# Atosan, a new exopolysaccharide secreted by a Vibrio alginolyticus strain isolated from a deep-sea hydrothermal vent environment

Agata Zykwinska, Méline Calatraba, Sandrine Bonnetot, Laëtitia Marchand, Mathieu Fanuel, Simon Ollivier, David Ropartz, Sophie Le Gall, Loric Thoulouze, Bérengère Marais, et al.

► To cite this version:

Agata Zykwinska, Méline Calatraba, Sandrine Bonnetot, Laëtitia Marchand, Mathieu Fanuel, et al. Atosan, a new exopolysaccharide secreted by a Vibrio alginolyticus strain isolated from a deep-sea hydrothermal vent environment. Carbohydrate Research, 2025, 556, pp.109609. 10.1016/j.carres.2025.109609. hal-05206165

HAL Id: hal-05206165

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# Atosan, a new exopolysaccharide secreted by a Vibrio alginolyticus strain isolated from a deep-sea hydrothermal vent environment

Zykwinska Agata <fnref n="1" />,*, Calatraba Meline <fnref n="1" />, Bonnetot Sandrine <fnref n="1" />, Marchand Laetitia <fnref n="2" />, Fanuel Mathieu <fnref n="3" />,<fnref n="4" />, Ollivier Simon <fnref n="3" />,<fnref n="4" />, Ropartz David <fnref n="3" />,<fnref n="4" />, Le Gall Sophie <fnref n="3" />,<fnref n="4" />, Thoulouze Loric <fnref n="3" />,<fnref n="4" />, Marais Bérengère <fnref n="3" />,<fnref n="4" />, Cuenot Stéphane <fnref n="5" />, Cerantola Stéphane <fnref n="6" />, Collicec Jouault Sylvia <fnref n="1" />, Delbarre-Ladrat Christine <fnref n="1" />

## Abstract :

Bacteria are able to colonize various environments, even the most hostile ones, due to their ability to synthesize an extracellular protective matrix. Bacteria isolated from deep-sea hydrothermal vent environments have been identified as very effective producers of polysaccharide-rich matrix. In particular, exopolysaccharides (EPS) synthesized by Vibrio marine species have shown to be composed of uronic acid and hexosamine residues, similarly to glycosaminoglycans, components of mammalian tissues. In the present study, the fine structure of a new high-molecular weight EPS, named atosan, secreted by the deep-sea hydrothermal vent bacterium Vibrio alginolyticus AT1219 strain, was determined. A complementary analytical approach, including Nuclear Magnetic Resonance spectroscopy and mass spectrometry was applied to elucidate the repeating unit of the polysaccharide. A linear pentasaccharide with a unique monosaccharide sequence: [→3)-α-d-Galp-(1→3)-β-d-GalpA-(1→3)-β-d-GlcpNAc-(1→4)-α-d-GalpNAc-(1→4)-β-d-GlcpA-(1→] was described. In parallel, the genome sequence was investigated to get insight into the genetic mechanisms of atosan biosynthesis. Amongst eight clusters assigned to carbohydrate biosynthesis, three of them were closely related to the biosynthesis of the extracellular polysaccharide. Determination of the fine structure of atosan is a prerequisite step to better understand the relationship between its structure and resulting functional properties.

<footnote n="1">¹ Ifremer, MASAE Microbiologie Aliment Santé Environnement, F-44000 Nantes, France</footnote>

<footnote n="2">² Ifremer, HALGO Halieutique Grand Ouest, F-44000 Nantes, France</footnote>

<footnote n="3">³ INRAE, UR1268 BIA, F-44300 Nantes, France</footnote>

<footnote n="4">⁴ INRAE, PROBE research infrastructure, BIBS facility, F-44300 Nantes, France</footnote>

<footnote n="5">⁵ Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel, IMN, F-44000 Nantes, France</footnote>

<footnote n="6">⁶ Plateforme RMN-RPE, Service Général des Plateformes/Université de Bretagne Occidentale (UBO), Brest, France</footnote>

<footnote>* Corresponding author : Agata Zykwinska, email address : Agata.Zykwinska@ifremer.fr</footnote>
**Graphical abstract**

<figure><img src="image_2.png" /></figure>

**Highlights**

► A fine structure of a new bacterial exopolysaccharide (EPS) was determined using complementary analytical approach. ► Nuclear Magnetic Resonance spectroscopy and mass spectrometry revealed a unique pentasaccharide repeating unit. ► Glycosaminoglycan-mimetic chemical composition of the EPS was evidenced. ► The investigation of the genome sequence led to identification of main clusters responsible for polymer biosynthesis.

**Keywords** : exopolysaccharide, Vibrio alginolyticus, fine structure, biosynthesis, Nuclear Magnetic Resonance, Mass Spectrometry
## 1. Introduction

Polysaccharides are natural polymers constitutive of plant and algal cells walls, animal tissues, being also secreted by some microorganisms. Although polysaccharides are ubiquitous, they present a huge diversity in term of their chemical compositions and structures, and their molecular weights. All these features are determinant for the functional properties that the polysaccharides fulfill at different levels, from the single cell to the whole organism, in both physiological and pathological processes. Bacteria are well-known producers of extracellular polysaccharides, providing a protecting microenvironment favorable for their growth [1]. The ability of bacteria to produce exopolysaccharides (EPS), such as xanthan (Xanthomonas campestris), gellan (Sphingomonas paucimobilis) and hyaluronic acid (Streptococcus sp) has been exploited for decades to obtain several valuable actives and ingredients widely used in food, cosmetics and biomedical applications [2-5]. In contrast to other polysaccharide sources (plant, algae, animal), requiring complex extractions and purifications, bacteria excrete their polysaccharides outside their cells directly into the culture medium, thus facilitating their recovery. Therefore, polysaccharide production by bacterial fermentation constitutes a renewable and reproducible way for their recovery, leading to highly conserved structures. An excellent example constitutes hyaluronic acid, a glycosaminoglycan (GAG) essential for biological functions in mammalian tissues, initially extracted from animal sources and now obtained at industrial scale through biotechnological production [6].

Marine ecosystems and particularly extreme habitats, such as deep-sea hydrothermal vents, constitute a rich source of innovative bacterial EPS [7]. Marine bacteria belonging to *Alteromonas*, *Pseudoalteromonas*, *Halomonas*, *Cobetia* and *Vibrio* genera have been reported as the main EPS producers [8-11]. For instance, *Alteromonas* sp. mainly synthesize highly branched high molecular weight (HMW) EPS composed of neutral sugars and uronic acids, frequently substituted by inorganic (sulfate) and/or organic (pyruvate, lactate) acids [12-14]. In
contrast to *Alteromonas, Vibrio* strains excrete linear unsulfated HMW EPS, particularly rich
in uronic acids and hexosamines. *Vibrio diabolicus* HE800 strain (CNCM I-1629), a deep-sea
hydrothermal vent bacterium, produces diabolican, a linear EPS with a tetrasaccharide
repeating unit composed of two adjacent glucuronic acid residues (GlcA) and two types of N-
Acetyl hexosamines: N-Acetyl-glucosamine (GlcNAc) and N-Acetyl-galactosamine (GalNAc)
linked in the following sequence: [→3)-β-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→4)-β-D-GlcpA-
(1→4)-α-D-GalpNAc-(1→] [15,16]. Diabolican shows some structural similarities with
hyaluronic acid composed of alternating GlcNAc and GlcA residues linked by β-(1→4) and β-
(1→3) bonds [17]. A marine bacterium *V. alginolyticus* (CNCM I-4994) strain has been shown
to secrete a linear EPS consisting of a tetrasaccharide repeating unit composed of three adjacent
galacturonic acid residues (GalA) and one GlcNAc residue: [→3)-α-D-GalA-(1→4)-α-D-GalA-
(1→3)-α-D-GalA-(1→3)-β-GlcNAc(1→] [18]. Another marine *V. alginolyticus* strain (CNCM
I-4151) was able to produce a linear EPS displaying a tetrasaccharide repeating unit composed
of four distinct monosaccharides, including, besides uronic acid and hexosamine residues
classically described for Vibrio species, one galactose (Gal) residue: [→4)-β-D-GlcA-(1→3)-
α-D-Gal-(1→3)-α-D-GalA-(1→3)-β-GlcNAc(1→] [19].

These few examples clearly highlight a high diversity in both monosaccharide compositions
and structures of hyaluronic acid-like EPS secreted by marine bacteria belonging to *Vibrio*
genus. High chemodiversity offers a large panel of functional, physicochemical and biological,
e.g. GAG-mimetic properties that can be exploited for several applications, similarly to widely
known EPS produced by non-marine bacteria, *i.e.* xanthan and hyaluronic acid. For instance,
native diabolican has been shown to enhance *in vivo* bone repair in a rat model [20] and to
stimulate collagen structuring as well as extracellular matrix settle by fibroblasts in
reconstructed dermis [21]. Recently, one low molecular weight and highly sulfated diabolican
derivative (5,000 g/mol and 40 wt% sulfate) has been shown to partly inhibit heparanase
97 activity, thus leading to reduced accumulation of intracellular heparan sulfate fragments in
98 mucopolysaccharidosis IIIA (MPSIIIA) mouse fibroblasts. When administrated in MPIIIA
99 mouse model, diabolican treatment preserved myelination and white matter metabolic
100 alterations in the brain [22]. In addition to its biological activities, diabolican displays
101 interesting physicochemical properties and forms physical gels in the presence of divalent
102 cations, mainly copper, allowing its formulation into microgels for protein delivery [23].

103 In this context, the objective of the present study was to determine the structure of a
104 novel EPS, named atosan, produced by a bacterial strain isolated from a deep-sea hydrothermal
105 vent environment. Its phylogeny was firstly described using genome sequence and genetic
106 clusters potentially involved in polysaccharide biosynthesis were identified. Nuclear Magnetic
107 Resonance (NMR) spectroscopy and mass spectrometry (MS) were then applied to elucidate
108 the polysaccharide fine structure. Although determination of the polysaccharide repeating unit
109 is a particularly complex task requiring specific analytical techniques, it remains a key step to
110 better understand the structure-function relationship allowing for polysaccharide optimal use in
111 various domains.
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## 2. Materials and Methods

### 2.1 Bacterial strain

AT1219 strain was isolated from a diluted vent fluid collected by Remote Operated Vehicle Victor 6000 during ATOS cruise in 2001 near active smokers in the deep-sea hydrothermal vent Rainbow site located in Mid Atlantic Ridge.

### 2.2 Genome sequencing and analysis

Bacteria were grown at 30°C under 150 rpm agitation in Zobell medium (4 g/L tryptone, 1 g/L yeast extract, 33.3 g/L sea salts for aquarium, Instant Ocean). Genomic DNA was extracted using the GeneJET Genomic DNA Purification kit (Fisher Scientific, Illkirch, France). Sequencing was sub-contracted at Eurofins (Köln, Germany) on an Illumina HiSeq NovaSeq 6000 S4 PE150 XP and produced a total of 7,486,990 paired-end 150 bp reads. Assembling was carried out using CELIA v1.0 and DATARMOR Ifremer calculator (https://gitlab.ifremer.fr/bioinfo/celia). Assembly resulted in a total of 84 contigs and 38 contigs >1000bp with an N50 value of 390,051 bp; the largest contig was 1,545,137 bp. The draft genome is 5,090,173 bp, with 44.58% GC content with an estimated coverage of 440X, uploaded on MicroScope (Microbial Genome Annotation & Analysis Platform) [24]. Gene prediction and annotation were done automatically. 4801 CDS were obtained.

Genome sequence together with sequencing reads were deposited at the European Nucleotide Archive under the ENA/EMBL bioproject PRJEB75743.

Genome of AT1219 strain was compared to nine Vibrio strain genomes using "genome clustering" on Microscope web interface based on Mash software; this comparison is based on average nucleotide identity (ANI) calculation. A distance matrix and a phylogram were obtained. Glycosyltransferases (GT) were identified using dbcan annotation tool implemented in the MicroScope platform; it allows the annotation of CAZymes (Carbohydrate active Enzymes) using three algorithmic tools (Diamond, HMMER, Hotpep). Genetic clusters
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potentially involved in the biosynthesis of EPS were identified based on the genomic context
of the GT and compared with other genomes using synteny in the Microscope Platform [24,
25]. Genetic clusters were drawn with the R package gggenes.

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## 2.3 Production of native EPS

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AT1219 strain was cultured at 25°C in 1L culture medium composed of yeast extract (15 g/L, Organotechnie, La Courneuve, France), wheat peptone (15 g/L, Organotechnie, La Courneuve, France), NaCl (10 g/L), MgSO4 (3 g/L) and CaCl2 (1 g/L) at pH 7.2 at 150 rpm in a 2L fermenter (Applikon Biotechnology, SystemC Bioprocess, Saint-Paul Trois Châteaux, France). Glucose at 30 g/L final concentration was added as a carbon source. After 24 h of fermentation, the culture medium was centrifuged at 8000 g for 30 min, the supernatant containing soluble EPS was filtrated on 0.7 µm Whatman filter and ultrafiltrated on a 100 kDa cut-off membrane (Pellicon®, Merck Millipore) before being freeze-dried. Residual glucose was monitored by enzymatic assay with Megazyme D-Glucose HK kit (Libios, Vindry-sur-Turdine, France). Fermentation culture was stopped when glucose was almost completely consumed. EPS amount of 931 mg was obtained yielding 31 mg EPS/g of glucose in final 1.2L. The EPS is soluble in water.

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## 2.4 Preparation of LMW EPS derivatives (EPS DR and EPS DR-SD)

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LMW EPS DR was prepared, as previously described [26]. Briefly, native EPS (100 mg) solubilized in water (150 mL) was depolymerized at 60°C for 45 min using H2O2 added dropwise (58 µL at 33 wt%). After overnight reduction by NaBH4 (20 mg) and purification on Chelex® 20 resin, the solution containing depolymerized EPS was filtrated on 0.7 µm Whatman filter, ultrafiltrated on a 10 kDa cut-off membrane and freeze-dried.

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LMW EPS DR-SD was obtained by Smith degradation [27]. Briefly, 25 mg of LMW EPS DR was oxidized for 5 days with 50 mM NaIO4 (25 mL) at 4°C under continuous stirring. The

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oxidized polysaccharide solution was reduced with 5 mL of NaBH₄ at 15 mg/mL, dialyzed against distilled water for 48 h (6000–8000 Mw cut-off) and freeze-dried. The reduced product (11.3 mg) was hydrolyzed with acetic acid (1.5%) for 2 h at 100°C, neutralized by co-evaporation with methanol and freeze-dried, leading to LMW EPS DR-SD (7.8 mg).

## 2.5 Preparation of EPS oligosaccharide fractions by acid hydrolysis (EPS OF)

EPS oligosaccharides were obtained from native EPS (25 mg) after 1M TFA hydrolysis for 20 min at 100°C. After evaporation, the sample was washed 3 times with methanol/water (50/50, v/v) and solubilized in 0.1 M ammonium bicarbonate prior to fractionation on Bio-Gel P2 column at a flow rate of 1 mL/min with ammonium bicarbonate as eluent buffer. Three fractions were obtained: EPS OF1 (5.6 mg), EPS OF2 (4.9 mg) and EPS OF3 (3 mg).

## 2.6 Monosaccharide analysis

Monosaccharide composition of native EPS, its LMW derivatives (EPS DR, EPS DR-SD) and EPS oligosaccharide fractions obtained after TFA hydrolysis (EPS OF) was determined according to Kamerling *et al.* [28] method, modified by Montreuil *et al.* [29]. Monosaccharide standards (200 µg) and samples (400 µg) were hydrolyzed using MeOH/HCl at 100°C for 4 h, followed by re-N-acetylation with acetic anhydride overnight at room temperature. Myositol was used as internal standard. The methyl glycosides obtained were then converted to trimethylsilyl derivatives using *N*,*O*-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA:TMCS) 99:1. Gas chromatography (GC-FID, Agilent 8860) was used for separation and quantification of the per-*O*-trimethylsilyl methyl glycosides formed. Each sample was analyzed in triplicate and the average value was then calculated. The retention times of monosaccharide standards were used to identify the monosaccharides present in different samples. Monosaccharide composition in wt% was obtained using the following equation: $(m_1/m_2)*100\%$, where m₁ is the mass (in µg) of the monosaccharide calculated from the chromatogram and m₂ is the mass of the sample (400 µg).
## 2.7 Permethylation analysis

The positions of glycosidic linkages were determined on native EPS by preparing partially methylated alditol acetates (PMAAs) according to Pettolino et al. [30]. The partially methylated monosaccharides were converted to alditol acetates and analyzed by gas chromatography/mass spectrometry (GC/MS) (TRACE-GC-ISQ, Thermo Scientific™) on a nonpolar Thermo Scientific™ TraceGOLD™ TG-1MS GC Column (30 m x 0.25 mm x 0.25 µm), using carrier gas H₂ at 1.5 mL/min, as previously described in Buffetto et al. [31].

## 2.8 Absolute configuration

The absolute configuration of monosaccharides in native EPS and EPS-DR was determined according to Gerwing et al. [32,33]. Monosaccharide standards (1 mg), native EPS and EPS-DR (2 mg) were hydrolyzed using 2N TFA (500 µL) for 4h at 100°C. After TFA evaporation, samples were incubated with 500 µL of (S)-(+)-2-butanol (Sigma-Aldrich) with a drop of 13N TFA for 8h at 80°C and re-N-acetylated with acetic anhydride overnight at room temperature. The butyl glycosides obtained were then converted to trimethylsilyl derivatives using *N*,*O*-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA:TMCS) 99:1 and analyzed by gas chromatography according to the protocol described for the monosaccharide analysis (section 2.6).

## 2.9 Molecular weight

Weight-average molecular weight (Mw), number-average molecular weight (Mn) and dispersity index (D = Mw/Mn) of native EPS and its derivative EPS-DR were determined by High-Performance Size-Exclusion Chromatography (HPSEC, Prominence Shimadzu) coupled with a multiangle light scattering (MALS, Dawn Heleos-II, Wyatt Technology) and a differential refractive index (RI) (Optilab Wyatt technology) detectors. Samples solubilized at 2 mg/mL in water were eluted with 0.1 M ammonium acetate at a flow rate of 1 mL/min. The
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molecular weight was calculated using a refractive index increment dn/dc of 0.145 used for
polysaccharides.

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**2.10 Organic and inorganic acid substitution.**

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Organic (lactate, pyruvate) and inorganic (sulfate) acid substituents in native EPS were determined using high-performance anion-exchange chromatography (HPAEC), as previously described [26]. Briefly, an aqueous solution of an internal standard, KNO3 (10 g/L), was added to an aqueous EPS solution (2 mg/mL). The resulting solution was hydrolyzed with HCl (1M) at 133°C for 4h20'. After cooling to room temperature and the addition of water (4.5 mL), 800 µL of the sample was analyzed by HPAEC (in triplicate). Before injection, all samples were filtered on 0.45 µm filters. Acids were attributed according to standard samples (sodium lactate, sodium pyruvate, sodium sulfate, sodium phosphate from Sigma-Aldrich), investigated over the concentration range 1-12 mM. HPAEC analyses were carried out using Dionex Integron HPIC (Thermo Scientific) instrument controlled using Chromeleon software. The chromatographic system was composed of AS-AP autosampler, Dionex EGC eluent generator cartridge, an electrochemical detector with ADRS 600 4 mm conductivity suppressor. Sample separation was performed on Dionex IonPacTM AS11-HC (4 x 250mm) analytical anion exchange column with a guard column IonPacTM AG11-HC (4 x 50mm).

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**2.11 Protein content**

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The protein content of native EPS was determined using bicinchoninic acid method (BCA-Kit, Sigma). Bovine Serum Albumin was used as standard.

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**2.12 ATR-FTIR Spectroscopy**

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Infrared spectra of native EPS and its derivatives were recorded with a FTIR VERTEX 70 spectrometer (Bruker) in ATR spectroscopy mode in the range 4000-500 cm⁻¹.
## 2.13 Nuclear Magnetic Resonance (NMR)

NMR analyses were performed on EPS DR and EPS DR-SD in D₂O at 298 K on a Bruker Advance III HD500 spectrometer equipped with an inverse TCI ¹H/¹³C/¹⁵N cryoprobe. ¹H spectra were recorded using a 30° pulse angle, 16 scans and a sweep width of 7500 Hz. Acquisition times and recycle delays were 2.18 s and 2 s respectively. Raw data were apodized using an exponential line broadening of 0.3 Hz prior to Fourier transformation. 2D experiments were performed using standard pulse sequences available in the Bruker software. Chemical shifts were expressed in ppm relative to TSP used as external.

## 2.14 Mass spectrometry

Ion-pairing reversed-phase chromatography separation was carried out on an ultra-high-performance liquid chromatography system (UHPLC, Acquity H-Class plus® Waters, Wilmslow, U.K), equipped with a BEH C18 column (100 mm × 1 mm, packed with 1.7 µm porosity particles) (Waters, Manchester, UK). The flow rate was 0.150 mL/min and the column was heated to 45 °C. A ternary gradient was performed (A: H₂O, B: MeOH and C: 20mM heptylammonium formate at pH 6), with the same gradient steps as described in Ropartz et al., [34]. Mass spectrometry measurements were performed by direct coupling the UHPLC with a SELECT SERIES Cyclic IMS (Waters, Wilmslow, U.K.). Analyses were carried out in negative ion mode over a mass range of 300-2000 m/z (mass-to-charge ratio). Tandem MS (MS/MS) analyses were carried out with upstream separation of isomers by ion mobility spectrometry (IMS) on a SELECT SERIES Cyclic IMS instrument, which operation was extensively described in Giles et al. [35]. EPS OF2 was infused in negative polarity over a mass range of 100-2000 m/z, at 10 µL/min. First, separation of the isomers in this fraction was optimized using high resolution IMS experiments [36]. The separated species were then fragmented in the transfer cell of the instrument, allowing the MS/MS spectrum of each isomer to be reconstructed. Additional information about the parameters applied on the mass spectrometer
are given as the Supporting Information (Text S1). For MS/MS experiments, the sample was labeled with heavy oxygen as described in Ollivier et al. [36]. Data were carried out and processed using MassLynx v. 4.2 (Waters, Wilmslow, U.K.). Arrival time distributions (ATDs) were smoothed in MassLynx 4.2 using the Savitzky-Golay algorithm (3 iterations over 2 bins). Gaussian fitting of the smoothed ATDs was performed using CIUSuite2 [37] with an expected peak width of 1.0 ± 0.1 ms.

## 3. Results and discussion

### 3.1. Bacterial strain and its phylogeny

The AT1219 genome sequence was compared to nine *Vibrio* sp. from the Harveyi clade available on the Microscope public database (Figure 1). The highest similarity was found between the AT1219 strain and *Vibrio alginolyticus* ATCC 33787 reaching 99.993%. Similarity with other *V. alginolyticus* strains was over 98.5%, while it reached 93.2% with its closest counterparts from the *V. diabolicus* subclade (*V. diabolicus* and *V. antiquarius*) [38]. These values well above the 95-96% cutoff for species delineation clearly indicate that the AT1219 strain is a member of *V. alginolyticus* sp. [39].

<table><thead><tr><th>Genomes</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th><th>I</th><th>J</th></tr></thead><tbody><tr><td>V. alginolyticus 12G01</td><td>A</td><td>0.0000</td><td>0.0148</td><td>0.0148</td><td>0.0177</td><td>0.0690</td><td>0.0692</td><td>0.1205</td><td>0.1522</td><td>0.1478</td><td>0.1723</td></tr><tr><td>V. alginolyticus ATCC 17749<sup>T</sup></td><td>B</td><td></td><td>0.0000</td><td>0.0134</td><td>0.0166</td><td>0.0676</td><td>0.0680</td><td>0.1209</td><td>0.1490</td><td>0.1515</td><td>0.1687</td></tr><tr><td>Vibrio sp. AT1219</td><td>C</td><td></td><td></td><td>0.0000</td><td>0.0066</td><td>0.0677</td><td>0.0676</td><td>0.1181</td><td>0.1481</td><td>0.1493</td><td>0.1723</td></tr><tr><td>V. alginolyticus ATCC 33787</td><td>D</td><td></td><td></td><td></td><td>0.0000</td><td>0.0687</td><td>0.0707</td><td>0.1226</td><td>0.1525</td><td>0.1493</td><td>0.1714</td></tr><tr><td>V. diabolicus CNCM I-1629<sup>T</sup></td><td>E</td><td></td><td></td><td></td><td></td><td>0.0000</td><td>0.0181</td><td>0.1086</td><td>0.1594</td><td>0.1579</td><td>0.1705</td></tr><tr><td>V. antiquarius EX25<sup>T</sup></td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>0.0000</td><td>0.1063</td><td>0.1594</td><td>0.1576</td><td>0.1709</td></tr><tr><td>V. parahaemolyticus ATCC 17802<sup>T</sup></td><td>G</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.0000</td><td>0.1522</td><td>0.1528</td><td>0.1653</td></tr><tr><td>V. campbellii ATCC 25920<sup>T</sup></td><td>H</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.0000</td><td>0.0943</td><td>0.1816</td></tr><tr><td>V. harveyi ATCC 14126<sup>T</sup></td><td>I</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.0000</td><td>0.1851</td></tr><tr><td>V. natriegens CCUG 16371</td><td>J</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.0000</td></tr></tbody></table>

<figure><img src="image_3.png" /></figure>
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Figure. 1 Genome relatedness between ten bacteria belonging to the Harveyi clade of *Vibrio* genus. (A)

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Pairwise distance (=1-ANI) data used to build the phylogram. (B) Phylogram based on pairwise

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distances between the genome sequences.

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**3.2 Physicochemical composition of native EPS and assessment of the glycosidic linkage positions**

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Native EPS, named atosan, synthesized by *V. alginolyticus* AT1219 strain is a HMW polysaccharide (Mw 3,000,000 g/mol, D 1.2) of original monosaccharide composition. Indeed, the monosaccharide analysis revealed the presence of five main osidic residues, namely Galp, two types of uronic acids, GlcpA and GalpA, and two types of hexosamines, GlcpNAc and GalpNAc (Table 1). Besides Galp residue, low quantities of other neutral sugars, such as mannose (Manp) and glucose (Glcp) were also detected. Molar ratio indicated that the EPS repeating unit could be constituted of five monosaccharides, including 1 Gal, 1 GlcA, 1 GalA, 1 GlcNAc and 1 GalNAc. The absolute configuration analysis of the monosaccharides constitutive of atosan revealed that they were all in D configuration. Native EPS contained low amount of proteins (14.9 wt%), which are usually co-synthesized by bacteria [11,40]. Except for acetate substitution on hexosamine residues (GlcNAc, GalNAc), no other organic (lactate, pyruvate) or inorganic (sulfate) acid groups were detected in atosan composition, similarly to other EPS from *Vibrio* sp. [18,19].

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Table 1. Monosaccharide composition (wt% and molar ratio) of native EPS from *V. alginolyticus* AT1219 strain, its LMW derivatives (EPS DR and EPS DR-SD) and EPS oligosaccharide fractions (EPS OF1, OF2, OF3).

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<table><thead><tr><th rowspan="3"></th><th colspan="9">Monosaccharide composition (wt%)</th></tr><tr><th colspan="9">Molar ratio</th></tr><tr><th>Rha</th><th>Fuc</th><th>Man</th><th>Gal</th><th>Glc</th><th>GlcA</th><th>GalA</th><th>GlcNAc</th><th>GalNAc</th></tr></thead><tbody><tr><td rowspan="2">Native EPS</td><td>0</td><td>0</td><td>0.7</td><td>10.7</td><td>2.4</td><td>12.0</td><td>8.4</td><td>8.2</td><td>8317</td></tr><tr><td>0</td><td>0</td><td>0.1</td><td>1.5</td><td>0.3</td><td>1.6</td><td>1.1</td><td>1.0</td><td>318</td></tr><tr><td rowspan="2">EPS DR</td><td>0</td><td>0</td><td>3.0</td><td>6.5</td><td>1.3</td><td>10.3</td><td>9.4</td><td>9.1</td><td>320</td></tr><tr><td>0</td><td>0</td><td>0.4</td><td>0.8</td><td>0.2</td><td>1.2</td><td>1.1</td><td>1.0</td><td>321</td></tr><tr><td rowspan="2">EPS DR-SD</td><td>0</td><td>0</td><td>0.4</td><td>9.3</td><td>0</td><td>0</td><td>8.4</td><td>15.7</td><td>322</td></tr><tr><td>0</td><td>0</td><td>0</td><td>0.8</td><td>0</td><td>0</td><td>0.6</td><td>1.1</td><td>323</td></tr><tr><td rowspan="2">EPS OF1</td><td>0</td><td>0</td><td>0.2</td><td>8.3</td><td>3.2</td><td>13.9</td><td>13.9</td><td>11.8</td><td>326</td></tr><tr><td>0</td><td>0</td><td>0</td><td>0.8</td><td>0.3</td><td>1.3</td><td>1.1</td><td>1.1</td><td>327</td></tr><tr><td rowspan="2">EPS OF2</td><td>0</td><td>0</td><td>11.3</td><td>6.8</td><td>3.7</td><td>10.6</td><td>10.2</td><td>9.3</td><td>328</td></tr><tr><td>0</td><td>0</td><td>1.3</td><td>0.8</td><td>0.4</td><td>1.1</td><td>1.1</td><td>0.9</td><td>329</td></tr><tr><td rowspan="2">EPS OF3</td><td>0</td><td>0</td><td>3.8</td><td>9.6</td><td>2.6</td><td>13.2</td><td>13.7</td><td>12.8</td><td>330</td></tr><tr><td>0</td><td>0</td><td>0.3</td><td>0.8</td><td>0.2</td><td>1.0</td><td>1.0</td><td>0.9</td><td>331</td></tr></tbody></table>

Rha: rhamnose, Fuc: fucose, Man: mannose, Gal: galactose, Glc: glucose, GlcA: glucuronic acid, GalA: galacturonic acid, GlcNAc: N-Acetyl-glucosamine, GalNAc: N-Acetyl-galactosamine.

Glycosyl linkage positions determined on native EPS enabled the identification of monosaccharides involved in polymeric structures, accounting for 7% of the initial mass of the native EPS sample, equivalent to 14% of the total native EPS content (Table 2, Figure S1 A-G). The remaining unidentified structures are likely inaccessible to methylation and/or hydrolysis. Among the identified linkages, (1→3)-linked Gal and (1→3)-linked GalA are the most abundant, representing 30.9% and 26.2% of the total linked monosaccharides, respectively. GlcA residue is detected as (1→4)-linked, accounting for 19% of the total. Additionally, the hexosamines GlcNAc and GalNAc are found as (1→3)- and (1→4)-linked residues, representing 10.2% and 8.3% of the total linked monosaccharides, respectively.
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<caption>Table 2. Glycosyl-linkage analysis on native EPS. Data were expressed in molar percentage of total sugar.</caption>

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<table><thead><tr><th>Number / RT</th><th>Linkages type</th><th>Molar ratio (%)</th><th>Mass fragments (m/z)</th></tr></thead><tbody><tr><td>1 / 42.18min</td><td>Glc-term</td><td>2.4</td><td>102, 118, 145, 162, 205 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>2 / 42.29min</td><td>Man-term</td><td>1.9</td><td>102, 118, 145, 162, 205 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>3 / 45.66min</td><td>(1,4)-linked Glc</td><td>1.0</td><td>102, 113, 118, 129, 131, 162, 173, 233 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>3 / 45.66min</td><td>(1,4)-linked GlcA</td><td>19.0</td><td>102, 113, 118, 129, 131, 162, 173, 233 (NaBH<sub>4</sub>/NaBD<sub>4</sub>) & 102, 113, 118, 129, 133, 162, 175, 235 (NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>4 / 45.95min</td><td>(1,3)-linked Gal</td><td>30.9</td><td>101, 118, 129, 161, 174, 234 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>4 / 45.95min</td><td>(1,3)-linked GalA</td><td>26.2</td><td>101, 118, 129, 161, 174, 234 (NaBH<sub>4</sub>/NaBD<sub>4</sub>) & 101, 118, 131, 163, 174, 234 (NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>5 / 54.27min</td><td>(1,4)-linked GalNAc</td><td>8.3</td><td>113, 117, 129, 159, 233 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td>6 / 55.13min</td><td>(1,3)-linked GlcNAc</td><td>10.2</td><td>117, 129, 159 (NaBH<sub>4</sub>/NaBD<sub>4</sub> & NaBD<sub>4</sub>/NaBD<sub>4</sub>)</td></tr><tr><td></td><td>Yield (wt%)</td><td>7.0</td><td></td></tr></tbody></table>

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## 3.3 Elucidation of the EPS structure

For structure elucidation, native HMW EPS was depolymerized using radical depolymerization process to obtain LMW derivative, EPS DR of Mw 275,000 g/mol. Its monosaccharide composition was similar to native EPS (Table 1) with five main monosaccharides present, namely Gal, GlcA, GalA, GlcNAc and GalNAc. Indeed, radical depolymerization was previously shown to efficiently decrease the molecular weight through the cleavage of the main chain, while the polysaccharide fine structure, either in branched or linear polysaccharides, was highly conserved [41,42]. No differences between native EPS and its LMW depolymerized derivative, EPS DR, were observed on ATR-FTIR spectra (Figure 2). Spectra were normalized with respect to the C=O stretching band of carboxylic acid group at 1650 cm⁻¹ to better visualize the differences between the samples. In addition to this band, a typical broad band corresponding to O-H at 3600-3000 cm⁻¹ was clearly observed, together with an important absorption band at 1100-1010 cm⁻¹ attributed to the C-O stretching vibration of pyranosyl rings.

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

Figure 2. ATR-FTIR normalized spectra with respect to the C=O band at 1650 cm⁻¹ of native EPS atosan from *V. alginolyticus* AT1219 strain, depolymerized LMW derivative (EPS DR) and LMW derivative after periodate oxydation (EPS Ox).
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Due to HMW of native EPS (3,000,000 g/mol) preventing from interpretation of its ¹H NMR spectrum (Fig. S2 A), NMR study was firstly performed on its LMW derivative, EPS DR of Mw 275,000 g/mol. The ¹H NMR spectrum of EPS DR showed the presence of five main signals in the region 4.4-5.5 ppm (Figure 3A), which were attributed to anomeric signals of five sugar residues, consistent with a regular polymer with a pentasaccharide repeating unit. Two additional signals at 2.01 and 2.04 ppm were indicative of the presence of two N-acetylated residues (Figure S2 B). However, low resolution of the NMR spectra of EPS DR prevented an unambiguous characterization of all the sugar residues and their glycosyl linkages for detailed structural study. Therefore, in order to obtain better resolved NMR spectra, EPS DR was subjected to a Smith degradation, which consists in periodate oxidation of polysaccharides, leading to specific oxidation of free vicinal hydroxyl groups to aldehydes with internal ring cleavage [27, 43]. Polyaldehydes thus formed are subsequently reduced to polyalcohols. Mild acidic conditions then applied lead finally to hydrolysis of the acetal linkage of the sugar oxidized during the reaction. Based on the glycosyl linkage analysis (Table 2), only 4-GlcA residue can undergo Smith degradation and form erythronic acid (EryA). Successful modification of LMW derivative leading to EPS DR-SD was confirmed by the monosaccharide analysis (Table 1), revealing that GlcA residue was totally absent in the sample. Moreover, a considerable increase in the intensity of the absorption band at 1400 cm⁻¹, corresponding to asymmetric stretching of CH₂ group was observed on ATR-FTIR spectrum of EPS that has undergone periodate oxidation (EPS Ox, Figure 2). This characteristic band indicated that polyalcohols were efficiently formed after reduction step applied to the oxidized sample.
A

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

B

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

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Figure 3. ¹H NMR spectra of (A) LMW EPS derivative (EPS DR) and (B) LMW EPS derivative obtained following Smith degradation (EPS DR-SD).

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The ¹H (Figure 3B) and ¹³C (Figure S3) NMR spectra of EPS DR-SD showed the presence of four anomeric signals labelled **A** to **D** in order of decreasing chemical shifts of their respective H-1 protons at 5.19/98.3 ppm (**A**), 5.09/100.7 ppm (**B**), 4.78/104.4 ppm (**C**) and 4.48/105.9 ppm (**D**) (Table S1). The concomitant presence of two signals for N-linked carbon at 52.9/57.3 ppm and signals at 2.02-2.03/177.2-177.7 ppm were consistent with the presence of two N-acetylated glycosylamine residues. Two other signals in the carbonyl region for acidic residues at 177 ppm and 180.5 ppm were also observed.

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Based on the monosaccharide composition of EPS DR-SD (Table 1), the ¹H/¹³C chemical shifts determined from HSQC experiments (Figure 4A, Table S1), **A** was readily attributed to a Gal residue at the non-reducing end position with an α configuration (³J₁,₂ = 3.9 Hz). The monosaccharide composition of EPS DR-SD indicated the presence of two hexosamine residues, which could be attributed to residues **B** and **C** based on the chemical shift of their C-2 carbons at 52.9 and 57.3 ppm, respectively. ¹H/¹³C chemical shifts of **B** were in good agreement with those expected for a GalNAc residue in a α configuration (J₁,₂ = 3.7 Hz, Table S1), whereas **C** was identified, in accordance with the sugar composition of EPS DR-SD, as a

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GlcNAc residue from its C-2, C-4 and C-5 chemical shifts. Moreover, the chemical shifts at 78.5 ppm for **B** C-4 and 86.5 ppm for **C** C-3 were indicative of a particular linkage on these positions with **B** 4-linked and **C** 3-linked. An intra-residual correlation on the HMBC spectrum (Figure 4B) between **D** H-5 and a carbon at 177.03 ppm allowed to identified **D** as an uronic acid residue with a β-anomeric configuration ($J_{1,2}$ = 8.1 Hz). Attribution of **D** as a GalA was done on the basis of the chemical composition of EPS DR-SD and the relative deshielded values observed for the H-4 and H-5 resonances [44]. An additional spin system composed of three ¹H at 4.24, 3.94 & 3.73 ppm and four ¹³C signals at 63.2, 83.9, 75.9 & 180.5 ppm was also characterized (Table S1) and was shown to correspond to expected EryA aglycon originating from the Smith degraded 4-linked GlcA unit. Glycosyl sequence of EPS DR-SD was established from the ¹H/¹³C correlations observed on the HMBC spectrum (Figure 4B). Inter-residual connectivities **A** H-1/ **D** C-3, **D** H1/ **C** C-3, **C** H-1/ **B** C4 and **B** H-1/ **E** C-3 allowed to assign the sequence **I** for EPS DR-SD:

I) α-D-Galp-(1→3)-β-D-GalpA-(1→3)-β-D-GlcpNAc-(1→4)-α-D-GalpNAc-(1→3)-EryA

The resistance to cleavage of the Gal residue after Smith degradation was indicative of a substitution on position 3, in agreement with permethylation analysis (Table 2). Furthermore, the anomeric configuration of the GlcA residue was deduced from the ¹H spectrum of EPS DR, showing the presence of two α and three β residues. Two α and two β units were also identified for EPS DR-SD, thus the GlcA residue was shown to be in a β configuration. Therefore, the sequence (II) for the repeating unit of EPS DR was shown to be:
438 **II)** →3)-α-D-Galp-(1→3)-β-D-GalpA-(1→3)-β-D-GlcNAc-(1→4)-α-D-GalpNAc-

439 (1→4)-β-D-GlcNAc-(1→

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

<figure><img src="image_8.png" /><figcaption>Figure 4. (A) HSQC and (B) HMBC NMR spectra of LMW EPS derivative obtained following Smith</figcaption></figure>

degradation (EPS DR-SD).

440 In order to confirm the polysaccharide repeating unit deduced from NMR analysis, mass

443 spectrometry was performed on the oligosaccharides released from native EPS after acid

444 hydrolysis. Three main fractions were recovered: EPS OF1, OF2 and OF3. Their

445 monosaccharide compositions were very similar (Table 1). Molar ratio indicated that the EPS

446 repeating unit is constituted of five monosaccharides, including 1 Gal, 1 GlcA, 1 GalA, 1

447 GlcNAc and 1 GalNAc. Intermediate fraction EPS OF2 was then selected for further analysis

448 by UHPLC-MS. Ion-pairing reversed-phase chromatography separation was used because it

449 allows efficient separation of molecules according to the number of carboxylic acid functions

450 present in their structure. Analysis of the fraction revealed the presence of more than 30 species

451 with degree of polymerization (DP), being a number of monosaccharide residues, from DP4 to
452 DP15 (Table S2).

453 To study in more depth the structure of the species present in the fraction OF2, ion mobility

454 spectrometry (IMS), instead of LC-MS, was used to separate isomers. IMS is a gas-phase

455 separation method (based on the interaction of the ionized species with a buffer gas and an
electric field) performed post-ionization in the mass spectrometer, and it therefore allows to acquire MS/MS data over extended periods of time in direct infusion, yielding better quality spectra and thus more structural information compared to LC-MS. Before the IMS separation, the first step consisted in oligosaccharide analysis through MS experiment. On the MS spectrum (Figure 5A), the major species that had already been observed in UHPLC-MS were identified.

<figure><img src="image_9.png" /><figcaption>Figure 5. IMS-MS experiment on EPS OF2 performed in negative ion mode. (A) MS spectrum of the fraction with mass-to-charge ratios annotated as m/z(z), with z being the charge of the ion. Major species are annotated according to the Symbol Nomenclature for Glycans (SNFG) [45]: circle: hexose, square: N-Acetylated hexosamine, divided diamond: hexuronate. Ac: acetylated form of an annotated species. Black star: ion corresponding to in-source fragmentation of larger species. (B) Zoom around the DP10 on the MS spectrum. (C) Arrival time distribution for the DP10 after high-resolution separation in the cyclic IMS cell (blue dot points) and result of the gaussian deconvolution (dotted lines and black line).</figcaption></figure>
Further mass spectrometry analysis was carried out on DP10 (Figure 5B), chosen for two reasons. Firstly, it is one of the largest oligosaccharides released by acid hydrolysis, which is long enough to characterize the EPS repeating unit. Secondly, this DP10 has sufficient intensity on the MS spectrum to consider IMS separation and to obtain informative MS/MS spectra.

After high-resolution separation through the ion mobility cell, a complex arrival time distribution (ATD) was obtained, corresponding to numerous unresolved IMS peaks (blue dot points, Fig. 5C), likely DP10 isomers. In order to assess the number of isomers and because the ion mobility peaks are expected to be Gaussians, a Gaussian Fitting deconvolution of the ATD was performed (dotted lines and black line, Figure 5C). The Gaussian fit distinguished ten peaks, three of which had sufficient intensities for MS/MS experiments (arrival times 88.5, 89.5 and 95.7 ms). To facilitate interpretation and avoid ambiguous interpretation of fragments, the sample EPS OF2 sample was labelled with heavy oxygen for IMS-MS/MS analysis. As a result, a ¹⁸O-isotopic tag is present at the reducing end of the oligosaccharides, inducing a mass shift of +2 Da. The DP10 measured doubly charged at m/z 928.29 in IMS-MS was therefore isolated at m/z 929.29 for IMS-MS/MS analysis (Figure S4). On the IMS-MS/MS spectra of the three isomers (Figures S5, S6 and S7), the fragments obtained confirm the repetition of the sequence Gal-GalA-GlcNAc-GalNAc-GlcA highlighted in NMR experiments. The structures of these three isomers presented in Table 3 reflect the random cuts expected during acid hydrolysis.

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Table 3. Summary of the DP10 characterized by IMS-MS/MS in EPS OF2 ($t_A$: arrival time). Structures were depicted according to the Symbol Nomenclature for Glycans (SNFG) [45]. The repeating unit was surrounded by red dotted lines.

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

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Linear pentasaccharide repeating unit described for atosan, [→3)-α-D-Galp-(1→3)-β-D-GalpA-(1→3)-β-D-GlcNAc-(1→4)-α-D-GalpNAc-(1→4)-β-D-GlcNAc-(1→], remains unique compared to other EPS already described for Vibrio sp. [8,18,19]. A very similar repeating unit was reported for EPS produced by another V. alginolyticus strain (CNCM I-4151) constituted of a tetrasaccharide: [→4)-β-D-GlcA-(1→3)-α-D-Gal-(1→3)-α-D-GalA-(1→3)-β-GlcNAc(1→] [19]. In comparison to this EPS, atosan has additional GalNAc residue between GlcNAc and GlcA residues. Uronic acid (GlcA) and hexosamines (GlcNAc, GalNAc) present in atosan structure are also characteristic of GAGs of mammalian tissues, such as hyaluronic acid (GlcA, GlcNAc) and unsulfated chondroitin sulfate (GlcA, GalNAc) [46,47]. In similar manner to GAGs, anionic nature of atosan and particular its monosaccharide sequence may provide some biological activities of interactions with enzymes, signaling proteins (e.g. growth factors, chemokines) and fibrillar proteins (e.g. collagen) that can be exploited for biomedical applications (regenerative medicine, cancer research). Contrary to GAGs, atosan presents a
GalA found mainly in polysaccharides of plant cell walls such as pectin or in some bacterial lipopolysaccharides and for which the biosynthetic origin is not totally understood in bacteria and even less in marine bacteria such as Vibrio sp [48].

### 3.4 Genetic clusters involved in polysaccharide biosynthesis

The genome sequence was investigated to get insight into the genetic mechanisms of atosan biosynthesis by V. alginolyticus AT1219 strain. GT encoded in the AT1219 genome were identified using dbcan tools implemented in Microscope platform (Table S3) [24,25]. Based on the genomic context and functions of GT, genetic clusters potentially involved in the biosynthesis were further identified (Table S4 and S5). Among eight clusters identified (Figure S8), three main genetic clusters, most probably related to the biosynthesis of the extracellular polysaccharide, were presented in Figure 6.

Two cazymes were identified in contig 10 (genes labeled AT1219_v1_100135 and 100136). They both belong to GT2 family and are annotated as cellulose synthase periplasmic and catalytic subunits, respectively (Figure 6). Cellulose synthase consists in two subunits, BcsA and BcsB, and catalyzes the formation of cellulose from nucleotide glucose. In this genetic cluster, other seven genes were identified; bscC encodes BcsC involved in the export and crystallization of bacterial cellulose microfibrils with the protein BcsD, absent from this gene cluster. The gene bcsZ encodes an endo-β-1-4-glucanase, which is required for bacterial cellulose production; it may degrade accumulated cellulose in the periplasm and cleave nascent cellulose chains to allow microfibril formation occurring outside the cell [49,50]. Similarly to Escherichia coli cellulose biosynthetic genetic cluster described by Krasteva et al. [51], the presence of bcsE and bcsG groups and absence of bcsD are characteristic of group II of bacterial cellulose clusters, as described by Römling et al. [50].

The cps biosynthetic genetic cluster was identified with two GT4 (AT1219_v1_10177 and AT1219_v1_10178). This cluster presents synteny with the cps locus of V. diabolicus which
was described as a capsular polysaccharide or a KLPS biosynthetic cluster. Indeed, the gene AT1219_v1_10179 is annotated as a Lipid A ligase which might suggest that the capsular polysaccharide of AT1219 strain could be linked to Lipid A (KLPS) [52].

The *syp* cluster contains genes assigned as symbiosis polysaccharide biosynthetic genes (*syp*), which are in synteny with the *syp* cluster of *V. diabolicus* and several other *V. alginolyticus* strains. It contains one priming GT which initiates the biosynthesis of the repeating unit by transferring the first nucleotide sugar to a membrane acceptor (AT1219_v1_90063), one GT2 (AT1219_v1_90064), and five GT4 (AT1219_v1_90065, AT1219_v1_90067, AT1219_v1_90070, AT1219_v1_90071, AT1219_v1_90072) probably involved in the chain elongation. By inactivating the flippase gene of *V. diabolicus* (homolog to AT1219_v1_90069), Goudenège et al. (2014) highlighted the role of the *syp* cluster in the biosynthesis of the EPS diabolican (HE800 strain).

<figure><img src="image_11.png" /></figure>

Figure 6. Schematic representation of *bcs*, *cps*, and *syp* loci in *V. alginolyticus* AT1219. Genes are represented by oriented arrows, encoded proteins are indicated in the arrows. GT are in blue (GT2) and green (GT4); other non-GT protein genes are indicated by gray arrows.

Five other loci containing GT were identified (glycogen, *mal*, *ara*, *lps*, and *mur-ftS*) (Figure S8). The glycogen locus shows that *V. alginolyticus* AT1219 might accumulate intracellular glycogen as a reserve polymer from Glc-1-P using *GlcC* and *GlcA* enzymes
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(AT1219_v1_20080 and AT1219_v1_20081); glycogen is composed of α-1,4-linked Glc
subunits with α-1,6-linked Glc branches [53]. The *ara* genes have been shown to link arabinose
to membrane polysaccharides such as arabinogalactan [54]. Then a long *lps* locus has been
identified (52918 bp and 53 genes); it includes genes for Lipid A, inner and outer core
oligosaccharide (GT25, GT9, GT30: AT1219_v1_50160, AT1219_v1_50161,
AT1219_v1_50162, AT1219_v1_50163), together with several GT2 involved in the O-antigen
biosynthesis, and enzymes catalyzing the production of nucleotide sugars (AT1219_v1_50168
to AT1219_v1_50172). This suggests that this locus includes the whole set of genes for
lipopolysaccharide (LPS) biosynthesis. LPS located in the outer cell membrane of Gram-
negative bacteria provide cell integrity, can be related to pathogenicity but are usually endowed
with low virulence activity [55-57]. The *mal* cluster contains genes potentially involved in the
growth on maltose as the substrate and/or maltodextrin biosynthesis from maltose
(AT1219_v1_60036 is MalQ protein and AT1219_v1_60037 is MalP). The locus also contains
the transcriptional regulator MalT (AT1219_v1_60039), that is exclusively activated by
maltotriose [58]. The last genetic cluster (*mur-fts*) contains one GT (AT1219_v1_80088) and
several genes involved in peptidoglycan metabolism (*mur*) and in cell division (*fts*). This clearly
indicates that this locus is not involved in EPS biosynthesis. By homology, it appeared that the
*syp* locus of *V. alginolyticus* AT1219 might be involved in the biosynthesis of atosan EPS. This
cluster is widely present in the strains of the *alginolyticus* clade and exhibits high homology
between strains [59]. Although *syp* loci of *V. diabolicus* and *V. alginolyticus* AT1219 are very
similar, both EPS structures are different. The genetic cluster includes a set of GT in excess
[52], and encodes several proteins involved in regulation, indicated a complex biosynthesis.

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4. Conclusion

V. alginolyticus AT1219 strain isolated from deep-sea hydrothermal vent environment was shown to synthesize a unique extracellular polysaccharide, named atosan. A large panel of complementary analytical techniques was applied to elucidate the polysaccharide fine structure. Monosaccharide composition revealed the presence of five osidic residues with the molar ratio 1. NMR spectroscopy allowed to determine the sequence of linked monosaccharides, in accordance with the glycosidic linkages revealed by permethylation analysis. Finally, mass spectrometry was used to confirm the pentasaccharide repeating unit of atosan: [→3)-α-D-Galp-(1→3)-β-D-GalpA-(1→3)-β-D-GlcpNAc-(1→4)-α-D-GalpNAc-(1→4)-β-D-GlcpA-(1→]. The presence of uronic acids and hexosamines in the EPS structure mimics the composition of GAGs, in particular hyaluronic acid. Negatively charged atosan may interact with positively charged proteins and find its use in biomedical applications, such as tissue engineering and cancer research. Functional, physicochemical and biological properties of this new polysaccharide will be explored to determine if the particular monosaccharide sequence revealed in this study confers to the polysaccharide some valuable properties.

V. alginolyticus AT1219 genome includes several genetic loci potentially involved in the biosynthesis of polysaccharide. Although some loci were assigned to cellulose, maltodextrin, and glycogen biosynthesis, these polysaccharides were not detected in the supernatant of the culture broth. Our study strongly suggests that the *syp* locus of *V. alginolyticus* AT1219 may be involved in the biosynthesis of atosan. However, the enzymes identified in the genetic clusters should further be investigated to uncover their function in the polysaccharide biosynthesis. This requires knock out studies based on genetic tools to transform and mutate wild type *V. alginolyticus* AT1219. This future research work would also expand our understanding of the polysaccharide biosynthesis and relationship between gene cluster and EPS structure, and could help in developing applications in various industrial fields.
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**Declarations of competing interest:** The authors declare no conflict of interest.

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**Acknowledgements**

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Financial support was provided by the French National Research Agency (ANR) within the framework of the *FunCapsul* project (ANR-17-CE08-0001). The LABGeM (CEA/Genoscope & CNRS UMR8030), France Genomique and French Institute of Bioinformatics (funded as part of Investissement d'Avenir program managed by ANR, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform. We acknowledge Corinne Sinquin and Véronique Verrez-Bagnis from Ifremer for her involvement in EPS production and separation, and bioinformatic analysis, respectively. Elodie Fonseca Nascimento (Ifremer) is acknowledged for the initial microbiological study. SEBIMER colleagues are acknowledged for their help in genome assembling (celia workflow) and sequence submission to ENA. Structural characterization of native EPS by linkage analysis and oligosaccharides by mass spectrometry were performed on the BIBS instrumental platform (DOI: 10.15454/1.5572358121569739E12; http://www.bibs.inrae.fr/bibs_eng/, UR1268 BIA, IBiSA, PROBE infrastructure, Biogenouest).

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A CC-BY public copyright license has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission, in accordance with the grant's open access conditions.

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615
616 **Author contribution**

617 **Agata Zykwinska:** Conceptualization, Methodology, Investigation, Formal analysis, 618 Validation, Funding acquisition, Writing – Original Draft, Writing – Review & Editing; **Méline 619 Calatraba:** Methodology, Investigation, Formal analysis; **Sandrine Bonnetot:** Methodology, 620 Investigation, Formal analysis; **Laëtitia Marchand:** Methodology, Investigation, Formal 621 analysis; **Mathieu Fanuel:** Methodology, Investigation, Formal analysis, Validation, Writing 622 – Original Draft, Writing – Review & Editing; **Simon Ollivier:** Methodology, Investigation, 623 Formal analysis, Validation, Writing – Review & Editing; **David Ropartz:** Methodology, 624 Investigation, Writing – Review & Editing; **Sophie Le Gall:** Methodology, Investigation, 625 Formal analysis, Validation, Writing – Original Draft, Writing – Review & Editing; **Loric 626 Thoulouze:** Methodology, Formal analysis, validation; **Bérengère Marais:** Methodology, 627 Formal analysis; **Stéphane Cuenot:** Methodology, Investigation, Formal analysis, Validation, 628 Writing – Review & Editing; **Stéphane Cerantola:** Methodology, Investigation, Formal 629 analysis, Validation, Writing – Original Draft, Writing – Review & Editing; **Sylvia Colliec- 630 Jouault:** Conceptualization, Investigation, Writing – Review & Editing; **Christine Delbarre- 631 Ladrat:** Conceptualization, Methodology, Investigation, Formal analysis, Validation, Writing 632 – Original Draft, Writing – Review & Editing.

633
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* A fine structure of a new bacterial exopolysaccharide (EPS) from marine origin was determined using complementary analytical approach

* Nuclear Magnetic Resonance spectroscopy and mass spectrometry revealed a unique pentasaccharide repeating unit

* Glycosaminoglycan-mimetic chemical composition of the EPS was evidenced

* The investigation of the genome sequence led to identification of main clusters responsible for polymer biosynthesis

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**Declaration of interests**

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Agata Zykwinska, a member of Carbohydrate Research Editorial Board

Given her role as Editorial Board Member, had no involvement in the peer review of this article and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to another journal editor. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.