# Kineothrix sedimenti sp. nov., a 3-hydroxybutyrate-producing bacterium isolated from sediment of the meromictic Lake Pavin

Corinne Biderre-Petit, Ayité Adama Hongleda, Gaëlle Marchal, Neha Mehta, Jean-Sébastien Guez, Eric Viollier, Maïlysia Scheepers, Damien Courtine, Khaled Fadhlaoui

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Corinne Biderre-Petit, Ayité Adama Hongleda, Gaëlle Marchal, Neha Mehta, Jean-Sébastien Guez, et al.. Kineothrix sedimenti sp. nov., a 3-hydroxybutyrate-producing bacterium isolated from sediment of the meromictic Lake Pavin. International Journal of Systematic and Evolutionary Microbiology, 2025, 75 (4), pp.006750. 10.1099/ijsem.0.006750. hal-05046305

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1 Kineothrix sedimenti sp. nov., a 3-hydroxybutyrate-producing bacterium isolated from

2 sediment of the meromictic Lake Pavin

3

4 Corinne Biderre-Petit¹*, Ayité Adama Hongleda¹,², Gaëlle Marchal¹, Neha Mehta³, Jean-

5 Sébastien Guez⁴, Eric Viollier⁵, Maïlysia Scheepers¹, Damien Courtine¹, and Khaled

6 Fadhlaoui¹,⁶

7 ¹Université Clermont Auvergne, CNRS, Laboratoire Microorganismes : Génome et

8 Environnement, F-63000, Clermont-Ferrand, France.

9 ²UMR GMPA, AgroParisTech, INRAE, Université Paris-Saclay, F-78850 Thiverval-Grignon, 10 France (current address).

11 ³Université libre de Bruxelles, Faculty of Science Biogeochemistry and Modeling of Earth

12 System, Avenue FD Roosevelt, 501050 Brussels, Belgium

13 ⁴Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, 63000

14 Clermont-Ferrand, France

15 ⁵Laboratoire des Sciences du Climat et de l'Environnement and Université Paris Cité, CEA

16 Saclay - l'Orme des Merisiers - bat. 714, France 91191 Gif-sur-Yvette Cedex

17 ⁶Université Clermont Auvergne, UMR 454 MEDIS UCA-INRAE, F-63000 Clermont-
Ferrand, France

19 *For correspondence: Corinne Biderre-Petit, 1 Impasse Amélie Murat - Bat BioA 63178

20 Aubière (France). E-mail: [corinne.petit@uca.fr](mailto:corinne.petit@uca.fr), Tel: +33(0)473177959;

21 Fax+33(0)473407670.

22 **Orcid:**

23 Corinne Biderre-Petit: https://orcid.org/0000-0001-7962-4171

24 Damien Courtine: https://orcid.org/0000-0002-9162-0111

25 Khaled Fadhaloui: https://orcid.org/0000-0001-9166-5857
26 Jean-Sébastien Guez: https://orcid.org/0000-0002-8736-7758

27 Neha Mehta: https://orcid.org/0000-0002-7312-6398

28 Maïlysia Scheepers: https://orcid.org/0009-0009-5148-626X

29 Eric Viollier: https://orcid.org/0000-0002-8863-4563

30

31 **Keywords:** Kineothrix, freshwater sediment, anaerobe, 3-hydroxybutyrate-producing bacterium

33

34 ** Repositories: The Genbank/ENA/DDBJ accession numbers for the 16S rRNA sequence and genome sequence of strain IPX_CK<sup>T</sup> are CP146256.1 and PP429277, respectively.**

36

37 **Abstract**

38 An anaerobic, spore-forming, 3-hydroxybutyrate (3-HB)-producing bacterium, strain IPX_CK<sup>T</sup>, was isolated from sediment of a meromictic lake located in Massif Central (France). Cells were rods, forming filamentous chains which were observed moving under the microscope. Strain IPX_CK<sup>T</sup> utilized a wide variety of carbohydrates, but not raffinose, rhamnose, and starch. Hydrogen (H<sub>2</sub>), 3-HB, acetate, and ethanol were the main fermentative end-products from growth in medium containing glucose. Strain IPX_CK<sup>T</sup> grew optimally at 37°C and pH 7. Its closest phylogenetic relative was *Kineothrix alysoides* (16S rRNA gene sequence identity 98.7%, *isDDH* 34.6%, ANIb 87.4%). The genomic DNA G+C content was 43.0 mol%. Like for *K. alysoides*, whole-genome sequencing suggested that strain IPX_CK<sup>T</sup> is capable of fixing nitrogen (N<sub>2</sub>). However, strain IPX_CK<sup>T</sup> carried a five-*nif*-gene-set (*nifHDKEB*), not present in *K. alysoides*. Genome sequence also showed a high number of encoded chemotaxis receptors (42 genes, the second highest in the family *Lachnospiraceae* after *K. alysoides*). Based on phenotypic, genomic, phylogenetic and chemotaxonomic
analyses, it is proposed that a novel species, *Kineothrix sedimenti* sp. nov., be created, with
strain IPX_CK<sup>T</sup> (DSM 118044<sup>T</sup>, CIP 112511<sup>T</sup>) as the type strain.

53

## 54 Introduction

Three-hydroxybutyrate (3-HB) is a crucial metabolite found across all domains of life.

Among bacteria, acetogens, a group of ecologically significant, strictly anaerobic bacteria, are
key 3-HB producers. For most of these microorganisms, 3-HB serves as a vital building block
in the synthesis of compounds such as polyhydroxybutyrate (PHB), which is used as source of
carbon and energy [1]. Most acetogens are mixotrophic, meaning they employ both glycolysis
and Wood-Ljungdahl Pathway (WLP) within a single organism to convert various substrates
into acetyl-CoA, the primary precursor for 3-HB biosynthesis from either organic (e.g.
carbohydrates) or inorganic (e.g. carbon dioxide (CO₂), hydrogen (H₂)) substrates,
respectively [2]. Moreover, acetogens utilize WLP to recapture CO₂ from pyruvate
decarboxylation, enabling them to generate three acetyl-CoA molecules per glucose,
compared to the two typically produced by heterotrophic glycolysis. As a result, although
acetogens produce theoretically 36% more 3-HB when grown on carbohydrate alone, they can
achieve complete carbon conversion with the addition of H₂ [2].

Recently, 3-HB, and also PHB, have garnered significant attention from both the socio-economic sector and biotech industry due to their diverse applications and properties: 1) in animals and humans, they demonstrate therapeutic effects on colorectal cancer, diseases of nervous and circulatory systems [1,3,4], ii) in the environment, they contribute to the reduction of overall greenhouse gas CO₂ emissions [2], and iii) in industry, they serve as precursors for the production of valuable compounds (e.g. antibiotics, pheromones, bioplastics) [5,6]. Recent studies have also shown that PHB biosynthesis from CO₂ could potentially be cost-competitive due to its current high market price [7].
3-HB is a chiral molecule that exists in two enantiomeric forms: *R/D* and *S/L*. Both forms are synthetized by microorganisms, with the specific enantiomer produced depending on the species. Bacteria can produce 3-HB using acetyl-CoA as a precursor, with some of them leading to (*S*)-3-HB and others, to (*R*)-3-HB [8–12]. All these pathways require a thiolase to convert two acetyl-CoA molecules into acetoacetyl-CoA. From this point, the pathways diverge and exhibit varying degrees of efficiency. For example, the (*R*)-3-HB pathway which utilizes acetyl-CoA acetyltransferase/(*R*)-3-HB-CoA dehydrogenase (PhaAB) enzymes has demonstrated relatively poor 3-HB production compared to the other pathways. In contrast, the (*S*)-3-HB pathway involving thiolase (Thl)/ (*S*)-3-HB-CoA dehydrogenase (Hbd)/thioesterase (TesB) results in high 3-HB titers when using syngas as a substrate [13]. In the present study, a novel 3-HB-producing acetogen, designated IPX_CK<sup>T</sup> was isolated from the sediment of Lake Pavin, a meromictic lake in Massif Central (France). This bacterium belongs to the genus *Kineothrix* within the family *Lachnospiraceae* (phylum *Bacillota*). The identification of this strain employed a comprehensive polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences, phylogenomics based on genome sequence, average nucleotide identity (ANI), average amino-acid identity (AAI), *in silico* DNA-DNA hybridization (*isDDH*), phenotypic characterization, and fatty acid analyses. Based on these analyses, the strain is proposed to represent a novel species named *Kineothrix sedimenti* sp. nov. Currently, the genus *Kineothrix* includes a single species, *K. alysoides*, whose type strain was isolated from a switchgrass seeded with forest soil. Unlike the newly discovered strain, *K. alysoides* KNHs209<sup>T</sup> produces butyrate rather than 3-HB [14]. Another strain, *Kineothrix* sp. M12-C1, was isolated from the faeces of black soldier fly larvae and deposited in the Bioresource Collection and Research Center (strain BCRC 81406) [15]. Recent research has highlighted the potential health benefits of the species *K. alysoides*. Its administration to mice showed promise in restoring gut health and improving lipid
metabolism, potentially preventing and treating metabolic dysfunction-associated steatotic
liver disease (MASLD) [16]. Also, increasing the relative abundance of this bacterium in mice
gut correlated with reduced anxiety-like behavior in the most anxious mice [17].

104

105 Isolation and 16S rRNA phylogeny

Strain IPX_CK<sup>T</sup> was isolated under anaerobic conditions from a 30 cm core sediment
collected at 22 m depth in the water column of Lake Pavin (45°29.740 N, 2°53.280 E). The
temperature of the overlying water was 15.6°C. Oxygen (O<sub>2</sub>) measurements showed depletion
within 3.5-4.5 mm depth into the core sediments. The core was cut every centimeter and the
first slice, which had a redox potential of up to -33 mV, was used for bacteria isolation
(Supplementary Table 1). This slice was transferred in serum vial containing 50 ml of
Anaerobic Vital (AV) medium. The AV medium contained per liter 0.5 g KH<sub>2</sub>PO<sub>4</sub>, 0.4 g
MgSO<sub>4</sub> x 7 H<sub>2</sub>O, 1 g NaCl, 1 g NH<sub>4</sub>Cl, 0.05 g CaCl<sub>2</sub> x 2 H<sub>2</sub>O, 1.5 g C<sub>2</sub>H<sub>3</sub>NaO<sub>2</sub>, 0.5 g L-
cysteine-HCl x H<sub>2</sub>O, 1 g yeast extract, 1 g casa-amino acids, 1 ml 0.1% resazurin solution,
0.1% SL10 Widdel trace element solution. The SL10 trace element solution contained per
liter: 0.25 g HCl, 1.5 g FeCl<sub>2</sub> x 4 H<sub>2</sub>O, 190 mg CoCl<sub>2</sub> x 6 H<sub>2</sub>O, 70 mg ZnCl<sub>2</sub>, 62 mg H<sub>3</sub>BO<sub>3</sub>, 36
mg Na<sub>2</sub>MoO<sub>4</sub> x 2 H<sub>2</sub>O, 24 mg NiCl<sub>2</sub> x 6 H<sub>2</sub>O, 17 mg CuCl<sub>2</sub> x 2 H<sub>2</sub>O and 100 mg MnCl<sub>2</sub> x 4
H<sub>2</sub>O. The vitamin solution contained per liter: 2 mg folic acid, 10 mg pyridoxine-HCl, 5 mg
riboflavin, 5 mg nicotinic acid, 5 mg vitamin B5, 0.1 mg vitamin B12, 5 mg 4-aminobenzoic
acid, 5 mg lipoic acid.

The mixed culture was supplied with glucose (20 mM final) and cultivated anaerobically at
37°C with 100% N<sub>2</sub> atmosphere. The mixed culture was monitored for growth (optical density
OD<sub>600</sub>) using a spectrophotometer (Jenway 7415 Scanning UV/Visible Spectrophotometer,
Fischer Scientific, France), and formation of gaseous end products (CO<sub>2</sub>, H<sub>2</sub>, CH<sub>4</sub>), using gas
chromatography (3000A MicroGC, Agilent Technologies, Santa Clara, CA, USA). Isolation
126 was performed by serial dilutions and the Hungate roll-tube technique [18]. Single colonies 127 were picked and transferred to the same medium and incubated at 37°C. The roll-tube 128 procedure was repeated several times until a pure culture was obtained. Culture purity was 129 checked by microscopic examination (Leica DM IRB inverted microscope equipped with a 130 Hamamatsu C13440 camera and Zen Blue v3.1 software). The strain was routinely cultivated 131 under anaerobic conditions using Hungate tubes sealed with rubber stoppers at 37°C under a 132 gaseous atmosphere of 100% N₂. The culture medium used was AV-glucose broth with 133 glucose at a final concentration of 20 mM. 134

134 DNA was extracted from a 5 ml culture using a standard phenol-chloroform method as 135 previously described [19] and quantified on a Qubit Fluorometer (Thermo Fisher Scientific, 136 Waltham, MA, USA). The 16S rRNA gene was amplified using primers 27F/1492R [20] and 137 sequenced by Sanger method (Eurofins Genomics, Cologne, Germany). After assembly 138 (Staden Package software [21]), 16S rRNA sequence was used to infer the nearest 139 phylogenetic relatives using BLASTN [22] against nucleotide database (NCBI, National 140 Center for Biotechnology Information). Sequences of strain IPX_CK<sup>T</sup> as well as its closest 141 relatives retrieved from GenBank (www.ncbi.nlm.nih.gov/genbank) [23], were aligned using 142 MUSCLE [24] allowing for the reconstruction of maximum-likelihood algorithm 143 phylogenetic tree (JTT model, discrete Gamma distribution (five categories), 1,000 bootstrap 144 replicates) as implemented in the program Mega v11 [25].

145 Phylogenetic analyses of 16S rRNA gene sequences revealed that strain IPX_CK<sup>T</sup> is most 146 closely affiliated with members of the genus *Kineothrix* and shows the highest sequence 147 identities with strains *K. alysoides* KNHs209<sup>T</sup> (98.7%) and *Kineothrix* sp. MB12-C1 (97.1%) 148 (Fig. 1). The percentage of identity with other genera of the family *Lachnospiraceae* was less 149 than 94% (data not shown).

150
151 **Phenotypic characterization**

152 The temperature range for growth was determined by incubation at 4, 19, 30, 37, 40, 45, and
153 55°C. The pH range for growth was assessed over the range 5.0-10.0 (increments of 0.5 pH
154 unit). The salinity range was tested with sodium chloride (NaCl) concentrations of 0, 1, 5, 7,
155 10, 20, 50, 75, and 100 g/L. End products of metabolism from culture grown in AV-glucose
156 broth were analysed using a liquid chromatograph (1260 HPLC, Agilent, Santa Clara, CA,
157 USA). The HPLC apparatus was equipped with two columns (Rezex ROA 300 x 7.8 nm,
158 Phenomenex, Torrance, CA, USA) mounted in serial in an oven (50°C) and coupled with a
159 refractive index detector. The mobile phase was a 2 mM sulfuric acid in ultra-pure water
160 pumped at 0.7 mL x min⁻¹ and 70 bars. For the analysis, 2 mL of sample were mixed with 125
161 µL of Ba(OH)₂ x 8 H₂O (0.3 M) and 125 µL of ZnSO₄ x 7 H₂O (5% w/v) before a 5 min
162 centrifugation at 10,000 g. Samples were filtered through 0.2 µm nylon filters before being
163 injected in the HPLC apparatus. Cellular fatty acid analyses were carried out by the DSMZ
164 services (Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und
165 Zellkulturen GmbH, Braunschweig, Germany), using the pellet from a 200 ml cell
166 suspension, resuspended in isopropanol.

167 Strain IPX_CK<sup>T</sup> exhibited Gram-negative staining, rod-shaped cells that were motile via
168 flagella (Table 1, Supplementary Fig. 1C). Like the type species of the genus *Kineothrix*, i.e.
169 *K. alysoides*, strain IPX_CK<sup>T</sup> tended to sediment at the bottom of the tube (Supplementary
170 Fig. 1B). Irregular colonies (0.2 mm in diameter) were white and raised with filiform margin
171 (Supplementary Fig. 1A) and were produced within 72 h of growth under anaerobic
172 conditions at 37°C on AV-glucose medium containing agar. Growth occurred from 19 to
173 37°C with the optimum growth temperature being 37°C. The tolerance of NaCl was from 0 to
174 30 g/L with the optimum growth concentration being 1 g/L. pH tolerance was from pH 6 to 9
175 with optimal growth occurring at pH 7. Cells of strain IPX_CK<sup>T</sup> were strictly anaerobic.
Strain IPX_CK<sup>T</sup> showed growth using the substrates fructose, galactose, glucose, lactose, maltose, cellobiose, saccharose, xylose, and yeast extract but was not able to utilize the substrates inositol, lactate, starch, rhamnose, and raffinose (Table 1). During fermentation of glucose (20 mM, initial concentration), strain IPX_CK<sup>T</sup> produced mainly 3-HB (4.8 mM), acetate (13.5 mM), ethanol (13.1 mM), and H<sub>2</sub> (4% of total gas in the headspace) as end-products, after 48 h of growth in AV-glucose broth (0.1 mM remaining glucose after 48 h).
Sulfate, thiosulfate, sulfite, nitrate and nitrite were not used as terminal electron acceptors.
The major components in the cellular fatty acids fraction of strain IPX_CK<sup>T</sup> cells grown using glucose were C14:0 (15.3%), C14:0 dimethylacetal (DMA) (8.7%), C16:0 (28.6%), C16:0 DMA (6.6%), C16:1 cis 9 DMA (13.8%), and C18:1 cis 11 DMA (9.2%).
Oxidase activity was determined using oxidase test disks (70439, Merck), while catalase activity was assessed by adding a 3% hydrogen peroxide solution to a Hungate tube containing bacterial cells and observing the formation of bubbles. Urease, gelatinase, and aesculin hydrolyse activities were evaluated using the API<sup>R</sup> 20 A gallery (bioMérieux). The indole test was performed using Kovac's reagent (738210301, bioMérieux), with resazurin omitted from the medium for this test. All tests yielded negative results, consistent with K. alysoides, the type species of the genus Kineothrix [14].

<table><caption>**Table 1:** Distinguishing characteristics of strain IPX_CK<sup>T</sup> compared to the type strain of its closest related species.</caption><thead><tr><th>Species</th><th>Kineothrix sedimenti</th><th>Kineothrix alysoides</th><th>Eisenbergiella tayi</th><th>"Eisenbergiella massiliensis"</th></tr><tr><th>Strain</th><th>IPX_CK<sup>T</sup>l</th><th>KNHs209<sup>T</sup> *</th><th>B086562<sup>T</sup> ¥</th><th>AT11<sup>T</sup>‡</th></tr></thead><tbody><tr><td><strong>Genomic data</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Genome size (Mb)</td><td>4.7</td><td>4.7</td><td>7.5</td><td>7.1</td></tr><tr><td>G+C content (%)</td><td>43.0</td><td>42.5</td><td>46.5</td><td>48.0</td></tr><tr><td>N50 (Mb)</td><td>-</td><td>1.9</td><td>1.2</td><td>3.3</td></tr><tr><td>L50</td><td>-</td><td>2</td><td>3</td><td>2</td></tr><tr><td>CDS number</td><td>4377</td><td>4176</td><td>6421</td><td>6114</td></tr><tr><td>rRNA operon</td><td>4</td><td>5</td><td>1</td><td>2</td></tr></tbody></table>
<table><tbody><tr><td>tRNA</td><td>61</td><td>63</td><td>56</td><td>56</td></tr><tr><td><strong>Morphology</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell length (µm)</td><td>1.8-3.6</td><td>3.3-6.7</td><td>3.4-7.3</td><td>1.0-3.0</td></tr><tr><td>Cell width (µm)</td><td>0.35-0.45</td><td>0.5-0.6</td><td>0.4-0.7</td><td>0.4-0.5</td></tr><tr><td>Motility</td><td>yes</td><td>yes</td><td>no</td><td>no</td></tr><tr><td>Flagella</td><td>yes</td><td>single polar</td><td>no</td><td>no</td></tr><tr><td>Colony size (mm)</td><td>≈0.18</td><td>≈1</td><td>0.17-0.6</td><td>0.5-1.5</td></tr><tr><td>Colony color and aspect</td><td>white, very small, convex, entire margins</td><td>white, translucent shiny, convex, entire margins</td><td>flat, opaque, rhizoid margins</td><td>light gray</td></tr><tr><td>Colony shape</td><td>circular</td><td>circular</td><td>irregular</td><td>irregular</td></tr><tr><td><strong>Culture and growth conditions</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature range (°C)</td><td>19-37</td><td>≤15 - 40</td><td>15-45</td><td>28-45</td></tr><tr><td>Optimal temperature (°C)</td><td>37</td><td>35-40</td><td>30-37</td><td>37</td></tr><tr><td>pH range</td><td>6.0-9.0</td><td>6.0-8.5</td><td>NA</td><td>NA</td></tr><tr><td>Salt concentration range (g/L)</td><td>0-30</td><td>NA</td><td>NA</td><td>0-5</td></tr><tr><td><strong>Physiology</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Ability of spore formation</td><td>yes</td><td>yes</td><td>no</td><td>no</td></tr><tr><td>Spore description</td><td>round subterminal, resulting in a lightbulb shape</td><td>round subterminal, resulting in a lightbulb shape</td><td>-</td><td>-</td></tr><tr><td>Major fatty acid profile</td><td>C14:0, C16:0, C16:1 cis 9 DMA</td><td>C14:0, C16:0</td><td>C16:0, C18:1 n9, DMA</td><td>C16:0, C18:1 n9, C18:1 n5, C18:0</td></tr><tr><td><strong>Enzymes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Catalase</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Oxidase</td><td>-</td><td>-</td><td>NA</td><td>-</td></tr><tr><td>Aesculin hydrolysis</td><td>-</td><td>NA</td><td>+</td><td>NA</td></tr><tr><td><strong>Acid from</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Cellobiose</td><td>+</td><td>+</td><td>-</td><td>NA</td></tr><tr><td>Fructose</td><td>+</td><td>+</td><td>-</td><td>NA</td></tr><tr><td>Galactose</td><td>+</td><td>+</td><td>-</td><td>NA</td></tr><tr><td>Glucose</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Lactose</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Maltose</td><td>+</td><td>+</td><td>-</td><td>v</td></tr><tr><td>Raffinose</td><td>-</td><td>NA</td><td>NA</td><td>+</td></tr><tr><td>Rhamnose</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Saccharose</td><td>+</td><td>+</td><td>-</td><td>NA</td></tr><tr><td>Starch</td><td>-</td><td>+</td><td>-</td><td>NA</td></tr><tr><td>Xylose</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Major end product of carbohydrate metabolism</td><td>A, Hb, E, H<sub>2</sub>, CO<sub>2</sub> (glucose metabolism)</td><td>A, B, F (cellobiose metabolism)</td><td>A, B, L, S (glucose metabolism)</td><td>NA</td></tr></tbody></table>

A : acetic acid, B : butyric acid, Hb: hydroxybutyric acid, E : ethanol, F : formic acid, S : succinic acid.
NA: not available, v: variable

N50 is the length of the shortest contig that together with all the contigs of the assembly that are the same length or longer than it covers 50% of the genome

L50 is defined as the count of smallest number of sequences whose length sum makes up 50% of the assembly

Data from this study

* Data from Haas and Blanchard [14]

¥ Data from Amir et al. [26]

‡ Data from Togo et al. [27]

194

## 195 Genome features

196 Genomic DNA (gDNA) from strain IPX_CK<sup>T</sup> was extracted for sequencing using a

197 Nucleobond high molecular weight DNA kit (MACHERY-NAGEL, Germany) and quantified

198 on a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was checked by Agilent High Sensitivity DNA Kit on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

201 bp) was carried out by Eurofins Genomics services (Constance, Germany). Purified gDNA

202 was also sequenced on a MinION Mk1C sequencing device (Oxford Nanopore Technologies, Oxford, UK) according to manufacturer instructions. Genome assemblies were carried out using Unicycler version 0.4.3 software [28].

205 (OGRI) was used as minimal standards to determine the degree of similarity between strain IPX_CK<sup>T</sup> genome and those of its closest related strains [29]. ANIb and AAI values were calculated between strains IPX_CK<sup>T</sup> and its closest related species using pyANI v0.2.12 and

207 compareM v0.1.2 ([30]; https://github.com/dparks1134/CompareM) and isDDH, using

209 Genome-to-Genome Distance Calculator v3 (GGDC) with formula 2 as previously recommended [31]. For genome-based phylogenetic analysis, the GToTree v1.8.2 program

211 was used to generate the phylogenomic tree (Fig. 2) [32].

212 Hybrid sequencing results in a complete, single chromosome of 4.7 Mb with a G+C content of 43.0% (Table 1). The mean depth of coverage was 860X and 132X for Illumina and

214 Nanopore, respectively. The assembled sequences were deposited in the NCBI database under

19

20

10
215 the accession number CP146256.1. The genome sequence contains four ribosomal gene
216 operons, 61 tRNA genes and 4,377 coding DNA sequences (CDS). Genomic comparisons
217 between strain IPX_CK<sup>T</sup> and its closest relatives within the *Lachnospiraceae* family revealed
218 that the ANIb and AAI scores reached up to 87.4% and 89.2%, respectively (Fig. 3). The
219 *isDDH* scores were 34.6 and 21.1% when compared with *K. alysoides* KNHs209<sup>T</sup> and
220 *Kineothrix* sp. MB12-C1, respectively (data not shown). These values fall below the
221 established threshold for species definition, i.e. 95-96% for ANIb [28] and 70% for *isDDH*
222 [33].

223

224 **Ecology**

225 Currently, there is limited ecological and genomic data available for members of the genus
226 *Kineothrix*. However, existing results indicate that *Kineothrix* bacteria are adapted to diverse
227 biological niches, including both host-associated environments such as the gut microbiome of
228 mice [34, 35] and black soldier fly larvae [15], as well as non-host-associated environments
229 like soil (*K. alysoides* [14]) and remote lake sediments (this study). In the mouse gut
230 microbiome, *Kineothrix* sp. were found to be significantly more abundant in laboratory
231 animals compared to wild rodents, where their prevalence is low [35]. Moreover, strain
232 KNHs209<sup>T</sup> was identified as a member of the rare biosphere in its original soil habitat,
233 comprising only about 0.001% of all reads [14]. Given this diverse ecological distribution,
234 exploring the potential ecological functions of *Kineothrix* and its genomic adaptability to
235 various lifestyles remains a challenging yet important area of research.

236 Comparison of non-host-associated strains reveals that the strain IPX_CK<sup>T</sup> genome, similar to
237 that of strain *K. alysoides* KNHs209<sup>T</sup>, contains most genes encoding proteins involved in
238 flagellum substructures (filament, hook, rod formation) and chemotaxis (e.g. *motA*, *motB*,
239 *cheY*). This genomic evidence aligns with the presence of flagella (Supplementary Fig. 1C).
240 However, the outer-membrane traversing FlgF, FlgH, and FlgI proteins were lacking in both.

241 Similarly to *K. alysoides* KNHs209<sup>T</sup>, the genome of strain IPX_CK<sup>T</sup> dedicates over 3% of its
242 genes to chemotaxis and motility functions. Notably, it contains 42 chemoreceptors

243 (COG0840, methyl accepting chemotaxis proteins). This number is particularly significant
244 within the *Lachnospiraceae* family since it presents the second-highest number of

245 chemoreceptors among all sequenced members of the family. While the range for other
246 *Lachnospiraceae* members is 0-32 genes, only *K. alysoides* KNHs209<sup>T</sup> surpasses this count

247 with 45 genes. The number of chemoreceptors has been shown to be an indicator of the

248 breadth of chemosensory capacities [36]. The large number observed in the *Kineotrix* strains
249 could suggest a lifestyle adapted to variable environments and may indicate a wide range of
250 metabolic versatility.

251 Moreover, annotation results showed that the chromosome of strain IPX_CK<sup>T</sup> harbored an
252 operon containing at least seven proteins involved in N<sub>2</sub> fixation: three genes for the

253 molybdenum (Mo)-dependent nitrogenase structural proteins (NifH, NifD, and NifK), two
254 proteins involved in the biosynthesis of MoFe protein cofactors (NifB and NifE), and two
255 proteins of the PII family involved in posttranslational nitrogenase regulation (NifI1 and

256 NifI2) (Fig. 4A). NifN was not found (Fig. 4B). This kind of operon (i.e. absence of *nifN*
257 gene) was shown to allow *Caldicellulosiruptor* spp. to fix N<sub>2</sub> [37]. Only 53 bacterial species

258 are known to carry a five-*nif*-gene-set (*nifHDKEB*) out of 3,485 containing at least one of the
259 *nifH*, *nifD*, *nifK*, *nifE*, *nifN*, and *nifB* genes [38]. This genomic locus contained an ABC

260 transporter system for Mo uptake directly located upstream of the Mo-nitrogenase genes.

261 None of these genes have homologs in the genome of *K. alysoides* KNHs209<sup>T</sup> (Fig. 4A). The
262 genome of strain IPX_CK<sup>T</sup> encoded two additional copies of NifDK. Only one of them has
263 high sequence similarity (up to 98% amino acid identity) and genomic organization

264 conservation with a copy found in the genome of *K. alysoides* KNHs209<sup>T</sup> (Fig. 4A) [14].
These results suggest that strains K. alysoides KNHs209T and IPX_CK^T are both capable of fixing N₂, thus contributing to nitrogen input in their respective habitats.

The genomes of these strains also showed the presence of genes encoding the bcs operon (butyryl-coenzyme A synthesis) additionally with the gene encoding thiolase (thlA) in the same gene cluster. This operon consists of genes crt, bcd, etfAB, and hbd encoding the enzymes crotonase (Crt) [EC 4.2.1.17], butyryl-CoA dehydrogenase Bcd-EtfAB [EC 1.3.8.1] and 3-hydroxyacyl-CoA dehydrogenase (Hbd) [EC 1.1.1.157]. A locus containing a phosphate butyryltransferase (Ptb) [EC 2.3.1.19] and a butyrate kinase (Buk) [EC 2.7.2.7], both involved in butyrate formation (Fig. 4), was detected upstream to this operon in both strains. In addition, their genomes comprised a complete gene cluster encoding the Rnf (Rhodobacter nitrogen fixation), assumed to function as a ferredoxin:NAD⁺ oxidoreductase to oxidize Fd²⁻ formed by the pyruvate:ferredoxin oxidoreductase or by the Bcd-Etf complex [39]. The ability of strain KNHs209T to produce butyrate and form spores led Haas and Blanchard [14] to suggest it could be a gastrointestinal-tract-associated bacterium. Similarly, strain IPX_CK^T has the capacity to form spores, as evidenced by the presence of genes encoding proteins required for engulfment, spore coat formation, and spore germination [40]. Although it does not produce butyrate under the conditions used in this study (growth on glucose), its similarities with strain KNHs209T and the low abundance, or even absence, of sequences affiliated with the genus Kineothrix in "omics" data from natural environments could suggest a similar origin for strain IPX_CK^T. Unlike strain KNHs209T, strain IPX_CK^T produces 3-HB, which is in accordance with the presence in its genome of genes encoding enzymes involved in (S)-3-HB enantiomer biosynthesis from acetyl-CoA precursor [Fig. 5]. This pathway is characterized by its critical gene hbd1 ((S)-3-hydroxybutyryl-CoA dehydrogenase [EC 1.1.1.57]). The process involves the formation of acetoacetyl-CoA by a thiolase (Thl, [EC 2.3.19]), followed by Hbd1 reducing acetoacetyl-CoA to (S)-3-
290 hydroxybutyryl-CoA, which is subsequently converted to 3HB *via* a thioesterase [EC
291 3.1.2.20] through removal of CoA. The hbd1 pathway has shown (S)-3HB high titers from
292 syngas [13]. (S)-3HB is a product of interest as it can be used as chiral building blocks for the
293 synthesis of fine chemicals such as antibiotics, vitamins, perfumes, and pheromones [41].
294 Additionally, strain IPX_CK<sup>T</sup> can produce formate, acetate and ethanol from acetyl-CoA (Fig.
295 5).

296

297 Conclusions

298 Strain IPX_CK<sup>T</sup> is a novel bacterial strain isolated from the sediment of a pristine meromictic
299 lake (Lake Pavin, France). Its characterization was conducted using a polyphasic taxonomic
300 approach which integrates multiple lines of evidence to determine taxonomic placement.
301 Phylogenetic analysis of 16S rRNA gene, combined with phylogenomic position confirmed
302 that this novel strain belongs to the genus *Kineothrix*. Furthermore, OGRI values between the
303 genome of strain IPX_CK<sup>T</sup> and those of its closest relatives fall below the species delineation
304 threshold. Based on this comprehensive analysis, strain IPX_CK<sup>T</sup> is proposed as a novel
305 species of the genus *Kineothrix*, for which the name *Kineothrix sedimenti* sp. nov. is
306 suggested. The genus *Kineothrix* currently comprises only a limited number of members.
307 Aside from strain IPX_CK<sup>T</sup>, only two strains have been isolated: one from insect faeces
308 (MB12-C1) and another from soil (*K. alysoides*, KNHs209<sup>T</sup>). Other data on this genus come
309 exclusively from metagenome-assembled genomes derived from intestinal microbiota
310 sequencing in mice, indicating strains associated with host environments. The very low
311 abundance of sequences affiliated with *Kineothrix* in environmental databases, combined with
312 the observation that many members of the *Lachnospiraceae* family producing butyrate and
313 forming spores are typically gut-associated, suggests that the two strains isolated from soil
314 (KNHs209T) and sediment (IPX_CKТ) may also have a connection to gastrointestinal
315 environments.

316

317 **Description of *Kineothrix sedimenti* sp. nov.**

318 *Kineothrix sedimenti* (se.di.men'ti. L. gen. n. sedimenti, of sediment).

319 Cells are rod-shaped, anaerobic, Gram-stain-negative, spore-forming, motile and 3-HB-
320 producing. Colonies growing on AV-glucose agar in Hungate tubes are small (<0.2 mm),
321 circular and white. Cells are 1.8-3.6 µm long and 0.35-0.45 µm wide. Growth of cells occurs
322 in a temperature range of 19-37°C with an optimum at 37°C (no growth was observed at 4°C
323 and from 40 to 55°C), and in a pH range of 6-9, optimum being pH 7. Organic acids are
324 produced from carbohydrates fructose, galactose, glucose, lactose, maltose, cellobiose,
325 saccharose, and xylose as well as from yeast extract. No cell growth occurs using the
326 substrates inositol, lactate, starch, rhamnose, and raffinose. Fermentative end-products from
327 growth on glucose are 3-HB, acetate, ethanol, and H2. The cell contains a chromosome of an
328 estimated size of 4.7 Mb containing four rRNA operons. No plasmids were detected. The
329 genomic data were deposited in NCBI/ENA/DDBJ under the accession number CP146256.1,
330 and the 16S rRNA sequence, at the Genbank database, under accession number PP429277.

331 The genome harbors genes forming a *bcs* operon, and a complete gene cluster encoding the
332 Rnf complex. It also carries a five-*nif*-gene-set (*nifHDKEB*) involved in N2 fixation.

333 Furthermore, genes encoding proteins involved in flagellum apparatus and chemotaxis are
334 present. The major components of the cellular fatty acid fraction, when cell grown using
335 glucose, are C14:0, C14:0 DMA, C16:0, C16:0 DMA, C16:1 cis 9 DMA, and C18:1 cis 11
336 DMA. The type strain of *Kineothrix sedimenti* is IPX_CKТ. (=DSM 118044T =CIP 112511T).

337

338 **Conflicts of interest**
339 The authors declare that there are no conflicts of interest.

340

341 **Funding sources**

342 This work received no specific grant from any funding agency.

343

344 **Author contributions**

345 **Corinne Biderre-Petit**: conceptualization, supervision, data curation, investigation, writing-

346 original draft; **Damien Courtine**: investigation, data curation, writing-review and editing;

347 **Khaled Fadhlaoui**: conceptualization, supervision, investigation, writing-review and editing;

348 **Jean-Sébastien Guez**: investigation, writing-review and editing; **Ayité Adama Hongleda**:

349 investigation, writing-review and editing; **Gaëlle Marchal**: investigation; **Neha Metha**:

350 resource; **Maïlysia Scheepers**: investigation, **Eric Viollier**: resource.

351

352 **Acknowledgements**

353 The authors are grateful to the Mésocentre Clermont-Auvergne of the Université Clermont

354 Auvergne (UCA) for providing help, computing, and storage resources. We also thank Pr.

355 Cécile Lepère for her thorough reviewing of the manuscript.

356

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471 **Figure legends**

472 **Figure 1:** Maximum-likelihood phylogenetic tree based on 16S rRNA sequences showing the
473 relationship between strain IPX_CK<sup>T</sup> and its closest related taxa within the family
474 *Lachnospiraceae*. Bar, 0.02 substitution per nucleotide position. Bootstrap values (≥ 50) are
475 shown at branching point as percentage of 1,000 replicates. *Defluviitalea phaphyphila*,
476 belonging to the family *Defluviitaleaceae*, is used as an outgroup. The NCBI accession
477 numbers are shown into parentheses. Strain IPX_CK<sup>T</sup> appears in red.

478 **Figure 2:** Phylogenomic tree including strain IPX_CK<sup>T</sup> and its closest relatives generated
479 with the program GToTree v1.8.2 [29] using a set of 74 markers spanning all bacterial
480 genomes. Bar, 0.1 substitution per nucleotide position. Circles shown at branching point
481 indicate Shimodaira–Hasegawa [SH] approximate likelihood ratio test [aLRT] values ≥80%.
482 Strain IPX_CK<sup>T</sup> appears in red.

483 **Figure 3:** Heatmap showing pairwise average nucleotide identity (ANIb, lower matrix) and
484 pairwise average amino acid identity (AAI, upper matrix) values calculated for the genome of
485 strain IPX_CK<sup>T</sup>, and those of other *Kineothrix* strains (data from metagenome-assembled
486 genomes are indicated with an asterisk) as well as other representatives of genera belonging to
487 the *Lachnospiraceae* family.

488 **Figure 4:** Schematic representation of nitrogenase cofactor biosynthesis, and the genomic loci
489 carrying the genes involved in this biosynthesis in *Kineothrix* strains. A) Loci containing *nif*
490 genes found in the genome of *K. sedimenti* strain IPX_CK<sup>T</sup> and *K. alysoides* strain
491 KNHs209<sup>T</sup>. B) Biosynthesis of molybdenum (FeMoco), vanadium (FeVco) and iron (FeFeco)
492 cofactors of nitrogenases. All pathways start with the formation of (4Fe-4S) clusters serving
493 as basic modules for NifB-mediated synthesis of NifBco, the (8Fe-9S-C) precursor of
494 FeMoco, FeVco and FeFeco (modified from [42]). Proteins present in the strain IPX_CK<sup>T</sup> are
495 depicted in black and those absent, in grey.
**Figure 5:** Schematic representation of pathways involved in formate, acetate, ethanol, 3-hydroxybutyrate, and butyrate formation. Proteins encoded in the genome of *Kineothrix sedimenti* strain IPX_CK<sup>T</sup> are depicted in black and bold and those absent, in grey. The main precursor for 3-HB production, i.e. acetyl-CoA, is indicated in red.

**AckA:** acetate kinase; **ADH:** alcohol dehydrogenase; **Ato:** butyryl-CoA:acetoacetate CoA transferase (a, b subunits); **Bcd:** butyryl-CoA dehydrogenase (including electron transfer protein a, b subunits); **Buk:** butyrate kinase; **But:** butyryl-CoA:acetate CoA transferase; **Cro:** crotonase; **Hbd1 and 2:** hydroxybutyryl-CoA dehydrogenase; **PFL:** pyruvate-formate lyase; **PhaA:** Acetyl-CoA acetyltransferase; **PhaB:** acetoacetyl-CoA reductase; **PhaEC:** poly[(R)-3-hydroxyalkanoate] polymerase; **PhaJ:** enoyl-CoA hydratase 2; **PhaZ:** poly(3-hydroxybutyrate) depolymerase; **Pta:** phosphotransacetylase; **Ptb:** phosphate butyryltransferase; **TesB:** thioesterase II; **Thl:** thiolase; **(S)-3-HB:** 3-hydroxybutyrate enantiomer S; **(R)-3-HB:** 3-hydroxybutyrate enantiomer R, **CoA:** coenzyme A; **P:** phosphate.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
A

<figure><img src="image_8.png" /></figure>

**A:** ABC transporter binding; **B:** Cystathione β-lyase; **C:** Cysteine synthase A; **D:** DUF4418; **E-F:** ABC transporter permease and binding proteins; **G:** NifD; **H:** NifK, **I:** NifX; **J:** NifB; **K:** radical SAM protein; **L-N:** NitT/TauT transport system; **O:** NifH; **P:** DUF4250; **Q:** DUF4445, **HP:** hypothetical protein

B

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /></figure>

Pyruvate

PFL
EC 2.3.1.54

Formate

ADH

EC 1.1.1.1

Ethanol

[H]

Acetyl-CoA

Thl/PhaA

EC 2.3.1.9

Acetate

AckA

EC 2.7.2.1

Pta

EC 2.3.1.8

Acetyl-P

ATP

ADP

Pi

Acetoacetate

EC 3.1.2.11

Acetoacetyl-CoA

NAD(P)H

Ptb

EC 2.3.1.19

(R)-3HB-CoA

(R)-3HB-P

Buk

EC 2.7.2.7

TesB

EC 3.1.2.20

(R)-3-HB

PhaZ

EC 3.1.1.75

PHB

PhaB

EC 1.1.1.36

NAD(P)H

Hbd1/Hbd2

EC 1.1.1.57

(S)-3HB-CoA

Cro

EC 4.2.1.17

Crotonoyl-CoA

Bcd-(αβ)

EC 1.3.8.1

Butanoyl-CoA

Acetate

EC 2.8.3.8

But

Butyrate

Ato

EC 2.8.3.8

TesB

EC 3.1.2.20

Acetate

Acetyl-CoA

(S)-3-HP

Ptb

EC 2.3.1.19

Butanoyl-P

Buk

EC 2.7.2.7
<figure><img src="image_11.png" /><figcaption>**Supplementary Table 1.** Physico-chemical parameters of the top centimeters of sediment.</figcaption></figure>

<table><thead><tr><th>Depth</th><th>pH</th><th>F<sup>-</sup> (µM)</th><th>NO<sub>3</sub><sup>-</sup> (µM)</th><th>SO<sub>4</sub><sup>2-</sup> (µM)</th><th>PO<sub>4</sub><sup>3-</sup> (µM)</th><th>H<sub>2</sub>S (µM)</th><th>Redox</th></tr></thead><tbody><tr><td>0 cm</td><td>6.8</td><td>7.2</td><td>3.0</td><td>12.1</td><td>1.1</td><td>0.1</td><td>192.9</td></tr><tr><td>1 cm</td><td>6.9</td><td>7.9</td><td>1.4</td><td>0.0</td><td>29.1</td><td>1.7</td><td>-32.8</td></tr><tr><td>2 cm</td><td>6.9</td><td>9.6</td><td>59.7</td><td>1.2</td><td>25.3</td><td>1.0</td><td>-74.7</td></tr><tr><td>3 cm</td><td>6.8</td><td>8.6</td><td>2.0</td><td>0.0</td><td>22.0</td><td>1.0</td><td>-68.3</td></tr></tbody></table>

The red frame indicates the depth used to isolate the strain IPX_CK<sup>T</sup>.