Genomic attributes and characterization of novel exopolysaccharide-producing bacterium Halomonas piscis sp. nov. isolated from jeotgal

Halophilic bacterial strains, designated SG2L-4T, SB1M4, and SB2L-5, were isolated from jeotgal, a traditional Korean fermented food. Cells are Gram-stain-negative, aerobic, non-motile, rod-shaped, catalase-positive, and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain SG2L-4T is closely related to Halomonas garicola KACC 18117T with a similarity of 96.2%. The complete genome sequence of strain SG2L-4T was 3,227,066 bp in size, with a genomic G + C content of 63.3 mol%. The average nucleotide identity and digital DNA–DNA hybridization values between strain SG2L-4T and H. garicola KACC 18117T were 90.5 and 40.7%, respectively. The optimal growth conditions for strain SG2L-4T were temperatures between 30 and 37°C, a pH value of 7, and the presence of 10% (w/v) NaCl. The polar lipids identified included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid, an unknown glycolipid, and an unknown polar lipid. The major cellular fatty acids were C16:0, summed features 8 (C18:1ω6c and/or C18:1ω7c), C19:0 cyclo ω8c, and summed features 3 (C16:1ω6c and/or C16:1ω7c). The predominant respiratory quinone was ubiquinone with nine isoprene units (Q-9). Based on the phenotypic, genotypic, and chemotaxonomic results, strain SG2L-4T represents a novel species within the genus Halomonas, for which the name Halomonas piscis sp. nov. is proposed. The type strain is SG2L-4T (=KCTC 92842T = JCM 35929T). Functional annotation of the genome of strain SG2L-4T confirmed the presence of exopolysaccharide synthesis protein (ExoD) and capsular polysaccharide-related genes. Strain SG2L-4T also exhibited positive results in Molisch’s test, indicating the presence of extracellular carbohydrates and exopolysaccharides (EPS) production. These findings provide valuable insights into the EPS-producing capabilities of H. piscis sp. nov. isolated from jeotgal, contributing to understanding its potential roles in food and biotechnological applications.


Introduction
Jeotgal is a traditional Korean fermented food similar to kimchi, doenjang, and makgeolli.It is typically prepared by salting and fermenting seafood in brine for an extended period, resulting in a highly savory and pungent condiment that is used to enhance the flavor of various Korean dishes.Comprising primary ingredients such as eggs, gut, or whole body of fish or shellfish, jeotgal serves both as a standalone dish or sauce and as a flavor and fermentation enhancer when incorporated into other fermented foods like kimchi.The complex microbial community developed throughout jeotgal fermentation and its metabolites significantly affect the taste, flavor, and texture of the final product (Jung et al., 2018).The bacterial communities usually include the genera Bacillus, Lactobacillus, Pseudomonas, Tetragenococcus, Sporosarcina, Virgibacillus, and Halomonas (Guan et al., 2011;Song et al., 2018).Among these, Halomonas species are prominent halophilic bacteria that thrive under the high-salinity conditions characteristic of jeotgal, and are expected to contribute to promoting jeotgal fermentation.
Halomonas, a genus initially discovered by Vreeland in solar salterns, was reported with the description of Halomonas elongate (Vreeland et al., 1980).The genus Halomonas encompasses 121 species isolated from various saline environments such as saline lakes, salt flats, seawater, fermented fish sauce, and saline-alkali soils (Poli et al., 2007;Jiang et al., 2014;Li et al., 2022;Yoo et al., 2022;Lin et al., 2023).These bacteria, characterized by Gram-stain-negative, halophilic, rod-shaped, and catalase-positive, can grow under aerobic or facultative anaerobic conditions and exhibit a wide range of physiological and metabolic features.
One notable metabolic characteristic among Halomonas species is their capacity to produce exopolysaccharides (EPS).For instance, H. rifensis HK31 T , H. ventosae Al12 T , and H. maura S31 T have been reported to possess the capability of EPS production, as confirmed by the presence of EPS on the cell surfaces through scanning electron microscope (SEM) analysis (Bouchotroch et al., 2001;Martínez-Cánovas et al., 2004;Amjres et al., 2011).EPS are high-molecularweight, long-chain carbohydrates secreted by microorganisms into their extracellular environment.Depending on their constituent sugars, these biopolymers can be categorized as homopolysaccharides, which consist of a single type of monosaccharide, or heteropolysaccharides, which consist of multiple monosaccharide types (Ruas-Madiedo et al., 2002).EPS play a pivotal role in various biological processes and have diverse applications (Poli et al., 2007;Joulak et al., 2019Joulak et al., , 2022)).In microbial communities, EPS contribute significantly to biofilm formation, providing structural integrity and protection against environmental stressors (Ghafoor et al., 2011).From an industrial perspective, microbial EPS have substantial commercial potential because of their natural occurrence, ease of extraction, low purification costs, and safety for both humans and the environment (Qi et al., 2022).For instance, Lactobacillus fermentum Lf2-extracted EPS improves yogurt texture and water-holding capacity, being a valuable natural ingredient with technological benefits (Ale et al., 2016).H. saccharevitans AB2, isolated from algae and rocks in the Red Sea, produces EPS with antimicrobial and antitumor activities (Abdrabo et al., 2018).Furthermore, administration of the EPS-enriched fraction obtained from the culture of Bacillus subtilis J92 improved intestinal barrier integrity and reduced inflammation in a dextran sulfate sodium-induced colitis mouse model, suggesting its potential as a pharmaceutical agent for inflammatory bowel disease management (Chung et al., 2021).These reports highlight the versatile applications of EPS across multiple fields, including food and pharmaceutical industries.
In this study, we have characterized three strains of the novel species Halomonas piscis sp.nov., isolated from jeotgal, using a polyphasic approach.Furthermore, we highlighted the potential uses of these novel strains by confirming the EPS production capability of H. piscis.

Isolation and culture conditions of Halomonas strains
Jeotgal samples were purchased from a traditional market in Jeongeup-si, Jeollabuk-do, Republic of Korea (35°33′47.0"N,126°51′19.0″E).Strains SG2L-4 T , SB1M-4, and SB2L-5 were isolated from yellow corvina (Collichthys lucidus) jeotgal or mixed (shrimp, yellow corvina, pilchards, and anchovy) jeotgal and ripened for approximately 2 years with a final salinity of about 50% (w/v).The supernatants of the jeotgal samples were serially diluted 10 −1 and 10 −2fold using sterile phosphate-buffered saline (Bioneer, Republic of Korea).One hundred microliters of diluted samples were spread onto Trypticase Soy Agar (TSA; Becton, Dickinson and Company, NJ, United States), Brain Heart Infusion Agar (Becton, Dickinson and Company), Gifu anaerobic medium (Nissui Pharmaceutical Co., Ltd., Japan), R2A Agar (Becton, Dickinson and Company), Marine Broth (Becton, Dickinson and Company), and Lactobacilli MRS Broth (Becton, Dickinson and Company) with 10% (w/v) NaCl, and then incubated at 25°C for 5 days.Colonies were randomly selected based on their morphological features and transferred to a fresh medium to purify a single colony.The isolated pure colonies underwent 16S rRNA gene sequence analysis and were preserved at −80°C in 40% glycerol stock.Reference strains for comparative analysis included three species of the genus Halomonas: H. garicola KACC 18117 T and H. cibimaris KACC 14932 T obtained from the Korean Agricultural Culture Collection (KACC), and H. jeotgali KCTC 22487 T obtained from the Korean Collection for Type Cultures (KCTC).

Genomic features
Whole-genome sequencing was performed at a Macrogen facility (Macrogen, Republic of Korea).Genomic DNA was extracted from strain SG2L-4 T and sequenced using the PacBio Sequel system and Illumina HiSeq sequencing platform.The PacBio reads were used for de novo assembly via Microbial Genome Assembly application.Reads were mapped against assembled contigs, and polishing was performed using Racon, resulting in a consensus sequence of higher quality.Illumina reads were applied after assembly to enhance the genome sequence's accuracy, utilizing Pilon software for polishing and correcting errors as well as filling gaps.The genomic DNA of strains SB1M-4, SB2L-5, and H. garicola KACC 18117 T was employed for draft genome sequencing using the Illumina HiSeq platform.The G + C content was determined using genomic DNA sequencing data.A phylogenomic tree was constructed using the Up-To-data Bacterial Core Genes pipeline and FastTree program (Na et al., 2018).Genome sequences of species closely related to the novel strains were obtained from the publicly available NCBI GenBank website.The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolated genomes and closely related strains were calculated using the OrthoANI tool4 (Lee et al., 2016) and Genome-to-Genome Distance Calculator v3.0, on the DSMZ server5 (Meier-Kolthoff et al., 2013), respectively.Functional annotation of the genome sequences was performed using the Rapid Annotation using Subsystem Technology (RAST) server6 (Aziz et al., 2008).A circular map of the annotated genome was generated using the CGView online service (Stothard et al., 2019).

Morphological and physiological test
Phenotypic characterizations of strains SG2L-4 T , SB1M-4, and SB2L-5, and reference strains were determined by culturing them in TSA containing 10% (w/v) NaCl at 30°C for 2 days.Cell morphology and the presence of flagella were determined using a JEOL JSM 7600F field emission SEM (JEOL Ltd., Japan) after fixation with 3% (w/v) glutaraldehyde at 4°C for 24 h (Li et al., 2019).A motility test was performed by culturing cells in 0.3% (w/v) semi-solid TSA with 10% (w/v) NaCl.Gram reactions were determined using 3% KOH (Powers, 1995).Catalase activity was examined for bubble production using 3% (v/v) hydrogen peroxide.Oxidase activity was tested by applying 1% (w/v) tetramethyl-p-phenylenediamine (bioMérieux, France) onto the colonies on filter paper (Kovacs, 1956).A positive reaction resulted in dark purple colonies within 5-10 s.Lactose fermentation ability was assessed on MacConkey agar (Becton, Dickinson and Company) with 10% (w/v) NaCl.Anaerobic growth was examined on TSA with 10% (w/v) NaCl under anaerobic conditions at 30°C for 20 days in an anaerobic chamber (Bactron 300; Sheldon Manufacturing, Inc., OH, United States) with an atmosphere of N 2 /CO 2 /H 2 (90:5:5, v/v/v).The ability to perform anaerobic respiration using nitrate or nitrite as an alternative electron acceptor was determined by adding 0.2 g/L of potassium nitrate or potassium nitrite to TSB with 10% (w/v) NaCl medium under anaerobic conditions, respectively.

Biochemical characterization
Metabolic activity was assessed using Biolog GEN III MicroPlate™ (Biolog Inc., CA, United States) containing 71 carbon sources and 23 chemical sensitivities.Cell suspensions were inoculated and incubated at 30°C for 24 h.Plates were monitored using a Biolog Microstation (Biolog Inc.) at a wavelength of 590 nm.Biochemical features and enzyme activities were determined using API 20NE and API ZYM (bioMérieux) according to the manufacturer's instructions.The Voges-Proskauer reaction was tested using VP medium (7 g/L peptone, 5 g/L dextrose, and 5 g/L dipotassium phosphate) containing 10% (w/v) NaCl.After incubation for 24 h, 5% (w/v) 1-naphthol and 40% (w/v) potassium hydroxide were added sequentially to the culture broth.The gluconate oxidation test was performed on the following medium: 1.5 g/L peptone; 1.0 g/L yeast extract; 1.0 g/L K 2 HPO 4 ; and 40 g/L potassium gluconate.After incubation at 30°C for 48 h, Benedict's reagent was added to the culture, and the mixture was boiled for 5-10 min.Tyrosine hydrolysis was tested on TSA supplemented with 10% (w/v) NaCl and 5 g/L tyrosine by observing a clear zone of disappearance of tyrosine crystals (Gordon and Smith, 1955).

Respiratory quinone
Respiratory quinone was extracted from freeze-dried cells using chloroform:methanol (2:1, v/v) according to a previously described method (Collins and Jones, 1981).Quinone was separated and analyzed using an HPLC system (Shimadzu Corp., Japan) equipped with a CBM-20A communication bus module, DGU-20A degassing unit, LC-20 AD pumps, a CTO-20A column oven, and an SPD-20A UV/VIS detector.The extracted quinone samples were isocratically eluted on ZORBAX SB-C18 ODS column (4.6 mm × 250 mm, 5 μm; Agilent Technologies, CA, United States) with degassed methanol:diisopropyl ether (3:1, v/v) as the eluent at a flow rate of 1 mL/min.Ubiquinone was detected at a UV detection wavelength of 275 nm.The quinone from H. garicola KACC 18117 T , known to contain ubiquinone 9 (Q-9), was analyzed as a standard to compare retention times.All samples were filtered through a 0.45 μm Advantech membrane filter.

Cellular fatty acid
Cellular fatty acid methyl esters were isolated, saponified, methylated, extracted, and washed according to the standard MIDI protocol (Sasser, 1990).Fatty acids were detected using gas chromatography-mass spectrometry and identified using the Microbial Identification Sherlock software package with the aerobic database version 6.1.

Examination of EPS production
To confirm EPS production, the novel strains were grown on a modified MY agar medium (10 g/L glucose, 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, and 100 g/L NaCl) at 30°C for 7 days (Quesada et al., 1993) and underwent SEM analysis.To obtain EPS from strains SG2L-4 T , SB1M-4, and SB2L-5, broth cultures incubated at 150 rpm at 30°C for 7 days were centrifuged at 7,000 rpm for 10 min.The cell-free supernatant was mixed with three volumes of cold absolute ethanol and kept at 4°C overnight.Subsequently, 4% (w/v) trichloroacetic acid was added to the pellet after centrifugation and stored at 4°C for 2 h to induce protein precipitation.After removing the protein precipitate, the remaining soluble protein was filtered through a 0.2 μm membrane filter.The obtained supernatant was treated with two volumes of cold absolute ethanol overnight at 4°C to separate the precipitate, which was then dried.The dried sample was dissolved in hot distilled water, dialyzed at 4°C for 24 h using dialysis tubing (MW cut off 12-14 kDa, Thermo Fisher Scientific), and lyophilized.Crude EPS yield was measured, and the presence of carbohydrates was determined by Molisch's test using glucose as the standard (Teekanam et al., 2023).
The general genomic features of strains SG2L-4 T , SB1M-4, and SB2L-5 and the reference strains are summarized in Table 4. Strain SG2L-4 T had a single circular chromosome of 3,227,066 bp in size, comprising a single contig containing 3,135 genes, including 3,055 protein-coding genes (CDS), 62 tRNA genes, and 18 rRNA genes.The estimated genome sizes of the strains SB1M-4 and SB2L-5 were 3,295,832 bp and 3,385,314 bp, respectively.The genome of strain SB1M-4 was assembled into 135 contigs with 2,871 CDS, 57 tRNA, and 6 rRNA genes, whereas that of strain SB2L-5 comprised 156 contigs, including 3,095 CDS, 61 tRNA, and 6 rRNA genes.The genomic G + C content of strains SG2L-4 T , SB1M-4, and SB2L-5 was 63.3, 64.0, and 63.3 mol%, respectively, similar to that of the closely TABLE 2 Differential biochemical characteristics between strains SG2L-4 T , SB1M-4, SG2L-5, and their closely related type strains.Strains: 1, SG2L-4 T ; 2, SB1M-4; 3, SB2L-5; 4, H. garicola KACC 18117 T ; 5, H. jeotgali KCTC 22487 T ; 6, H. cibimaris KACC 14932 T .All data were obtained from this study.Data are expressed as percentages of the total fatty acids.Major components (>10%) are highlighted in bold.Fatty acids constituting <0.5% of the total content in all strains are not displayed.tr, trace amount (<1%); −, not detected.*Summed features represent groups of two or three fatty acids that could not be separated via GLC using the MIDI system.Summed feature 3 contains C16 : 1 ω6c and/or C16 : 1 ω7c; summed feature 8 contains C18 : 1 ω6c and/or C18 : 1 ω7c. 10.3389/fmicb.2023.1303039 Frontiers in Microbiology 07 frontiersin.orgrelated strains (61.7-63.7 mol%).Annotated functional information for strains SG2L-4 T , SB1M-4, SB2L-5, and the reference strains is provided in Supplementary Figure S4, allowing for a comparison of similarities and peculiarities.Strain SG2L-4 T had 2,921 coding sequences and 299 subsystems, with the most represented subsystem features being "Amino Acid and Derivatives, " "Protein metabolism, " "Cofactors, Vitamins, Prosthetic Groups, Pigments, " and "Carbohydrates." Strain SG2L-4 T exhibited enrichment in genes associated with protein and RNA metabolism compared to other strains.These features maintain cellular functions, and adapt to environmental changes, contributing to cell health and survival (Anantharaman et al., 2002).In addition, their diversity enhances organismal functionality and adaptability, playing crucial roles in cell signaling and development processes.An annotated circular map including the genomic features of strain SG2L-4 T is shown in Figure 4.The outermost ring of the circular map highlights the presence of EPS synthesis-related gene, ExoD, which contributes to the formation of a protective film on the microbial cell surface.Similarly, it was observed that the reference strain H. garicola KACC 18117 T possesses both ExoD and capsular polysaccharide genes, whereas these genes were notably absent in H. jeotgali KCTC 22487 T .This finding demonstrates the EPS production capability of the strain SG2L-4 T and suggests that the produced EPS can be utilized environmentally friendly and  economically in various applications as a potential carbon source (Notararigo et al., 2013;Raghavan et al., 2023).In addition, genomic analysis of the strain SG2L-4 T revealed the presence of genes associated with capsular polysaccharide production, which forms a thick protective layer on the exterior of the microorganisms, preventing microbial attachment to the surface.KpsC, KpsD, and KpsS are responsible for encoding enzymes and proteins involved in the biosynthesis and export of capsular polysaccharides (Karlyshev et al., 2000).KpsC generates key components of the outer polysaccharide layer, whereas KpsD and KpsS contribute to the generation of polysaccharide units and their assembly into the outer layer.The function of these genes can vary among different bacterial species and strains, but they are essential for determining the composition and structural characteristics of the capsular polysaccharides.Additionally, the presence of WcbQ and WcbR genes in the genome indicates the involvement of SG2L-4 T in capsular polysaccharide biosynthesis (Warawa et al., 2009).These genome annotation results suggest the potential EPS-producing capability of H. piscis sp.nov.SG2L-4 T .

Production of exopolysaccharides
SEM analysis revealed the presence of overlaid microstructured EPS in strain SG2L-4 T (Figure 1B).To confirm EPS production in strains SG2L-4 T , SB1M-4, and SB2L-5, Molisch's test was conducted according to the method of Sudhesh and Sumathy (2016).A violet ring was observed in the cultures of the novel strains, indicating the presence of extracellular carbohydrates (Supplementary Figure S5).H. vantosae and H. anticariensis were reported to secrete 283.5 and 289.5 mg/L of EPS, respectively (Mata et al., 2006).Notably, the strains SG2L-4 T , SB1M-4, and SB2L-5 exhibited substantial EPS production, producing 420, 320, and 480 mg/L of crude EPS, respectively.These findings not only validate the EPS-producing potential of these isolated strains but also highlight their remarkable capability, surpassing previously reported Halomonas species in terms of yield.
Microbial-derived EPS has gained prominence for commercial applications in various industries including food, agriculture, and pharmaceuticals (Küçükaşik et al., 2011;Srivastava and Sharma, 2023).For instance, crude EPS derived from Phellinus baumii exhibited anti-diabetic effects in streptozotocin-induced diabetes (Hwang et al., 2005).EPS produced by acetic acid-producing bacteria improved the rheological properties of fermented foods, and the addition of EPS derived from Lactobacillus plantarum to cheese improves the moisture content, proteolysis, and textural properties, increasing cheese yield (Broadbent et al., 2001).Starter cultures such as L. delbruecki subsp.bulgaricus and Streptococcus thermophiles, which yielded 60-150 and 30-890 mg/L of EPS, respectively, improved the texture, consistency, and flavor of the final yogurt product (Jurášková et al., 2022).Although the effect of these novel strains on jeotgal fermentation is unclear, previous studies have suggested that Halomonas-derived EPS could exert beneficial effects on the taste and flavor of fermented foods such as jeotgal.Consequently, this study emphasizes the potential of these novel strains as natural food additives.Further research is needed to understand the effects of crude EPS produced by H. piscis sp.nov.during jeotgal ripening.

Conclusion
Three Gram-stain-negative halophilic bacterial strains, designated SG2L-4 T , SB1M-4, and SB2L-5, were isolated from jeotgal.Based on phylogenetic, genomic, phenotypic, and chemotaxonomic characteristics, these strains were identified as a novel species belonging to the genus Halomonas, for which the name Halomonas piscis sp.nov. is proposed.Genomic annotation of the novel type strain exhibited the presence of EPS biosynthesis-related genes, including ExoD (exopolysaccharide synthesis), KpsD (capsular polysaccharide export system periplasmic protein), KpsS (capsular polysaccharide export system protein), KpsC (capsular polysaccharide export system protein), WcbR (capsular polysaccharide biosynthesis fatty acid synthase), and WcbQ (capsular polysaccharide biosynthesis protein), in the whole-genome sequence.Furthermore, the EPS production capability of the novel strains was confirmed by detecting extracellular carbohydrates using Molisch's test.Our results suggest the potential of H. piscis sp.nov.as a novel candidate for the application of microbial EPS in various fields.Cells are Gram-stain-negative, aerobic, non-motile, positive for catalase, negative for oxidase, and rod-shaped (0.6-0.8 μm in width and 1.8-9.7 μm in length) without flagella.The optimal growth occurs , N-acetyl-β-glucosaminidase, ɑ-mannosidase, and ɑ-fucosidase.

FIGURE 2
FIGURE 2 Genomic relatedness of the novel strains and closely related Halomonas species.(A) Heatmap of pairwise similarity based on 16S rRNA gene sequences (lower left), dDDH values based on whole genome sequence (upper right), and (B) OrthoANI values between strains SG2L-4 T , SB1M-4, SB2L-5, and closely related Halomonas species.The pairwise similarity and dDDH values are visualized using Heatmapper tool.The gray color represents the absence of the genome sequence for H. cibimaris KACC 14932 T .OrthoANI values calculated using OAT software.

FIGURE 3 A
FIGURE 3A phylogenetic consensus tree based on 16S rRNA gene sequences, reconstructed using the neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) algorithms.The filled circles indicate the corresponding branches generated by all three algorithms.Node numbers represent bootstrap values (NJ/MP/ML) as a percentage of 1,000 replicates.Values higher than 70% are shown at the branch points.Oceanospirillum linum ATCC 11336 T served as an outgroup.Scale bar: 0.01 accumulated changes per nucleotide position.