t. Strain F1F22 was chosen for further studies.

Physiological optimal growth conditions were determined in duplicate experiments conducted in basal medium where glucose (20 mM) was added, except when substrates utilization were determined. For testing pH on growth, the culture medium was adjusted to the desired pH using anaerobically prepared stock solutions of NaHCO₃ (10 %) or Na₂CO₃ (2 %). The temperature range for growth was determined using the culture medium adjusted to the optimum growth pH. For studies of NaCl requirements, NaCl was weighed directly into the tubes at concentrations ranging from 0 to 40 g l⁻¹ before dispensing basal medium without NaCl, followed by autoclaving. The tubes were incubated at 55 °C. Growth was measured by inserting
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tubes directly into a model Cary 50 Scan spectrophotometer (Varian) and measuring the OD580 (nm).

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Substrate-utilization tests were carried out at 55 °C and an initial pH of 7.5 using the basal medium without glucose, as described by Ben Ali Gam *et al.* [20]. Substrates utilization was determined using a chromatograph equipped with a UV Detector (HP 1100, Hewlett Packard, Les-Ulis, France). Separation of metabolites was achieved using C₁₈-Symmetry (4.6x100 mm, 3.5 µm particle size) column (Waters Chromatography, Saint-Quentin-en-Yvelines, France). The tested substrates were added at a final concentration of 20 mM unless indicated : casamino acids (0.1%), tryptone (0.1%), peptone (0.1%), crotonate, H₂/CO₂ (80/20 v/v; ~100 kPa in the headspace) plus acetate (1 mM), betaine (10 mM), arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate (1 mM), acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate (5 mM), ethanol (10 mM), trimethylamine (10 mM), glycerol (5 mM). In addition, the following inorganic substances were tested as electron acceptors in the presence of 0.5 g/l yeast extract: nitrate (10 mM), nitrite (2 mM), sulphate, sulphite (2 mM), thiosulphate (20 mM), and elemental sulphur (1 % w/v).

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Genomic DNA was extracted using a phenol-chloroform-isoamyl alcohol method as previously described by Quéméneur et al. [21]. 16S rRNA genes were amplified by using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1531R (5'-AAGGAGGTGATCCAGCC-3'), then sequenced by GATC-Biotech (Konstanz, Germany). The 16S rRNA sequence was edited in BioEdit [22], a sequence alignment Editor software, and was manually corrected for errors. The 16S rRNA gene sequences were aligned with closest relative sequences from the NCBI nr database using the MUSCLE tool [23] implemented in MEGA7 package [24]. Pairwise evolutionary distances based on 1306 unambiguous nucleotides were computed by the Jukes and Cantor [25] method. Evolutionary analyses were conducted from 24 nucleotide sequences
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tubes directly into a model Cary 50 Scan spectrophotometer (Varian) and measuring the OD580 (nm).

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Substrate-utilization tests were carried out at 55 °C and an initial pH of 7.5 using the basal medium without glucose, as described by Ben Ali Gam *et al.* [20]. Substrates utilization was determined using a chromatograph equipped with a UV Detector (HP 1100, Hewlett Packard, Les-Ulis, France). Separation of metabolites was achieved using C₁₈-Symmetry (4.6x100 mm, 3.5 µm particle size) column (Waters Chromatography, Saint-Quentin-en-Yvelines, France). The tested substrates were added at a final concentration of 20 mM unless indicated : casamino acids (0.1%), tryptone (0.1%), peptone (0.1%), crotonate, H₂/CO₂ (80/20 v/v; ~100 kPa in the headspace) plus acetate (1 mM), betaine (10 mM), arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate (1 mM), acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate (5 mM), ethanol (10 mM), trimethylamine (10 mM), glycerol (5 mM). In addition, the following inorganic substances were tested as electron acceptors in the presence of 0.5 g/l yeast extract: nitrate (10 mM), nitrite (2 mM), sulphate, sulphite (2 mM), thiosulphate (20 mM), and elemental sulphur (1 % w/v).

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Genomic DNA was extracted using a phenol-chloroform-isoamyl alcohol method as previously described by Quéméneur et al. [21]. 16S rRNA genes were amplified by using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1531R (5'-AAGGAGGTGATCCAGCC-3'), then sequenced by GATC-Biotech (Konstanz, Germany). The 16S rRNA sequence was edited in BioEdit [22], a sequence alignment Editor software, and was manually corrected for errors. The 16S rRNA gene sequences were aligned with closest relative sequences from the NCBI nr database using the MUSCLE tool [23] implemented in MEGA7 package [24]. Pairwise evolutionary distances based on 1306 unambiguous nucleotides were computed by the Jukes and Cantor [25] method. Evolutionary analyses were conducted from 24 nucleotide sequences
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using three different methods: maximum-likelihood [26], maximum-parsimony, [27]
and neighbour-joining [28].

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Whole-genome sequencing was performed at the GeT-PlaGe platform (INRAe Toulouse, France) by combining long reads (Oxford Nanopore) and short reads (Illumina MiSeq) technologies, as described by Quéméneur *et al.* [21]. Genome assembly was performed with Unicycler v0.4.6 [29]. The genome sequence was deposited in the NCBI database under the accession number CP073355. Genome annotation was carried out online using bioinformatic tools available on three web platforms: MaGe (Microscope Genome Annotation) [30], Integrated Microbial Genomes [31], and Galaxy protologger [32]. Value of orthologous average nucleotide identity (OrthoANI) and Genome-to-Genome Distance was calculated with Orthologous Average Nucleotide Identity Tool (OAT) software (https://www.ezbiocloud.net/tools/orthoani) [33, 34].

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The fatty acid composition of the strain F1F22T was determined at the Identification Service of DSMZ [35]. The cellular fatty acid analysis was performed on a culture grown in basal medium containing 0.5 g/l (w/v) yeast extract and stopped in the exponential growth phase. Fatty acids were extracted using Miller's method [36] with modifications of Kuykendall *et al.* [37]. Then the cellular fatty acids profile was analysed using the MIDI Microbial Identification system (e.g. Method: TSBA40).

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Cells F1F22T from active cultures observed under phase-contrast microscopy never showed any motility. F1F22T cells were helical revealed the presence of hooked ends and spherical bodies (Fig. 1a) while negatively stained cells showed an helicoidal cell shape and a protoplasmic cylinder (Fig. 1b), the whole being typical of Spirochaetota morphology. The F1F22T cells stained Gram-reaction negative and electron microscopy revealed a Gram negative-type cell
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using three different methods: maximum-likelihood [26], maximum-parsimony, [27]
and neighbour-joining [28].

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Whole-genome sequencing was performed at the GeT-PlaGe platform (INRAe Toulouse, France) by combining long reads (Oxford Nanopore) and short reads (Illumina MiSeq) technologies, as described by Quéméneur *et al.* [21]. Genome assembly was performed with Unicycler v0.4.6 [29]. The genome sequence was deposited in the NCBI database under the accession number CP073355. Genome annotation was carried out online using bioinformatic tools available on three web platforms: MaGe (Microscope Genome Annotation) [30], Integrated Microbial Genomes [31], and Galaxy protologger [32]. Value of orthologous average nucleotide identity (OrthoANI) and Genome-to-Genome Distance was calculated with Orthologous Average Nucleotide Identity Tool (OAT) software (https://www.ezbiocloud.net/tools/orthoani) [33, 34].

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The fatty acid composition of the strain F1F22T was determined at the Identification Service of DSMZ [35]. The cellular fatty acid analysis was performed on a culture grown in basal medium containing 0.5 g/l (w/v) yeast extract and stopped in the exponential growth phase. Fatty acids were extracted using Miller's method [36] with modifications of Kuykendall *et al.* [37]. Then the cellular fatty acids profile was analysed using the MIDI Microbial Identification system (e.g. Method: TSBA40).

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Cells F1F22T from active cultures observed under phase-contrast microscopy never showed any motility. F1F22T cells were helical revealed the presence of hooked ends and spherical bodies (Fig. 1a) while negatively stained cells showed an helicoidal cell shape and a protoplasmic cylinder (Fig. 1b), the whole being typical of Spirochaetota morphology. The F1F22T cells stained Gram-reaction negative and electron microscopy revealed a Gram negative-type cell
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wall structure (Fig. 1c). Electron microscopy revealed also that the strain possessed a
periplasmic space, but periplasmic flagella could not be observed in any case.

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Strain F1F22T grew between 45 and 70 °C with optimal growth at 55 °C. The pH range for growth of the strain was pH 6.5–8.5, with an optimum at pH 7.5. The range of NaCl concentration for growth was 0–1 % (w/v), with the highest growth rate in the culture medium without NaCl. No growth was observed in a yeast extract-free medium. The strain was strictly anaerobic; growth was completely inhibited in the presence of trace quantities of oxygen (0.1% v/v O₂). Strain F1F22T used glucose, sucrose, and lactose with 0.5 g/l yeast extract in the culture medium. However, the following substrates did not support growth: Casamino acids, tryptone, peptone, crotonate, H₂/CO₂ (80:20) plus acetate, betaine, arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate, acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate, ethanol, trimethylamine, glycerol. In addition, the following inorganic substances were not reduced in the presence of 0.5 g/l yeast extract: nitrate, nitrite, sulphate, sulphite, thiosulphate and elemental sulphur (Table 1).

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The DNA G+C content of F1F22T is 43.15 mol%. Fatty acid methyl ester analysis (Table 2) showed that the strain F1F22T contained C₁₆:₀ (40.2%), iso-C₁₆:₀ (30.2%) and C₁₆:₀ DMA (14.5%) as the major fatty acids. Minor fatty acids upper than 1% were C₁₆:₁ DMA (4.2%), C₁₆:₀ 3-OH (3.7%), C₁₈:₀ (1.8%), anteiso-C₁₇:₀ (1.7%) and C₁₄:₀ (1.2%).

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The species *Brevinema andersonii*† affiliated to the family *Brevinemataceae*, order *Brevinematales*, class *Spirochaetia*, phylum *Spirochaetota* [38] was identified as the closest type species of strain F1F22T with 83% 16S rRNA gene sequence similarity (Fig. 2). The phylogenetic analysis based on its 16S rRNA gene sequence (Fig. 2 and Fig. S1) and the whole-proteome-based phylogeny (Fig. S2) show that strain F1F22T should be affiliated to a new genus and novel family belonging to the order *Brevinematales*, class *Spirochaetia*, phylum

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wall structure (Fig. 1c). Electron microscopy revealed also that the strain possessed a
periplasmic space, but periplasmic flagella could not be observed in any case.

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Strain F1F22<sup>T</sup> grew between 45 and 70 °C with optimal growth at 55 °C. The pH range for growth of the strain was pH 6.5–8.5, with an optimum at pH 7.5. The range of NaCl concentration for growth was 0–1 % (w/v), with the highest growth rate in the culture medium without NaCl. No growth was observed in a yeast extract-free medium. The strain was strictly anaerobic; growth was completely inhibited in the presence of trace quantities of oxygen (0.1% v/v O<sub>2</sub>). Strain F1F22<sup>T</sup> used glucose, sucrose, and lactose with 0.5 g/l yeast extract in the culture medium. However, the following substrates did not support growth: Casamino acids, tryptone, peptone, crotonate, H<sub>2</sub>/CO<sub>2</sub> (80:20) plus acetate, betaine, arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate, acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate, ethanol, trimethylamine, glycerol. In addition, the following inorganic substances were not reduced in the presence of 0.5 g/l yeast extract: nitrate, nitrite, sulphate, sulphite, thiosulphate and elemental sulphur (Table 1).

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The DNA G+C content of F1F22<sup>T</sup> is 43.15 mol%. Fatty acid methyl ester analysis (Table 2) showed that the strain F1F22<sup>T</sup> contained C<sub>16:0</sub> (40.2%), iso-C<sub>16:0</sub> (30.2%) and C<sub>16:0</sub> DMA (14.5%) as the major fatty acids. Minor fatty acids upper than 1% were C<sub>16:1</sub> DMA (4.2%), C<sub>16:0</sub> 3-OH (3.7%), C<sub>18:0</sub> (1.8%), anteiso-C<sub>17:0</sub> (1.7%) and C<sub>14:0</sub> (1.2%).

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The species *Brevinema andersonii*<sup>T</sup> affiliated to the family *Brevinemataceae*, order *Brevinematales*, class *Spirochaetia*, phylum *Spirochaetota* [38] was identified as the closest type species of strain F1F22<sup>T</sup> with 83% 16S rRNA gene sequence similarity (Fig. 2). The phylogenetic analysis based on its 16S rRNA gene sequence (Fig. 2 and Fig. S1) and the whole-proteome-based phylogeny (Fig. S2) show that strain F1F22<sup>T</sup> should be affiliated to a new genus and novel family belonging to the order *Brevinematales*, class *Spirochaetia*, phylum
191 *Spirochaetota*. Moreover, POCP values (obtained from Protologger) was 15.64% between
192 strain F1F22<sup>T</sup> and *Spirochaeta thermophila* GCF_000184345.1, and 13.76% with *Spirochaeta
193 africana* GCF_000242595.2 and 9.61% with *Brevinema andersonii* GCF_900112165.1, though
194 far below the threshold of 50% delineating genera [39]. The proposed name for the new genus
195 is *Thermospira* and for the novel family is *Thermospiraceae* that are both described below.

196 The complete genome (one single contig) of strain F1F22<sup>T</sup> consists of a circular chromosome
197 of 2.515.453 bp, with 43.15 % G+C content (Table 3). A circular view of the genome is shown
198 in Fig. S3. The size of the strain F1F22<sup>T</sup> genome is larger than that of *Brevinema andersonii*<sup>T</sup>
199 (ATCC 43811), 1.501.400 bp, smaller than that of *Treponema caldarium*<sup>T</sup> (DSM 7334),
200 3.239.340 bp, and in the range of *Spirochaeta thermophila*<sup>T</sup> (DSM 6578), 2.560.222 bp,
201 genome. The G+C content of F1F22<sup>T</sup> genome is close the one of *Treponema caldarium*<sup>T</sup>, but
202 higher than that of *Brevinema andersonii*<sup>T</sup> and lower than that of *Spirochaeta thermophila*<sup>T</sup>
203 (Table 3). The ANI and dDDH values between F1F22<sup>T</sup> and *Treponema caldarium*<sup>T</sup>, *Brevinema
204 andersonii*<sup>T</sup> and *Spirochaeta thermophila*<sup>T</sup> were respectively between 63.26% and 63.52% and
205 between 20% and 25% (Table 4), i.e., far below the species boundaries of 95~96 and 70 %,
206 respectively, recommended by Chun *et al*. [40].

207 The genome contains two copies of a 16S-23S-5S rRNA operon, 46 tRNA genes 2672 protein-
208 coding genes (CDS) (Table 3). A group of genes encoding a bacterial flagellar machinery and
209 chemotaxis was identified, consistent with the typical spirochete morphological characteristics.
210 In agreement with the culture-based conclusions on the metabolism, analysis of the potential
211 metabolic pathways showed that strain F1F22<sup>T</sup> metabolism is based on sugars utilization.
212 Searches against the carbohydrates-active enzymes (CAZYmes) database [41] identified 26
213 CAZymes including 6 alpha-amylases genes encoding both cytoplasmic and secreted extra-
214 cellular enzymes. This abundance and diversity of alpha-amylases suggest that the F1F22<sup>T</sup>
191 *Spirochaetota*. Moreover, POCP values (obtained from Protologger) was 15.64% between
192 strain F1F22<sup>T</sup> and *Spirochaeta thermophila* GCF_000184345.1, and 13.76% with *Spirochaeta
193 africana* GCF_000242595.2 and 9.61% with *Brevinema andersonii* GCF_900112165.1, though
194 far below the threshold of 50% delineating genera [39]. The proposed name for the new genus
195 is *Thermospira* and for the novel family is *Thermospiraceae* that are both described below.

196 The complete genome (one single contig) of strain F1F22<sup>T</sup> consists of a circular chromosome
197 of 2.515.453 bp, with 43.15 % G+C content (Table 3). A circular view of the genome is shown
198 in Fig. S3. The size of the strain F1F22<sup>T</sup> genome is larger than that of *Brevinema andersonii*<sup>T</sup>
199 (ATCC 43811), 1.501.400 bp, smaller than that of *Treponema caldarium*<sup>T</sup> (DSM 7334),
200 3.239.340 bp, and in the range of *Spirochaeta thermophila*<sup>T</sup> (DSM 6578), 2.560.222 bp,
201 genome. The G+C content of F1F22<sup>T</sup> genome is close the one of *Treponema caldarium*<sup>T</sup>, but
202 higher than that of *Brevinema andersonii*<sup>T</sup> and lower than that of *Spirochaeta thermophila*<sup>T</sup>
203 (Table 3). The ANI and dDDH values between F1F22<sup>T</sup> and *Treponema caldarium*<sup>T</sup>, *Brevinema
204 andersonii*<sup>T</sup> and *Spirochaeta thermophila*<sup>T</sup> were respectively between 63.26% and 63.52% and
205 between 20% and 25% (Table 4), i.e., far below the species boundaries of 95~96 and 70 %,
206 respectively, recommended by Chun *et al*. [40].

207 The genome contains two copies of a 16S-23S-5S rRNA operon, 46 tRNA genes 2672 protein-
208 coding genes (CDS) (Table 3). A group of genes encoding a bacterial flagellar machinery and
209 chemotaxis was identified, consistent with the typical spirochete morphological characteristics.
210 In agreement with the culture-based conclusions on the metabolism, analysis of the potential
211 metabolic pathways showed that strain F1F22<sup>T</sup> metabolism is based on sugars utilization.
212 Searches against the carbohydrates-active enzymes (CAZYmes) database [41] identified 26
213 CAZymes including 6 alpha-amylases genes encoding both cytoplasmic and secreted extra-
214 cellular enzymes. This abundance and diversity of alpha-amylases suggest that the F1F22<sup>T</sup>
strain is mostly used on starch or similar polysaccharides (glycogen, dextrin). Besides, genes
for converting of pyruvate to lactate (lactate dehydrogenase) or acetate (phosphate acetyl
transferase and acetate kinase) were found, representing main fermentation pathways in F1F22T
strain grown on sugars. Genes encoding typical membrane electron transporter (cytochromes,
quinones...) involved in aerobic or anaerobic respirations were not detected, while genes
encoding the complete Embden-Meyerhof pathway (glycolysis) are present in F1F22T genome.
The presence of genes for gluconeogenesis, and the pathways for glycogen and trehalose
biosynthesis and degradation, suggest that strain F1F22T utilizes these compounds either for
energy storage or as osmoprotectants or a protection against desiccation. A partial pentose-
phosphate pathway is also detected. The tricarboxylic acids cycle (TCA) pathway is atypical,
resembling *Helicobacter pylori* TCA but missing the genes for the acetoacetate CoA-
transferase, the fumarate reductase, and the malate dehydrogenase [42]. Like in *Helicobacter
pylori*, F1F22T possess an operon encoding a 2-oxo-glutarate oxidoreductase (OOR; EC
1.2.7.3), instead of the classical α-ketoglutarate dehydrogenase [43]. This OOR uses ferredoxin
(Fd) as electron acceptor cofactor, instead of NAD(P) for the dehydrogenase. We hypothesize
that the oxidative branch of this TCA pathway is crucial in energy conservation in F1F22T
strain. Indeed, in anaerobic bacteria, ferredoxin, which has a very low redox potential (-420 to
-500 mV) and thus is a very potent reductant for electron transport chains, can be utilized to
power several processes, including hydrogen evolution and a transmembrane ion gradient [44].
The F1F22T genome encodes a RnF complex, a respiratory enzyme that catalyzes the oxidation
of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this
reaction is used to generate a transmembrane ion gradient (either Na+ or H+). Moreover, an
operon for a cytoplasmic [FeFe] hydrogenase (*hndACD*) was found. Hnd is a flavin-based
electron-bifurcating (FBEB) hydrogenase, coupling an exergonic redox reaction to an
endergonic one allowing energy conservation in anaerobic microorganisms. Hnd hydrogenase
catalyze the simultaneous exergonic oxidation of ferredoxin (Fd) and the endergonic oxidation
strain is mostly used on starch or similar polysaccharides (glycogen, dextrin). Besides, genes
for converting of pyruvate to lactate (lactate dehydrogenase) or acetate (phosphate acetyl
transferase and acetate kinase) were found, representing main fermentation pathways in F1F22T
strain grown on sugars. Genes encoding typical membrane electron transporter (cytochromes,
quinones...) involved in aerobic or anaerobic respirations were not detected, while genes
encoding the complete Embden-Meyerhof pathway (glycolysis) are present in F1F22T genome.
The presence of genes for gluconeogenesis, and the pathways for glycogen and trehalose
biosynthesis and degradation, suggest that strain F1F22T utilizes these compounds either for
energy storage or as osmoprotectants or a protection against desiccation. A partial pentose-
phosphate pathway is also detected. The tricarboxylic acids cycle (TCA) pathway is atypical,
resembling *Helicobacter pylori* TCA but missing the genes for the acetoacetate CoA-
transferase, the fumarate reductase, and the malate dehydrogenase [42]. Like in *Helicobacter
pylori*, F1F22T possess an operon encoding a 2-oxo-glutarate oxidoreductase (OOR; EC
1.2.7.3), instead of the classical α-ketoglutarate dehydrogenase [43]. This OOR uses ferredoxin
(Fd) as electron acceptor cofactor, instead of NAD(P) for the dehydrogenase. We hypothesize
that the oxidative branch of this TCA pathway is crucial in energy conservation in F1F22T
strain. Indeed, in anaerobic bacteria, ferredoxin, which has a very low redox potential (-420 to
-500 mV) and thus is a very potent reductant for electron transport chains, can be utilized to
power several processes, including hydrogen evolution and a transmembrane ion gradient [44].
The F1F22T genome encodes a RnF complex, a respiratory enzyme that catalyzes the oxidation
of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this
reaction is used to generate a transmembrane ion gradient (either Na+ or H+). Moreover, an
operon for a cytoplasmic [FeFe] hydrogenase (*hndACD*) was found. Hnd is a flavin-based
electron-bifurcating (FBEB) hydrogenase, coupling an exergonic redox reaction to an
endergonic one allowing energy conservation in anaerobic microorganisms. Hnd hydrogenase
catalyze the simultaneous exergonic oxidation of ferredoxin (Fd) and the endergonic oxidation
of NADH to produce H₂ (electron confurcation) [45] or the reverse reaction in which the
exergonic reduction of NAD+ is coupled to the endergonic reduction of the Fd in the presence
of dihydrogen (electron bifurcation). The genome of strain NST lacked. The genome of strain
F1F22T does not encode typical F1F0-type ATP synthase that uses transmembrane proton
gradient for ATP production; only genes for a V-type ATPase were found. V-ATPases are
typically used in anaerobic bacteria to generate a transmembrane ion gradient at the expense of
ATP. However, it has been demonstrated that the reverse reaction, c.a., synthesis of ATP by V-
type ATPase is possible, although energetically less favorable, depending on the
electrochemical proton gradient (ΔμH) [46]. Therefore, we assume strain F1F22T uses the
proton-motive force generated by the RnF complex to produce ATP by the V-Type ATPase,
contributing to energy conservation in addition to the substrate-level phosphorylation for ATP
synthesis.

As a strict anaerobic bacterium, the strain F1F22T genome also encodes a protection system to
oxidative stress: a superoxide reductase an enzyme that catalyzes the conversion of highly
reactive and toxic superoxide (O₂⁻) into less toxic hydrogen peroxide which in turn is reduced
to water by rubrerythrin.

The morphology of strain F1F22T cells is typical of spirochete species. For example, strain
F1F22T cells had an outer envelope, a protoplasmic cylinder, and hook-shaped ends. Similar
cell structures have also been found for members of the genus *Leptospira* [47,48] and
*Brevinema* [38], both exclusively mesophilic genera. Moreover, strain F1F22T cells showed the
formation of spherical bodies in late-exponential phase cultures, a characteristic observed in
members of three different genera *Exilispira*, *Salinispira* and *Treponema*, where two of them
*Exilispira* and *Treponema* included thermophilic species [13, 49, 50, 51]. Contrarily to its
closest type species *Brevinema andersonii* (strain CT11616T), possessing two wave-shaped cell
structures of irregular length [38], strain F1F22T harbors more than two wave-shaped cell
of NADH to produce H₂ (electron confurcation) [45] or the reverse reaction in which the
exergonic reduction of NAD+ is coupled to the endergonic reduction of the Fd in the presence
of dihydrogen (electron bifurcation). The genome of strain NST lacked. The genome of strain
F1F22T does not encode typical F1F0-type ATP synthase that uses transmembrane proton
gradient for ATP production; only genes for a V-type ATPase were found. V-ATPases are
typically used in anaerobic bacteria to generate a transmembrane ion gradient at the expense of
ATP. However, it has been demonstrated that the reverse reaction, c.a., synthesis of ATP by V-
type ATPase is possible, although energetically less favorable, depending on the
electrochemical proton gradient (ΔμH) [46]. Therefore, we assume strain F1F22T uses the
proton-motive force generated by the RnF complex to produce ATP by the V-Type ATPase,
contributing to energy conservation in addition to the substrate-level phosphorylation for ATP
synthesis.

As a strict anaerobic bacterium, the strain F1F22<sup>T</sup> genome also encodes a protection system to
oxidative stress: a superoxide reductase an enzyme that catalyzes the conversion of highly
reactive and toxic superoxide (O<sub>2</sub><sup>-</sup>) into less toxic hydrogen peroxide which in turn is reduced
to water by rubrerythrin.

The morphology of strain F1F22<sup>T</sup> cells is typical of spirochete species. For example, strain
F1F22<sup>T</sup> cells had an outer envelope, a protoplasmic cylinder, and hook-shaped ends. Similar
cell structures have also been found for members of the genus *Leptospira* [47,48] and
*Brevinema* [38], both exclusively mesophilic genera. Moreover, strain F1F22<sup>T</sup> cells showed the
formation of spherical bodies in late-exponential phase cultures, a characteristic observed in
members of three different genera *Exilispira*, *Salinispira* and *Treponema*, where two of them
*Exilispira* and *Treponema* included thermophilic species [13, 49, 50, 51]. Contrarily to its
closest type species *Brevinema andersonii* (strain CT11616<sup>T</sup>), possessing two wave-shaped cell
structures of irregular length [38], strain F1F22<sup>T</sup> harbors more than two wave-shaped cell
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structures of regular length. Additionally, *Brevinema andersonii* type strain was mesophilic and pathogenic for humans, contrary to strain F1F22<sup>T</sup>. However, contrasting with most spirochetes, cells of strain F1F22<sup>T</sup> are always non-motile and never exhibit any flexing, rotational or translational movements or periplasmic flagella.

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Strain F1F22<sup>T</sup> was isolated from a water sample of a Tunisian hot spring and we showed it is thermophilic like only three species among the phylum *Spirochaetota: Spirochaeta thermophila* (strain Z-1203<sup>T</sup>), *Treponema caldarium* (strain H1<sup>T</sup>), and *Exilispira thermophila* (strain RASEN<sup>T</sup>). All strains are non-pathogenic for humans and phylogenetically distant to strain F1F22<sup>T</sup> (< 79%). These three thermophilic strains were described as helical, highly motile, and with an outer membrane enclosing a periplasmic flagellum, at the difference of strain F1F22<sup>T</sup>.

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Moreover, various physiological features of strain F1F22<sup>T</sup> differ from those of other members of the phylum *Spirochaetota*, including substrate utilization, optimum growth temperature, and NaCl growth requirement (Table 1). Regarding the lifestyle of strain F1F22<sup>T</sup>, we consider that the strain is derived from a free-living microbial population in the freshwater of a hot spring.

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The water sample for isolation contained no animal individuals and no specific physical association with animals. Strain F1F22<sup>T</sup> also differs from the closest relatives in many physiological features: first, the range of substrate utilized by strain F1F22<sup>T</sup> for growth is limited to glucose and maltose with a slight growth when sucrose was utilized. In contrast, *Exilispira thermophila* (strain RASEN<sup>T</sup>) grew only with yeast extract while *Treponema caldarium* (strain H1<sup>T</sup>) and *Spirochaeta thermophila* (strain Z-1203<sup>T</sup>) utilized a larger range of sugar including glucose, sucrose, arabinose, xylose and cellobiose (although substrate utilization patterns have not been determined for the species belonging to the genus *Brevinema*).

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In addition, the range of temperature of growth exhibited by strain F1F22<sup>T</sup> is rare amongst members of the phylum *Spirochaetota*, starting not lower than 45°C and up to 70°C. Indeed, both strains (RASEN<sup>T</sup> and H1<sup>T</sup>) grow only up to 60°C (not 70°C) but also tolerate lower

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structures of regular length. Additionally, *Brevinema andersonii* type strain was mesophilic and pathogenic for humans, contrary to strain F1F22<sup>T</sup>. However, contrasting with most spirochetes, cells of strain F1F22<sup>T</sup> are always non-motile and never exhibit any flexing, rotational or translational movements or periplasmic flagella.

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270

Strain F1F22<sup>T</sup> was isolated from a water sample of a Tunisian hot spring and we showed it is thermophilic like only three species among the phylum *Spirochaetota: Spirochaeta thermophila* (strain Z-1203<sup>T</sup>), *Treponema caldarium* (strain H1<sup>T</sup>), and *Exilispira thermophila* (strain RASEN<sup>T</sup>). All strains are non-pathogenic for humans and phylogenetically distant to strain F1F22<sup>T</sup> (< 79%). These three thermophilic strains were described as helical, highly motile, and with an outer membrane enclosing a periplasmic flagellum, at the difference of strain F1F22<sup>T</sup>.

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Moreover, various physiological features of strain F1F22<sup>T</sup> differ from those of other members of the phylum *Spirochaetota*, including substrate utilization, optimum growth temperature, and NaCl growth requirement (Table 1). Regarding the lifestyle of strain F1F22<sup>T</sup>, we consider that the strain is derived from a free-living microbial population in the freshwater of a hot spring.

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The water sample for isolation contained no animal individuals and no specific physical association with animals. Strain F1F22<sup>T</sup> also differs from the closest relatives in many physiological features: first, the range of substrate utilized by strain F1F22<sup>T</sup> for growth is limited to glucose and maltose with a slight growth when sucrose was utilized. In contrast, *Exilispira thermophila* (strain RASEN<sup>T</sup>) grew only with yeast extract while *Treponema caldarium* (strain H1<sup>T</sup>) and *Spirochaeta thermophila* (strain Z-1203<sup>T</sup>) utilized a larger range of sugar including glucose, sucrose, arabinose, xylose and cellobiose (although substrate utilization patterns have not been determined for the species belonging to the genus *Brevinema*).

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In addition, the range of temperature of growth exhibited by strain F1F22<sup>T</sup> is rare amongst members of the phylum *Spirochaetota*, starting not lower than 45°C and up to 70°C. Indeed, both strains (RASEN<sup>T</sup> and H1<sup>T</sup>) grow only up to 60°C (not 70°C) but also tolerate lower

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temperatures than strain F1F22<sup>T</sup> as they can grow at 25°C and 37°C, respectively. Only strain F1F22<sup>T</sup> can grow up to 73°C, but with an optimum higher than strain F1F22<sup>T</sup>, at 66-68°C. Moreover, inside the phylum *Spirochaetota*, the NaCl requirement of *Thermospira aquatica* (strain F1F22<sup>T</sup>) differs from the three other validly named type species of thermophilic species (strains RASEN<sup>T</sup>, Z-1203<sup>T</sup> and H1<sup>T</sup>). NaCl is not required for strain F1F22<sup>T</sup>, and this is shared partly by *Exilispira thermophila* strain RASEN<sup>T</sup> but only when it grows under optimal conditions. Contrarily, the other two thermophilic species (strains H1<sup>T</sup> and Z-1203<sup>T</sup>), required less than 1.5% and 2.5-3% of NaCl respectively.

Regarding the G+C% content, strain F1F22<sup>T</sup> (43.15% mol) differs markedly from the thermophilic species *Exilispira thermophila* strain RASEN<sup>T</sup> (27%) and *Spirochaeta thermophila* strain H1<sup>T</sup> (60.92%), but it was closer to the other thermophilic *Treponema caldarium* strain Z-1203<sup>T</sup> (45.58%).

In addition to the physiological and biochemical characteristics, the phylogenetic analysis concluded that strain F1F22<sup>T</sup> is distinct from other members of the phylum *Spirochaetota*. Indeed, the 16S rRNA gene sequence-based phylogenetic analysis showed that strain F1F22<sup>T</sup> is only distantly related to other members of the phylum *Spirochaetota* (Fig. 2) and should represent a new genus of a novel family. This assumption is further supported by genome-based analyses (genomic indexes and phylogenomic trees) (Fig. 2 and Fig. S2).

Based on its overall phylogenetic, genomic, morphologic, physiologic, and metabolic features, distinct from any previously described species of the phylum *Spirochaetota*, we propose strain F1F22<sup>T</sup> represents the first species of a novel family, namely *Thermospiraceae* of a new genus, namely *Thermospira aquatica* gen. nov. sp. nov.

313
temperatures than strain F1F22<sup>T</sup> as they can grow at 25°C and 37°C, respectively. Only strain F1F22<sup>T</sup> can grow up to 73°C, but with an optimum higher than strain F1F22<sup>T</sup>, at 66-68°C. Moreover, inside the phylum *Spirochaetota*, the NaCl requirement of *Thermospira aquatica* (strain F1F22<sup>T</sup>) differs from the three other validly named type species of thermophilic species (strains RASEN<sup>T</sup>, Z-1203<sup>T</sup> and H1<sup>T</sup>). NaCl is not required for strain F1F22<sup>T</sup>, and this is shared partly by *Exilispira thermophila* strain RASEN<sup>T</sup> but only when it grows under optimal conditions. Contrarily, the other two thermophilic species (strains H1<sup>T</sup> and Z-1203<sup>T</sup>), required less than 1.5% and 2.5-3% of NaCl respectively.

Regarding the G+C% content, strain F1F22<sup>T</sup> (43.15% mol) differs markedly from the thermophilic species *Exilispira thermophila* strain RASEN<sup>T</sup> (27%) and *Spirochaeta thermophila* strain H1<sup>T</sup> (60.92%), but it was closer to the other thermophilic *Treponema caldarium* strain Z-1203<sup>T</sup> (45.58%).

In addition to the physiological and biochemical characteristics, the phylogenetic analysis concluded that strain F1F22<sup>T</sup> is distinct from other members of the phylum *Spirochaetota*. Indeed, the 16S rRNA gene sequence-based phylogenetic analysis showed that strain F1F22<sup>T</sup> is only distantly related to other members of the phylum *Spirochaetota* (Fig. 2) and should represent a new genus of a novel family. This assumption is further supported by genome-based analyses (genomic indexes and phylogenomic trees) (Fig. 2 and Fig. S2).

Based on its overall phylogenetic, genomic, morphologic, physiologic, and metabolic features, distinct from any previously described species of the phylum *Spirochaetota*, we propose strain F1F22<sup>T</sup> represents the first species of a novel family, namely *Thermospiraceae* of a new genus, namely *Thermospira aquatica* gen. nov. sp. nov.

313
314 **Description of Thermospiraceae fam. nov.**

315 *Thermospiraceae* (Ther.mo spi'ra.ce'ae N.L. fem. n. *Thermospira* type genus of the family; L.
316 suff. -aceae ending to denote a family; N.L. fem. pl. n. *Thermospiraceae* the family of the genus
317 *Thermospira*).

318 The family is defined by phylogenetic analysis based on 16S rRNA gene sequences obtained
319 currently from not sporulating thermophilic cultured strains, which come from hot
320 environments (spring water and deep biosphere).

321 The type genus is *Thermospira*.

322

323 **Description of *Thermospira* gen. nov.**

324 *Thermospira* (Ther.mo spi'ra. Gr. masc. adj. thermos, hot; L. fem. n. *spira* coil, spire; N.L. fem.
325 n. *Thermospira*, a hot spiral)

326 Gram-stain negative. Strictly anaerobic and thermophilic organisms. Cells are helical with a
327 cell structure composed of an outer membrane without periplasmic flagella.
328 Chemoorganoheterotrophic. Sugars are the preferred growth substrates. The G+C content of
329 the genomic DNA is 43.15 mol%. Its phylogenetic position is in a deeply branched lineage of
330 the phylum *Spirochaetota*. The type species is *Thermospira aquatica* and the genus description
331 is based on the type species, currently the only validly published name of species ascribed to
332 this genus.

333 **Description of *Thermospira aquatica* sp. nov.**

334 *Thermospira aquatica* (a.qua'ti.ca. L. fem. adj. aquatica aquatic).
314 **Description of Thermospiraceae fam. nov.**

315 *Thermospiraceae* (Ther.mo spi'ra.ce'ae N.L. fem. n. *Thermospira* type genus of the family; L.
316 suff. -aceae ending to denote a family; N.L. fem. pl. n. *Thermospiraceae* the family of the genus
317 *Thermospira*).

318 The family is defined by phylogenetic analysis based on 16S rRNA gene sequences obtained
319 currently from not sporulating thermophilic cultured strains, which come from hot
320 environments (spring water and deep biosphere).

321 The type genus is *Thermospira*.

322

323 **Description of *Thermospira* gen. nov.**

324 *Thermospira* (Ther.mo spi'ra. Gr. masc. adj. thermos, hot; L. fem. n. spira coil, spire; N.L. fem.
325 n. *Thermospira*, a hot spiral)

326 Gram-stain negative. Strictly anaerobic and thermophilic organisms. Cells are helical with a
327 cell structure composed of an outer membrane without periplasmic flagella.
328 Chemoorganoheterotrophic. Sugars are the preferred growth substrates. The G+C content of
329 the genomic DNA is 43.15 mol%. Its phylogenetic position is in a deeply branched lineage of
330 the phylum *Spirochaetota*. The type species is *Thermospira aquatica* and the genus description
331 is based on the type species, currently the only validly published name of species ascribed to
332 this genus.

333 **Description of *Thermospira aquatica* sp. nov.**

334 *Thermospira aquatica* (a.qua'ti.ca. L. fem. adj. aquatica aquatic).
Displays the following characteristics in addition to those listed in the genus description. Strain F1F22T cells are helical and 0.5×10⁻³² µm in size, often with hooked ends. Cells are non-motile. Growth occurs between 45 and 70 °C, with optimum growth at 55 °C. The pH range for strain growth was estimated at pH 6.5–8.5, with optimum growth at pH 7.5. The range of NaCl concentration for growth was 0–1% (w/v) but the optimum growth was without NaCl added in the culture medium. Uses glucose, maltose, and sucrose. Yeast extract is necessary for growth.

Growth was not observed on casamino acids, tryptone, peptone, crotonate, H₂/CO₂ (80:20) plus acetate, betaine, arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate, acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate, ethanol, trimethylamine and glycerol. The major cellular fatty acids were C₁₆:₀, iso-C₁₆:₀ and C₁₆:₀ DMA; minor fatty acids were C₁₆:₁ DMA, C₁₆:₀ 3-OH, C₁₈:₀, anteiso-C₁₇:₀ and C₁₄:₀.

The type strain is strain F1F22T (=JCM 31314T =DSM 101182T), isolated from hot spring water in the North of Tunisia.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F1F22T is KT767982 and the one for the whole genome sequence is CP073355.

## Funding information

This work received no specific grant from any funding agency.

## Acknowledgments

The authors acknowledge the MIO laboratory, Aix-Marseille University, where most experiments were performed, according to ISO9001-2019 norms.

## Conflicts of interest

The authors declare that there are no conflicts of interest.
Displays the following characteristics in addition to those listed in the genus description. Strain F1F22T cells are helical and 0.5×10⁻³² µm in size, often with hooked ends. Cells are non-motile. Growth occurs between 45 and 70 °C, with optimum growth at 55 °C. The pH range for strain growth was estimated at pH 6.5–8.5, with optimum growth at pH 7.5. The range of NaCl concentration for growth was 0–1% (w/v) but the optimum growth was without NaCl added in the culture medium. Uses glucose, maltose, and sucrose. Yeast extract is necessary for growth.

Growth was not observed on casamino acids, tryptone, peptone, crotonate, H₂/CO₂ (80:20) plus acetate, betaine, arabinose, fructose, galactose, xylose, cellobiose, lactate, formate plus acetate, acetate, propionate, butyrate, malate, fumarate, succinate, pyruvate, benzoate, ethanol, trimethylamine and glycerol. The major cellular fatty acids were C₁₆:₀, iso-C₁₆:₀ and C₁₆:₀ DMA; minor fatty acids were C₁₆:₁ DMA, C₁₆:₀ 3-OH, C₁₈:₀, anteiso-C₁₇:₀ and C₁₄:₀.

The type strain is strain F1F22T (=JCM 31314T =DSM 101182T), isolated from hot spring water in the North of Tunisia.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F1F22T is KT767982 and the one for the whole genome sequence is CP073355.

## Funding information

This work received no specific grant from any funding agency.

## Acknowledgments

The authors acknowledge the MIO laboratory, Aix-Marseille University, where most experiments were performed, according to ISO9001-2019 norms.

## Conflicts of interest

The authors declare that there are no conflicts of interest.
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*brevicauda*) and the white-footed mouse (*Peromyscus leucopus*). *Int J Syst Bacteriol* 1995;

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[41] Drula E, Garron ML, Dogan S, Lombard V, Henrissat B et al. The carbohydrate-active enzyme database: functions and literature. *Nucleic Acids Research* 2022; 50:571–577.

466

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[42] Pitson SM, Mendz GL, Srinivasan L, Hazell SL. The tricarboxylic acid cycle of Helicobacter pylori. *Euro J Biochem* 1999; 260:258-267.

468

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**Table 1.** Comparison of the morphological and physiological properties of strain F1F22T and other thermophilic spirochete species. Taxa: 1, F1F22T (data from this study); 2, *Exilispira thermophila* [13]; 3, *Treponema caldarium* [12,49]; 4, *Spirochaeta thermophila* [11]; +, Positive; -, Negative; NR, not reported.

<table><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>Isolation source</td><td>Hot spring</td><td>Deep sea</td><td>Hot spring</td><td>Thermal site</td></tr><tr><td>Cell morphology</td><td>Helical</td><td>Helical</td><td>Helical</td><td>Helical</td></tr><tr><td>Cell size (µm)</td><td>0.5x10-32</td><td>0.23-0.28x15-27</td><td>0.2-0.3x15-45</td><td>0.2-0.25x16-50</td></tr><tr><td>Temperature Range (°C)</td><td>45-70</td><td>37-60</td><td>25-65</td><td>40-73</td></tr><tr><td>Optimum Temperature (°C)</td><td>55</td><td>50</td><td>48-52</td><td>66-68</td></tr><tr><td>pH range</td><td>6.5-8.5</td><td>6-7.5</td><td>5.8-8.5</td><td>5.9-7.7</td></tr><tr><td>Optimum pH</td><td>7.7</td><td>7.0</td><td>7.2-7.5</td><td>7.5</td></tr><tr><td>NaCl range (%)</td><td>0-1</td><td>2.5-3.5</td><td>0-0.4</td><td>0.5-4.5</td></tr><tr><td>Optimum NaCl (%)</td><td>0</td><td>2.5-3</td><td>NR</td><td>1.5</td></tr><tr><td colspan="5">Substrate utilization (with 0.5 g/l yeast extract)</td></tr><tr><td>Glucose</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Sucrose</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Arabinose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Fructose</td><td>-</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Galactose</td><td>-</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Xylose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Maltose</td><td>+</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Cellobiose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Butyrate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Nitrate</td><td>-</td><td>NR</td><td>NR</td><td>-</td></tr><tr><td colspan="5">Inorganic substance reduction (with 0.5 g/l yeast extract)</td></tr><tr><td>Sulphate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Sulphite</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Thiosulphate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Elemental sulfur</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>DNA G+C content (mol %)</td><td>43.15</td><td>27</td><td>45.58</td><td>60.92</td></tr></tbody></table>

500

501

502
**Table 1.** Comparison of the morphological and physiological properties of strain F1F22T and other thermophilic spirochete species. Taxa: 1, F1F22T (data from this study); 2, *Exilispira thermophila* [13]; 3, *Treponema caldarium* [12,49]; 4, *Spirochaeta thermophila* [11]; +, Positive; -, Negative; NR, not reported.

<table><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>Isolation source</td><td>Hot spring</td><td>Deep sea</td><td>Hot spring</td><td>Thermal site</td></tr><tr><td>Cell morphology</td><td>Helical</td><td>Helical</td><td>Helical</td><td>Helical</td></tr><tr><td>Cell size (µm)</td><td>0.5x10-32</td><td>0.23-0.28x15-27</td><td>0.2-0.3x15-45</td><td>0.2-0.25x16-50</td></tr><tr><td>Temperature Range (°C)</td><td>45-70</td><td>37-60</td><td>25-65</td><td>40-73</td></tr><tr><td>Optimum Temperature (°C)</td><td>55</td><td>50</td><td>48-52</td><td>66-68</td></tr><tr><td>pH range</td><td>6.5-8.5</td><td>6-7.5</td><td>5.8-8.5</td><td>5.9-7.7</td></tr><tr><td>Optimum pH</td><td>7.7</td><td>7.0</td><td>7.2-7.5</td><td>7.5</td></tr><tr><td>NaCl range (%)</td><td>0-1</td><td>2.5-3.5</td><td>0-0.4</td><td>0.5-4.5</td></tr><tr><td>Optimum NaCl (%)</td><td>0</td><td>2.5-3</td><td>NR</td><td>1.5</td></tr><tr><td colspan="5">Substrate utilization (with 0.5 g/l yeast extract)</td></tr><tr><td>Glucose</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Sucrose</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Arabinose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Fructose</td><td>-</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Galactose</td><td>-</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Xylose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Maltose</td><td>+</td><td>-</td><td>+</td><td>NR</td></tr><tr><td>Cellobiose</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Butyrate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Nitrate</td><td>-</td><td>NR</td><td>NR</td><td>-</td></tr><tr><td colspan="5">Inorganic substance reduction (with 0.5 g/l yeast extract)</td></tr><tr><td>Sulphate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Sulphite</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Thiosulphate</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>Elemental sulfur</td><td>-</td><td>-</td><td>NR</td><td>-</td></tr><tr><td>DNA G+C content (mol %)</td><td>43.15</td><td>27</td><td>45.58</td><td>60.92</td></tr></tbody></table>

500

501

502
<table><caption>**Table 2.** Comparison of the cellular fatty acid methyl ester content of strain F1F22T and *Exilispira thermophila* strain RASEN<sup>T</sup> (Method TSBA40; Calcul method ANAER6). Strains: 1, F1F22T (data from this study); 2, *E. thermophila* [13]; 3, *B. andersonii* [38]; Values are percentage (w/v) of total fatty acids; c, cis; NR, not reported.</caption><thead><tr><th rowspan="2">Fatty acids</th><th colspan="3">Percentage (w/v) of total fatty acids</th></tr><tr><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>C14:0</td><td>1.2</td><td>2</td><td>47.5</td></tr><tr><td>C15:0</td><td>0.4</td><td>1</td><td>2</td></tr><tr><td>C16:0</td><td>40.2</td><td>29</td><td>20.7</td></tr><tr><td>iso-C16:0</td><td>30.2</td><td>NR</td><td>NR</td></tr><tr><td>C16:0 DMA</td><td>14.5</td><td>NR</td><td>NR</td></tr><tr><td>C16:0 3-OH</td><td>3.7</td><td>NR</td><td>NR</td></tr><tr><td>C16:1 DMA</td><td>4.2</td><td>7</td><td>NR</td></tr><tr><td>iso-C17:0</td><td>0.3</td><td>3</td><td>NR</td></tr><tr><td>anteiso-C17:0</td><td>1.7</td><td>NR</td><td>NR</td></tr><tr><td>C18:0</td><td>1.8</td><td>14</td><td>5.3</td></tr><tr><td>iso-C18:0</td><td>0.5</td><td>2</td><td>NR</td></tr><tr><td>C18:1 ω9c</td><td>0.2</td><td>22</td><td>8.2</td></tr></tbody></table>

<table><caption>**Table 3.** Genomic features of strain F1F22T and the closest genomes of type species: *Treponema caldarium*<sup>T</sup> DSM 7334 (CP002868.1); *Brevinema andersonii*<sup>T</sup> ATCC 43811 (FOKY00000000) and *Spirochaeta thermophila*<sup>T</sup> DSM 6578 (CP002903.1).</caption><thead><tr><th>Characteristics</th><th>F1F22<sup>T</sup></th><th>Treponema caldarium<sup>T</sup> (DSM 7334)</th><th>Brevinema andersonii<sup>T</sup> (ATCC 43811)</th><th>Spirochaeta thermophila<sup>T</sup> (DSM 6578)</th></tr></thead><tbody><tr><td>Size (base pair)</td><td>2.515.453</td><td>3.239.340</td><td>1.501.400</td><td>2.560.222</td></tr><tr><td>G+C content</td><td>43.15</td><td>45.58</td><td>35.24</td><td>60.92</td></tr><tr><td>Contig nb</td><td>1</td><td>1</td><td>39</td><td>1</td></tr><tr><td>Gene</td><td>2.678</td><td>2.928</td><td>1581</td><td>2.350</td></tr><tr><td>Protein-coding genes (CDS)</td><td>2.672</td><td>2.869</td><td>1442</td><td>2.296</td></tr><tr><td>rRNA genes (16S, 23S, 5S)</td><td>6 (2, 2, 2)</td><td>9 (3, 3, 3)</td><td>2 (1, 1, 0)</td><td>6 (2, 2, 2)</td></tr><tr><td>tRNA</td><td>46</td><td>48</td><td>48</td><td>46</td></tr></tbody></table>
**Table 2.** Comparison of the cellular fatty acid methyl ester content of strain F1F22T and *Exilispira thermophila* strain RASEN<sup>T</sup> (Method TSBA40; Calcul method ANAER6). Strains: 1, F1F22T (data from this study); 2, *E. thermophila* [13]; 3, *B. andersonii* [38]; Values are percentage (w/v) of total fatty acids; c, cis; NR, not reported.

<table><thead><tr><th rowspan="2">Fatty acids</th><th colspan="3">Percentage (w/v) of total fatty acids</th></tr><tr><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>C14:0</td><td>1.2</td><td>2</td><td>47.5</td></tr><tr><td>C15:0</td><td>0.4</td><td>1</td><td>2</td></tr><tr><td>C16:0</td><td>40.2</td><td>29</td><td>20.7</td></tr><tr><td>iso-C16:0</td><td>30.2</td><td>NR</td><td>NR</td></tr><tr><td>C16:0 DMA</td><td>14.5</td><td>NR</td><td>NR</td></tr><tr><td>C16:0 3-OH</td><td>3.7</td><td>NR</td><td>NR</td></tr><tr><td>C16:1 DMA</td><td>4.2</td><td>7</td><td>NR</td></tr><tr><td>iso-C17:0</td><td>0.3</td><td>3</td><td>NR</td></tr><tr><td>anteiso-C17:0</td><td>1.7</td><td>NR</td><td>NR</td></tr><tr><td>C18:0</td><td>1.8</td><td>14</td><td>5.3</td></tr><tr><td>iso-C18:0</td><td>0.5</td><td>2</td><td>NR</td></tr><tr><td>C18:1 ω9c</td><td>0.2</td><td>22</td><td>8.2</td></tr></tbody></table>

**Table 3.** Genomic features of strain F1F22T and the closest genomes of type species: *Treponema caldarium*<sup>T</sup> DSM 7334 (CP002868.1); *Brevinema andersonii*<sup>T</sup> ATCC 43811 (FOKY00000000) and *Spirochaeta thermophila*<sup>T</sup> DSM 6578 (CP002903.1).

<table><thead><tr><th>Characteristics</th><th>F1F22<sup>T</sup></th><th>Treponema caldarium<sup>T</sup> (DSM 7334)</th><th>Brevinema andersonii<sup>T</sup> (ATCC 43811)</th><th>Spirochaeta thermophila<sup>T</sup> (DSM 6578)</th></tr></thead><tbody><tr><td>Size (base pair)</td><td>2.515.453</td><td>3.239.340</td><td>1.501.400</td><td>2.560.222</td></tr><tr><td>G+C content</td><td>43.15</td><td>45.58</td><td>35.24</td><td>60.92</td></tr><tr><td>Contig nb</td><td>1</td><td>1</td><td>39</td><td>1</td></tr><tr><td>Gene</td><td>2.678</td><td>2.928</td><td>1581</td><td>2.350</td></tr><tr><td>Protein-coding genes (CDS)</td><td>2.672</td><td>2.869</td><td>1442</td><td>2.296</td></tr><tr><td>rRNA genes (16S, 23S, 5S)</td><td>6 (2, 2, 2)</td><td>9 (3, 3, 3)</td><td>2 (1, 1, 0)</td><td>6 (2, 2, 2)</td></tr><tr><td>tRNA</td><td>46</td><td>48</td><td>48</td><td>46</td></tr></tbody></table>
**Table 4.** OrthoANI (lower left) and Genome-to-Genome Distance Calculator (GGDC) (upper right) values between the genome of strain F1F22<sup>T</sup> (CP073355, this study) and those of *Treponema caldarium*<sup>T</sup> DSM 7334 (CP002868.1); *Brevinema andersonii*<sup>T</sup> ATCC 43811 (FOKY00000000) and *Spirochaeta thermophila*<sup>T</sup> DSM 6578 (CP002903.1). Orthologous Average Nucleotide Identity Tool (OAT) software (https://www.ezbiocloud.net/tools/orthoani) was used for calculations.

<table><thead><tr><th>OrthoANI \ GGDC</th><th>Strain F1F22<sup>T</sup></th><th>Treponema caldarium<sup>T</sup></th><th>Brevinema andersonii<sup>T</sup></th><th>Spirochaeta thermophila<sup>T</sup></th></tr></thead><tbody><tr><td>Strain F1F22<sup>T</sup></td><td>\</td><td>0.25</td><td>0.20</td><td>0.24</td></tr><tr><td>Treponema caldarium<sup>T</sup></td><td>63.36</td><td>\</td><td>0.22</td><td>0.19</td></tr><tr><td>Brevinema andersonii<sup>T</sup></td><td>63.52</td><td>62.11</td><td>\</td><td>0.26</td></tr><tr><td>Spirochaeta thermophila<sup>T</sup></td><td>63.26</td><td>64.03</td><td>61.51</td><td>\</td></tr></tbody></table>

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## Legends of figures

**Fig. 1:** Micrographs of strain F1F22<sup>T</sup>. (a) Phase-contrast micrographs of cells with arrows indicating hooked ends (HE) and spherical bodies (SB). Bar, 10 µm. (b) Transmission electron micrograph of a negatively stained cell without any periplasmic flagella shows wave-shaped cell structures of regular length and the central axis. Bar, 200 nm. (c) Electron microscopy: cross-section of cells, showing the outer membrane typical of Gram-negative but no periplasmic flagella. Bar, 100 nm.

**Fig. 2.** Phylogenetic dendrogram based on 1193 unambiguous bp of 16S rRNA gene sequence data, showing the position of the new genus *Thermospira aquatica* F1F22<sup>T</sup> including the different families and orders of the class *Spirochaetia*. Bootstrap values (expressed as percentages of 2000 replications) greater than 40% are shown at branch points. Constructed
**Table 4.** OrthoANI (lower left) and Genome-to-Genome Distance Calculator (GGDC) (upper right) values between the genome of strain F1F22<sup>T</sup> (CP073355, this study) and those of *Treponema caldarium*<sup>T</sup> DSM 7334 (CP002868.1); *Brevinema andersonii*<sup>T</sup> ATCC 43811 (FOKY00000000) and *Spirochaeta thermophila*<sup>T</sup> DSM 6578 (CP002903.1). Orthologous Average Nucleotide Identity Tool (OAT) software (https://www.ezbiocloud.net/tools/orthoani) was used for calculations.

<table><thead><tr><th>OrthoANI \ GGDC</th><th>Strain F1F22<sup>T</sup></th><th>Treponema caldarium<sup>T</sup></th><th>Brevinema andersonii<sup>T</sup></th><th>Spirochaeta thermophila<sup>T</sup></th></tr></thead><tbody><tr><td>Strain F1F22<sup>T</sup></td><td>\</td><td>0.25</td><td>0.20</td><td>0.24</td></tr><tr><td>Treponema caldarium<sup>T</sup></td><td>63.36</td><td>\</td><td>0.22</td><td>0.19</td></tr><tr><td>Brevinema andersonii<sup>T</sup></td><td>63.52</td><td>62.11</td><td>\</td><td>0.26</td></tr><tr><td>Spirochaeta thermophila<sup>T</sup></td><td>63.26</td><td>64.03</td><td>61.51</td><td>\</td></tr></tbody></table>

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## Legends of figures

**Fig. 1:** Micrographs of strain F1F22<sup>T</sup>. (a) Phase-contrast micrographs of cells with arrows indicating hooked ends (HE) and spherical bodies (SB). Bar, 10 µm. (b) Transmission electron micrograph of a negatively stained cell without any periplasmic flagella shows wave-shaped cell structures of regular length and the central axis. Bar, 200 nm. (c) Electron microscopy: cross-section of cells, showing the outer membrane typical of Gram-negative but no periplasmic flagella. Bar, 100 nm.

**Fig. 2.** Phylogenetic dendrogram based on 1193 unambiguous bp of 16S rRNA gene sequence data, showing the position of the new genus *Thermospira aquatica* F1F22<sup>T</sup> including the different families and orders of the class *Spirochaetia*. Bootstrap values (expressed as percentages of 2000 replications) greater than 40% are shown at branch points. Constructed
533 from the Maximum Likelihood method based on the Tamura-Nei model. The tree with the
534 highest log likelihood (-16807.01) is shown. Initial tree(s) for the heuristic search were obtained
535 automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
536 distances estimated using the Maximum Composite Likelihood (MCL) approach, and then
537 selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch
538 lengths measured in the number of substitutions per site. The analysis involved 59 nucleotide
539 sequences. All positions containing gaps and missing data were eliminated. There were a total
540 of 1087 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. the
541 numbers in brackets are Genbank/EMBL/DDBJ accession numbers for the 16S rRNA gene
542 sequences. Bar, 5% estimated sequence divergence.
533 from the Maximum Likelihood method based on the Tamura-Nei model. The tree with the
534 highest log likelihood (-16807.01) is shown. Initial tree(s) for the heuristic search were obtained
535 automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
536 distances estimated using the Maximum Composite Likelihood (MCL) approach, and then
537 selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch
538 lengths measured in the number of substitutions per site. The analysis involved 59 nucleotide
539 sequences. All positions containing gaps and missing data were eliminated. There were a total
540 of 1087 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. the
541 numbers in brackets are Genbank/EMBL/DDBJ accession numbers for the 16S rRNA gene
542 sequences. Bar, 5% estimated sequence divergence.