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Comparative genome analysis of the genus Marivirga and proposal of two novel marine species: Marivirga arenosa sp. nov., and Marivirga salinae sp. nov.

BMC Microbiology volume 24, Article number: 245 (2024) Cite this article

Abstract

Background

The phylum Bacteroidota represents a significant proportion of heterotrophic bacteria found in marine ecosystems. Members of the phylum Bacteroidota are actively involved in the degradation of biopolymers such as polysaccharides and proteins. Bacteroidota genomes exhibit a significant enrichment of various enzymes, including carbohydrate-active enzymes (CAZymes), carboxypeptidases, esterases, isomerases, peptidases, phosphatases, and sulfatases. The genus Marivirga, a member of the family Marivirgaceae within the phylum Bacteroidota, comprises six documented species. During a microbial diversity study, three novel Marivirga strains (BKB1-2 T, ABR2-2, and BDSF4-3 T) were isolated from the West Sea, Republic of Korea.

Results

To explore the taxonomic status and genomic characteristics of the novel isolates, we employed a polyphasic taxonomic approach, which included phylogenetic, chemotaxonomic and comprehensive genome analysis. The three isolates were Gram-stain-negative, aerobic, rod-shaped, moderately halophilic, and had a gliding motility. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among the two isolates, BKB1-2 T and BDSF4-3 T, and the six reference strains were 70.5–76.5% for ANI and 18.1–25.7% for dDDH. Interestingly, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the strains harbor genes for a comprehensive pathway for dissimilatory nitrate reduction to ammonium (DNRA), as well as other nitrogen pathways for the reduction of nitrite, nitric oxide, and nitrous oxide. Additionally, the antiSMASH analysis indicated that the strains contained three to eight biosynthetic gene clusters (BGCs) associated with the synthesis of secondary metabolites. Furthermore, the strains carried a high number of CAZyme ranging from 53 to 152, which was also demonstrated by an in vitro analysis of degradation of the polysaccharide cellulose, chitin, laminarin, starch, and xylan. Additionally, all the strains carried genes for the metabolism of heavy metals, and exhibited tolerance to heavy metals, with minimum inhibitory concentrations (MICs) in millimoles (mM) in ranges of Co2+ (3–6), Cu2+ (0.2–0.4), Ni2+ (3–5), Zn2+ (2–4), Mn2+ (20–50), and Hg2+ (0.3).

Conclusions

Based on polyphasic taxonomic approach, the three isolated strains represent two novel species names Marivirga arenosa sp. nov. (BKB1-2 T = KCTC 82989 T = InaCC B1618T), and Marivirga salinae sp. nov. (BDSF4-3 T = KCTC 82973 T = InaCC B1619T).

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Background

The phylum Bacteroidota constitutes a significant component of marine heterotrophic bacteria. After Pseudomonatoda and Cyanobacteria, members of the phylum Bacteroidota rank as the ocean's most abundant bacterial group [1]. Bacteroidota strains have been isolated from a wide range of marine environments, including tidal flats [2], coastal sediments [3], deep sea [4], and hydrothermal vents [5]. By analyzing metagenomic data obtained from global ocean sampling and conducting a CARD-FISH study across the North Atlantic Ocean it was revealed that, subsequent to Cyanobacteria and Pelagibacter, Bacteroidota are the prevailing bacteria in marine environments [6].

Members of the phylum Bacteroidota participate in the breakdown of biopolymers, including polysaccharides and proteins, in marine ecosystems [1]. Bacteroidota are abundant in algal and phytoplankton blooms, displaying a preference for the consumption of complex polysaccharides [7]. Deep-sea sediments, where marine plants and animals remains accumulate, also contain complex polysaccharides and proteins, along with a notable presence of Bacteroidota species [4]. Complex polysaccharides found in marine environments comprise a variety of compound including agar, alginate, chitin, carrageenan, cellulose, fucoidans, laminarin, pectin, porphyrin, and xylan [8]. The members of the phylum Bacteroidota efficiently degrade complex polysaccharides, thus contributing to global recycling of carbon [4].

A comparative genome analysis showed that the genomes of Bacteroidota are highly enriched for carbohydrate-active enzymes (CAZymes), suggesting their role in degrading complex polysaccharides [9, 10]. Second to CAZymes, the Bacteroidota genome harbors a significant number of enzymes, including carboxypeptidases, esterases, isomerases, peptidases, sulfatases, and phosphatases that are involved in the mineralization of high molecular weight (HMW) organic matter [11]. In addition, genome mining data revealed that strains within the phylum Bacteroidota are abundant in genes of secondary metabolite synthesis that have diverse pharmacological activities [12].

The phylum Bacteroidota consists of six classes, each with corresponding six orders. Among them, the order Cytophagales of the class Cytophagia comprise a total of 21 families (https://lpsn.dsmz.de/order/cytophagales). The family Marivirgaceae contains only one validly published genus, Marivirga. A total of six Marivirga species have been documented, namely, M. sericea, M. tractuosa [13, 14], M. lumbricoides [15], M. atlantica, M. harenae [16, 17], and M. aurantiaca [18]. The isolation sources include marine aquariums, sand, sea water, and sediments. They are Gram-negative rod, non-spore forming, and aerobic. The respiratory quinone is menaquinone 7.

In this study, we isolated three novel gliding bacterial strains (BKB1-2 T, ABR2-2, and BDSF4-3 T) from marine samples collected in the West Sea, Korea. Based on a polyphasic taxonomic approach employing phylogenetic and chemotaxonomic analyses and a comprehensive genome analysis, two new species in the genus Marivirga were proposed. The genomes of isolates and six existing strains of the genus Marivirga were comparatively analyzed. The analysis showed that the strains carried a high number of CAZymes and degraded various types of complex polysaccharides, including chitin, cellulose, laminarin, starch, and xylan, contributing to the nutrient cycle. Additionally, the strains harbor genes responsible for synthesizing various amino acids, vitamins, and secondary metabolites, managing heavy metal metabolism, and carrying various pathways crucial for the nitrogen cycle in coastal ecosystems. Overall, the genomics analysis emphasized the strains' contributions at both environmental and industrial levels.

Methodology

Sampling sites

As part of a microbial diversity study, multiple marine samples were collected at various locations in the West Sea, Republic of Korea, in June 2021. Three types of samples were primarily collected: sea sand, green alga of the genus Ulva, and microbial mats, each with distinct ecological importance. A sample from sea sand was collected from an estuary located on Docho Island (34° 41′ 21.40″ N, 125° 55′ 08.46″ E). The sea sand in the estuary is influenced by both marine and freshwater systems, experiencing varying salinity levels and fostering a unique microbial community. A sample from green alga belonging to the genus Ulva was collected on Aphae Island (34° 50′ 43.42" N, 126° 13′ 32.19" E). The algae provide a habitat for diverse microbial communities, particularly for polysaccharide degraders, contributing to nutrient cycling. A sample from microbial mats was collected from a saltern on Bigeum Island (34° 45′ 49.35" N, 125° 58′ 42.48" E). The microbial mats from the saltern, a highly saline environment, form complex communities of cyanobacteria, algae, and various bacteria and archaea. These varied environments highlight the rich microbial diversity and ecological significance of the samples collected. All the samples were promptly transferred and processed at the laboratory.

Sample processing

To isolate more novel strains, we aimed to replicate natural conditions: seawater from the sampling area was collected and used in the preparation of media. A small part of each sample was placed at the center of a low-nutrient solid medium composed of 60% seawater, 1.5% agar, and 50 mg/L filtrate-sterilized cycloheximide. The plates were kept in a 20 °C incubator for seven days and observed using a stereoscopic microscope (ZEISS Stemi 508). The gliding colonies were transferred onto rich media of marine agar (MA, BD) and modified VY/2 agar media [MVY; 60% (v/v) seawater, 5 g/L baker’s yeast (Sigma), and 25 mg/L vitamin B12] [19]. The colonies were further purified and preserved at -80 °C in 20% glycerol. Finally, the isolated strains BKB1-2 T, ABR2-2, and BDSF4-3 T were deposited at the Korean Collection for Type Cultures (KCTC) and in the Indonesian Culture Collection (InaCC).

16S rRNA gene sequencing

The 16S rRNA gene was amplified with the universal bacterial primer pairs, 27F and 1492R. The gene was sequenced using the Sanger sequencing method with universal bacterial primers and two additional primers, 518F and 518R [20]. The near-complete 16S rRNA gene sequences were assembled by Vector NTI software. The sequences were then searched on the EzBioCloud database to find similar sequences [21]. The sequences obtained from the EzBioCloud database were aligned, trimmed, and then edited using BioEdit software (version 7.2.5). The number of nucleotides used for the construction of the phylogenetic tree was 1334 bp. Finally, three phylogenetic trees were made using molecular evolutionary genetics analysis (MEGA X) software [22], employing the neighbor-joining (NJ) [23], the maximum-likelihood (ML) [24], and the maximum parsimony (MP) methods [25]. The robustness of the phylogenetic trees was assessed with the 1,000 bootstrap iterations method. Flexibacter flexilis NBRC 15060 T (AB680763) was used as an outgroup.

Physical, biochemical and chemotaxonomic analysis

To determine characteristics, three isolated strains were cultivated on MA. A BBL™ Gram staining kit (BD, USA) was used to perform Gram staining. The morphology and size of bacterial cells were determined using a scanning electron microscope (SEM) (Regulus 8100, Hitachi) [26]. All isolates were checked for gliding motility through the hanging-drop technique and using soft agar media [27]. The optimum temperature and salt concentration for growth were assessed by using MA as the growth medium [2], while the optimum pH was examined using pH adjusted marine broth (MB, BD) as the testing medium [28]. For the determination of growth under anaerobic conditions, all isolates were inoculated within an anaerobic chamber (Coy Labs, USA) and the cells were cultivated on MA in an anaerobic jar at 30 °C (BD GasPak Systems).

For biochemical characterization, the catalase and oxidase production were examined using 3% (v/v) H2O2 and 1% (w/v) tetramethyl- p-phenylenediamine dihydrochloride reagents, respectively [29]. The hydrolysis of Tweens 20, 40, 80, and casein were determined as described in the Cowan and Steel protocol [30]. The activities of the DNase enzyme were checked using DNase agar (BD). The presence of flexirubin pigments was assessed by using a 20% (w/v) KOH solution [31]. For the other biochemical characterizations, API 20E, API ZYM, API 50CH strips (bioMérieux), and GEN III MicroPlates (Biolog) were used [32]. To investigate dissimilatory nitrate/nitrite reduction to ammonium (DNRA) activities, MB was supplemented with 1.0 g/L (w/v) NaNO3. Three isolated strains, along with the reference strains in the genus Marivirga, were inoculated into media. After three days of incubation, MQuant colorimetric test strips (Merck, Darmstadt, Germany) were employed to quantify the ammonium content in the media [33]. Second to DNRA, the nitrite reduction capacity was assessed by utilizing MB supplemented with 5 mM NaNO2. The strains were inoculated into media and subsequently checked for the reduction of nitrite. Nitrite concentrations were measured using Griess reagent, which consisted of sulfanilamide and N-(1-naphthyl) ethylenediamine [34]. The assay was performed at three time points during the incubation period: at the onset (day 0), after three days, and after five days, with the aim of assessing changes in nitrite concentration within the media [18]. For the chemotaxonomic analysis, the fatty acid profiles of three isolated strains and reference strains were determined by cultivating cells on MA for two days The cellular fatty acid methyl esters were extracted following the MIDI protocol (Sherlock Microbial Identification System version 6.0) [35]. Subsequently, the extracted components were injected into a gas chromatography system (GC system 8890, Agilent) and then the components were identified based on the TSBA version 6.0 database. The quinones and polar lipid profiles were assessed using the Komagata and Suzuki protocol [36]. For the quinone determination, the compounds were extracted from freeze-dried cells using chloroform–methanol (2:1, v/v). The crude components were separated by thin layer chromatography (TLC) and then identified by an HPLC system. The compound was identified by comparing the retention time of the standard compound extracted from reference strains. For the polar lipids, the compounds were separated by a two-dimensional TLC plate (silica gel 60 F254, 10 × 10 cm, Merck) and then sprayed with different reagents: ninhydrin, molybdenum blue, α-naphthol, and phosphomolybdic acid for the detection of amino lipids, phospholipids, glycolipids, and total lipids, respectively [36].

Genome sequencing and phylogeny

The genome sequences of three isolated strains, BKB1-2 T, ABR2-2, BDSF4-3 T, and one of the reference strains M. harenae KCTC 92433 T were determined by Oxford Nanopore Technologies (ONT, United Kingdom). In the sequencing process, a ligation sequencing kit (SQK-LSK112), a barcoding kit (SQK-NBD112.24), an R10.4 FLO-MIN112 flow cell, and a MinION device were utilized. Basecalling was conducted using MinKNOW software version 22.10.7 and Guppy software version 6.3.8 [37]. The genome was then assembled with Flye version 2.9.1 [38]. The genome's contamination and completeness were evaluated using CheckM version 1.2.2 (github.com/Ecogenomics/CheckM) and Busco version 5.4.4 (busco.ezlab.org/) [39, 40].

The ANI and dDDH were determined using the ANI calculator and DSMZ’s genome to genome distance calculator version 3.0 [21, 41]. A phylogenomic tree was constructed using the up-to-date bacterial core gene (UBCG) pipeline, incorporating the 92 prokaryotic core genes [42]. Flexibacter flexilis DSM 6793 T (GCF900112255) was used as an outgroup.

Functional analysis of genome

The genomes of the three strains, BKB1-2 T, ABR2-2, and BDSF4-3 T, along with the reference strain M. harenae KCTC 92433 T, were deposited at NCBI and annotated utilizing NCBI’s prokaryotic genome annotation pipeline (PGAP) [43]. The metabolic pathways were identified using the KEGG and RAST databases. First, KEGG pathways were detected using the BlastKOALA (kegg.jp/blastkoala/) server. The results from the BlastKOALA server were then processed using a KEGG-decoder [44]. Furthermore, a heatmap was generated from the KEGG pathways employing GraphPad Prism version 8.0.2. The RAST server was used to further determine the metabolic diversity and functional capabilities of all species in the genus Marivirga [45]. The antiSMASH database was employed to detect the secondary metabolite genes called biosynthetic gene clusters (BGCs) [46]. The genomes were further analyzed for carbohydrate active enzymes (CAZymes) through the dbCAN2 database [47].

Polysaccharide degradation testing

The degradation of complex polysaccharides was tested in basal agar media [60% (v/v) seawater, 0.1% sodium acetate, 0.01% (w/v) peptone, 0.06% (w/v) HEPES (pH xx), 0.01% (w/v) K2HPO4, 0.02% (w/v) NH4NO3, 1 ml trace elements, and multivitamins (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1579.pdf), containing 1% (w/v)) of polysaccharides, including alginate, cellulose, chitin, κ-carrageenan, λ-carrageenan, ι-carrageenan, laminarin, starch, and xylan [48, 49]. All three newly isolated strains were inoculated on the test media and incubated for one week at 30 °C. The breakdown of complex polysaccharides was identified by the formation of a clear zone surrounding the colonies.

To confirm the degradation of complex polysaccharides, we conducted further analyses in basal broth, mirroring the composition of the basal agar media. However, the media were modified to include 0.2% (w/v) of each test polysaccharide, encompassing alginate, cellulose, chitin, κ-carrageenan, λ-carrageenan, ι-carrageenan, laminarin, starch, and xylan [48, 49]. The degradation of polysaccharide in the broth media was confirmed by using 3, 5 dinitrosalicylic acid (DNS) reagent, which detects the reducing sugar released from the breakdown of polysaccharide [50]. The DNS reagent is added to the supernatant, and upon heating, it reacts with reducing sugars, causing a color change from yellow to orange. The DNS assay was performed at three time points of incubation: at the start (day 0), after two days, and then four days. During each of these time points, the color changes were subsequently measured at 570 nm using a microplate reader (Synergy H1, BioTek) [50].

Determination of tolerance to heavy metals

Minimum inhibitory concentration (MIC) is the lowest concentration of an inhibitory substance at which bacterial growth is inhibited. Tolerance to heavy metals was measured by determining the lowest concentration of each metal that prevents the growth of the isolated strains [51]. All the strains were tested for tolerance of heavy metals including Cu2+, Co2+, Ni2+, Zn2+, Mn2+, and Hg2+. The CuSO4.5H2O, CoCl2.6H2O, NiCl2.6H2O, ZnCl2, MnCl2.4H2O, and HgCl2 were used as sources of Cu2+, Co2+, Ni2+, Zn2+, Mn2+, and Hg2. For the determination of MIC, the MA medium was supplemented with mM concentrations of Cu2+ (0.1–1), Co2+ (1–7), Ni2+ (1–5), Zn2+ (1–5), Mn2+ (10–50), and Hg2+ (0.05–0.5) [52, 53]. All three isolates were inoculated in duplicate and then incubated for one week at 30 °C.

Results and discussion

Isolation of novel strains

Many novel strains have been isolated from multiple marine samples. Some of the isolates have been characterized as belonging to genera Reichenbachiella [49], Chondrinema [54], Fulvivirga [19] Flavobacterium [28], and Vibrio [32]. In this study three more strains, namely BKB1-2 T, ABR2-2, and BDSF4-3 T, isolated from sea sand, green algae of the genus Ulva, and microbial mat, respectively, were characterized (Figs. 1A, B, C). All three strains grew optimally on MA and produced orange, smooth, and circular colonies. The SEM images showed the strains are long rods with a length of 3.0–3.7 μm and a diameter range from 0.2–0.3 μm (Figs. 1D, E, F).

Fig. 1
figure 1

Sources of isolation (A, B, C), and SEM images (D, E, F) of three isolated strains. Strain: BKB1-2 T (A, D); ABR2-2 (B, E); BDSF4-3.T (C, F). Scale bars: 2 μm (D, E, F)

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Phylogenetic analysis based on 16S rRNA sequence

A phylogenetic analysis using 16S rRNA gene sequence confirmed that the strains BKB1-2 T, ABR2-2, and BDSF4-3 T were associated with the genus Marivirga within the phylum Bacteroidota, with the closest strains of BKB1-2 T and ABR2-2 being M. tractuosa DSM 4126 T with similarity values of 97.78% and 97.92%, while the closest strain of BDSF4-3 T was M. sericea DSM 4125 T with a similarity value of 98.68%. The 16S rRNA gene similarity value between BKB1-2 T and ABR2-2 was 99.8%, indicating that strains BKB1-2 T and ABR2-2 belong to the same species (Table S1). The similarity values of BKB1-2 T and BDSF4-3 T against the reference strains in the genus Marivirga fell below the threshold value of species description of 98.7% [55]. Furthermore, we compared the sequence of the 16S rRNA gene obtained from Sanger sequencing with the sequences obtained from whole genome sequencing. The 16S rRNA sequences from both sources were aligned using BioEdit to assess similarity, which were ranged from 99.95–99.98%. For further analysis, the 16S rRNA sequence from Sanger sequencing was used.

The phylogenetic trees were constructed using 16S rRNA genes to find the position of new isolates in the family Marivirgaceae. The phylogenetic trees showed a monophyletic clustering of strains BKB1-2 T, ABR2-2, and BDSF4-3 T with M. lumbricoides JLT 2000 T, M. aurantiaca S37H4T, M. atlantica SM 1354 T, M. harenae JK11T, M. tractuosa DSM 4126 T, and M. sericea DSM 4125 T (Fig. 2). Based on the 16S rRNA similarity values and the phylogenetic trees, strains M. tractuosa KCTC 2958 T, M. harenae KCTC 92433 T, M. sericea KCTC 2899 T, M. atlantica KCTC 42392 T, and M. lumbricoides KCTC 92621 T were selected as reference strains for further taxonomic study. The sequences of the 16S rRNA gene of the strains BKB1-2 T, ABR2-2, and BDSF4-3 T were submitted to GenBank/EMBL/DDBJ having accession numbers of OR233632, OR233633, and OR233634.

Fig. 2
figure 2

Maximum likelihood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic positions of the solates. ML tree was made using the 16S rRNA sequence to illustrate the phylogenetic connections between the isolates and the type species of different genus within the order Cytophagales. Bootstrap values (> 70%) are indicated at the branch points ML/NJ/MP, based on 1000 replication methods. Flexibacter flexilis NBRC 15060 T was applied as an outgroup

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Physiological, biochemical, and chemotaxonomic characterization

All three isolated strains were Gram-stain-negative, rod-shaped, strictly aerobic, and exhibited gliding motility. All three isolates exhibited optimal growth within the temperature range of 25–30 °C, an optimal NaCl concentration range of 2–4% (w/v), and an optimum pH range of 6.5–7.5. The comprehensive physiological properties of the isolated strains and the existing species in the genus Marivirga are summarized in Table 1.

Table 1 Differential physiological properties of three isolates and the reference species within the genus Marivirga. Strains: 1, BKB1-2 T; 2, ABR2-2; 3, BDSF4-3 T; 4, M. tractuosa DSM 4126 T; 5, M. harenae KCTC 92433 T; 6, M. sericea DSM 4125 T; 7, M. atlantica SM 1354 T; 8, M. lumbricoides CGMCC 1.10832 T
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The strains BKB1-2 T, ABR2-2, and BDSF4-3 T hydrolyzed Tweens 20 and 40, and casein but could not hydrolyze Tween 80. No strains produced flexirubin-type pigments. In the API ZYM kit, all three isolates and reference strains were positive for the activities of acid phosphatase, alkaline phosphatase, cystine arylamidase, α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase, while negative for the activities of β-glucuronidase, α-fucosidase, and α-mannosidase. Among all the newly isolated strains and the reference strains, only M. lumbricoides KCTC 92621 T showed α-galactosidase and β-galactosidase activities. Furthermore, all strains were positive for β-glucosidase except M. harenae KCTC 92433 T. In API 50CH, all three strains utilized only esculin ferric citrate and potassium 5-ketogluconate. Moreover, in GEN III MicroPlates (Biolog), all three strains utilized acetic acid, α-D-glucose, D-maltose, sodium butyrate, and D-trehalose. Among the tested Marivirga strains, only strain BKB1-2 T utilized D-malic acid and bromo succinic acid. Additionally, strains BKB1-2 T and ABR2-2 exhibited a positive reaction to 1% sodium lactate and the three isolated strains could not utilize formic acid. Despite that the strains share certain biochemical characteristics, there were notable differences in various biochemical tests between the isolates and the reference strains, as shown in Table 2.

Table 2 Differential biochemical characteristics of three isolates and the reference species within the genus Marivirga
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For the chemotaxonomic characterization, the most abundant fatty acids detected in the three newly isolated strains and reference strains were iso-C15:0 and C15:1 G, which ranged from 19.1–40.8% and 15.2–20.5%, respectively. Among the three isolated strains, strain BKB1-2 T additionally had summed feature 3 (C16:1 ω7c/C16:1 ω6c) at a level of 20.8% as a prominent fatty acid. Although the fatty acid profiles among isolated strains and the existing species were similar, there were differences among all eight strains (Table S2). All three strains had menaquinone 7 (MK-7) as a respiratory quinone, which is consistent within the genus Marivirga. The polar lipid profiles of the three isolated strains were composed of phosphatidylethanolamine (PE), unidentified amino lipids (AL), and unidentified lipids (L). The strain BKB1-2 T had PE, one AL, and five unidentified lipids L1-L5, while the strain ABR2-2 had PE, one AL, and six unidentified lipids L1-L6. The strain BDSF4-3 T had PE, two AL1-AL2, and six unidentified lipids L1-L6 (Fig S1).

Genome sequencing, analysis and phylogeny

The complete genomes of the strains BKB1-2 T, ABR2-2, and BDSF4-3 T and M. harenae KCTC 92433 T were sequenced using Oxford Nanopore Technology (ONT). All genomes were assembled and checked for completeness and contamination. The CheckM values of the determined genomes were 99.7–100, showing that all genome sequences were obtained with a high quality. The genomes of strains ABR2-2 and BDSF4-3 T were assembled into a single contig, each formed a circular chromosome with sizes of 3.94 and 4.49 Mb, respectively. In contrast, the genome of strain BKB1-2 T consisted of two contigs: contig 1 formed a circular chromosome with a size of 4.02 Mb, while contig 2 was a plasmid 25,320 bp long with a G + C content of 35.5%. Overall, the G + C content in the genomes of the three newly isolated strains ranged from 33.7–34.5%, aligning with the genus Marivirga. The PGAP annotation from NCBI provided information on the total number of genes, coding sequences (CDS), tRNAs, and rRNAs in the genome, and this is summarized in Table 3. The genomes of the strains BKB1-2 T, ABR2-2, and BDSF4-3 T and M. harenae KCTC 92433 T were uploaded to NCBI with GenBank accession numbers CP129968, CP129970, CP129971, and CP130565, respectively.

Table 3 Genomic properties of three isolates and the reference strains within the genus Marivirga
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The ANI and dDDH values between the two novel isolates BKB1-2 T and BDSF4-3 T and the existing type strains within the genus Marivirga ranged from 70.5–76.5% and 18.1–25.7%, respectively. The ANI and dDDH values fell below the specified thresholds for species differentiation, which are typically 95–96% for ANI and 70% for dDDH [41, 56]. Among the three newly isolated strains, the ANI and dDDH values between the isolates BKB1-2 T and ABR2-2 were 97.8% and 79.5%, respectively (Table 4), which were higher than the threshold values. Thus, based on ANI and dDDH calculations, only the strains BKB1-2 T and BDSF4-3 T could be considered novel species. A genome-based phylogenetic tree showed similar monophyletic clustering of the three strains with M. atlantica SM1354T, M. harenae JK11T, M. sericea DSM4125T, M. tractuosa DSM 4126 T, M. lumbricoides JLT2000T, and M. aurantiaca S37H4T, which was also observed in the 16S rRNA gene-based trees (Fig. 3).

Table 4 ANI (upper triangle in green) and dDDH (lower triangle in yellow) between three isolates and the reference strains within the genus Marivirga
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Fig. 3
figure 3

Maximum-likelihood (ML) tree based on 92 core genes using the UBCG pipeline. ML tree were constructed from 92 core genes through the UBCG pipeline, confirming the relationships among the three isolates and the existing strains of the genus Marivirga. Flexibacter flexilis DSM 6793 T (GCF900112255) was used as an outgroup. The branch nodes are labeled with bootstrap values based on 1000 replicates, and the scale bar represents 0.1 substitutions per site

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Comparative genomes functional analysis

Metabolic pathways analysis using KEGG and RAST servers

The genomes of three new isolates and the reference strains were analyzed for metabolic pathways using KEGG and RAST servers. The metabolic pathways were constructed using the KEGG database and then plotted in a heatmap (Fig. 4). The heatmap showed that all strains in the genus Marivirga possessed central metabolic pathways such as aerobic respiration, sugar metabolism, and various amino acid biosynthesis pathways. The KEGG analysis emphasized that all strains in the genus Marivirga have the potential to synthesize certain essential amino acids including histidine, lysine, methionine, threonine, and tryptophan and non-essential amino acids including alanine, aspartate, asparagine, cysteine, glycine, glutamine, proline, and serine. Among three isolated strains, only strain BDSF4-3 T had a complete pathway for the synthesis of arginine and leucine and partial pathways for valine and isoleucine. Furthermore, all the Marivirga species possessed a complete pathway for the production of riboflavin and additional partial pathways for the synthesis of thiamin and cobalamin (Fig. 4). The KEGG pathways also showed that strains in the genus Marivirga contained genes of various hydrolytic enzymes. All strains harbored genes for the synthesis of α-amylase and β-glucosidase. Strain BDSF4-3 T harbored genes for D-galacturonate epimerase. Among the reference strains, M. lumbricoides CGMCC 1.10832 T carried genes for D-galacturonate isomerase and M. sericea DSM 4125 T had genes for β-N-acetyl hexosaminidase, while M. atlantica SM 1354 T and M. aurantiaca S37H4T carried genes for the production of pullulanase [57]. These enzymes have diverse applications across multiple sectors, including food, pharmaceutical industries, biofuel production, and waste treatment [58, 59].

Fig. 4
figure 4

Heatmap showing the distribution of various metabolic pathways identified through KEGG analysis among the Marivirga strains. The scale bar indicates that the intensity of color reflects the completeness of pathways

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The KEGG analysis further highlighted the role of Marivirga species in the nitrogen cycle of coastal ecosystems. Strains BDSF4-3 T, M. tractuosa DSM 4126 T, and M. harenae KCTC 92433 T had a completed pathway of DNRA and reduction of nitrite, nitric oxide, and nitrous oxide (Fig. 4). The in vitro investigation of DNRA activity was conducted using an MQuant colorimetric strip to determine ammonium production, providing additional support to the genome analysis. The results showed strong activities of ammonium production from nitrate for the strains BDSF4-3 T, M. tractuosa DSM 4126 T, and M. harenae KCTC 92433 T (Table 2). Second to DNRA, the reduction of nitrite was determined using Griess reagent. During incubation, the strains reduced nitrite, resulting in a decrease in its concentration in the media. The assay showed that strain BDSF4-3 T, M. tractuosa DSM 4126 T, and M. harenae KCTC 92433 T exhibited ability for nitrite reduction (Table 2). Microbes utilize the DNRA pathway to transform nitrate into ammonium, effectively preserving bioavailable nitrogen within the ecosystem [60]. DNRA occurs in various ecosystems, including estuary and Antarctica [61]. This pathway not only aids in preserving nitrogen within these ecosystems but also plays a role in reducing the emission of harmful greenhouse gases, such as nitrous oxide [62]. Additionally, the reduction of nitrite and nitrate by marine microbes further is important for marine ecosystems and for global nitrogen cycling [34].

Based on RAST annotation, the genomes of strains BKB1-2 T and ABR2-2 showed the presence of 243 and 244 functional sub systems, while the strain BDSF4-3 T had 247 subsystems. In the RAST analysis, the largest number of annotated genes were assigned to amino acids and derivatives, which ranged from (209–257 genes), carbohydrates (102–193 genes), and protein metabolism (154–171 genes). Furthermore, the RAST analysis showed that all the isolates and reference strains in the genus Marivirga carried genes for the metabolism of nitrogen (7–34 genes), potassium (11–14 genes), phosphorus (14–25 genes), and sulfur (7–11 genes). Moreover, the RAST system identified 22–29 stress response genes that are essential for the protection of bacteria from reactive oxygen species in the marine environment (Table 5).

Table 5 Overview of the RAST subsystem and the numbers of genes involved in each metabolism of three isolates and reference strains within the genus Marivirga
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Detection of biosynthetic gene clusters (BGCs) using antiSMASH server

The antiSMASH analysis found type III polyketide synthases (PKS) and the terpene classes of BGCs in the genome of the isolates and the type strains in the genus Marivirga. Notably, strain BKB1-2 T had an additional BGC for lanthipeptide class I. Compared to the three newly isolated strains, the strain M. sericea DSM 4125 T had additional ribosomally synthesized and post-translationally modified peptides (RiPP-like) BGCs, while the strain M. lumbricoides CGMCC 1.10832 T had type I PKS, resorcinol, and non-ribosomal peptide synthetase (NRPS-like) BGCs (Table S3). Polyketide synthases constitute a group of enzymes that play a role in the synthesis of polyketides, which have antimicrobial properties [63]. Meanwhile, lanthipeptide class I refer to ribosomally synthesized antibacterial molecules, which are often referred to as lantibiotics. These bioactive compounds have potential applications in the food industry and can be used against highly antibiotic resistant pathogens [64].

Detection of carbohydrate-active enzymes (CAZymes) using dbCAN server

The genomes of three newly isolated strains and six type strains in the genus Marivirga were analyzed for CAZyme gene clusters (CGCs) using the dbCAN database. The dbCAN analysis showed that the genome of strain BKB1-2 T harbors a total of 53 CAZymes, distributed into 28 glycosyltransferases (GTs), 17 glycoside hydrolases (GHs), four carbohydrate esterases (CEs), three auxiliary activities (AAs), and one carbohydrate binding module (CBM). The strain ABR2-2 had a total of 56 CAZyme distributed into 27 GTs, 21 GHs, three CEs, three AAs, and two CBMs, while the strain BDSF4-3 T had a total of 59 CAZyme distributed into 35 GTs, 16 GHs, three CEs, one AA, and three CBMs. Among three new isolates and existing strains in the genus Marivirga, strain M. lumbricoides CGMCC 1.10832 T had the highest CAZymes of 152, distributed into 48 GTs, 75 GHs, 14 CEs, four AAs, three polysaccharide lyases (PLs), and three CBMs (Table 6). Furthermore, the percentage of CAZymes and ratio of GH per Mb of genome were calculated for all the Marivirga strains. The percentage of CAZymes out of the total genes was 1.52, 1.64, and 1.55% for the strains BKB1-2 T, ABR2-2, and BDSF4-3 T, respectively. The percentage of CAZymes was 3.05 for the strain M. lumbricoides JCM 18012 T, which was higher in the genus Marivirga. Furthermore, the GHs per Mbp in the genomes of strains BKB1-2 T, ABR2-2, and BDSF4-3 T were 4.23, 5.33, and 3.56, respectively (Table S4). The number of CAZymes in the family Marivirgaceae (53–152 CAZymes) closely resembled that of neighboring families within the phylum Bacteroidota, including Reichenbachiellaceae (79–216 CAZymes) [49], Fulvivirgaceae (55–264 CAZymes) [19], and Flavobacteriaceae Zobellia sp (257–315 CAZymes) [65, 66]. The presence of high numbers of CAZymes in bacteria offers insights into their ecological roles, adaptability, and potential applications across various biotechnological processes [67].

Table 6 Determination of CAZymes in three isolates and reference strains within the genus Marivirga, using dbCAN2
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Detection of heavy metal metabolism genes using the KEGG database

The KEGG pathways showed that three isolates and the reference strains had pathways for reduction of arsenic and also carried transporter genes including CorA, CopA, and AfuA for transport of cobalt, copper, and ferric iron into the cell, respectively (Fig. 4) [68]. The CorA protein is typically known for its role in transporting magnesium ions, but some members of the CorA family could also facilitate the transportation of cobalt (Co2+) and nickel (Ni2+) [69]. CopA is the most important transporter protein for the transport of metals in microbes. The CopA functions by binding to two Cu2+ ions and thereafter transferring them to the periplasmic space with the help of the Cu chaperone (CusF) using ATP as an energy source [68]. Furthermore, the isolates and the type strains in the genus Marivirga carried AfuA, which is an ABC-type transporter responsible for ferric iron transport. Among all strains, only M. lumbricoides JCM 18012 T carried FeoB, a cytoplasmic membrane transporter protein that participated in ferrous iron transport [70, 71].

In vitro polysaccharide degradation

The degradation of complex polysaccharides, including agar, alginate, cellulose, chitin, κ-carrageenan, λ-carrageenan, ι-carrageenan, laminarin, starch, and xylan, was tested in both solid and liquid media. First, the degradation of polysaccharides was assessed on solid media by identifying a clear zone around the colonies. The degradation of these complex polysaccharides was further tested by detecting reducing sugar using the 3, 5-dinitrosalicylic acid assay. The results showed the strain BKB1-2 T degraded cellulose, chitin, laminarin, and starch, the strain ABR2-2 degraded chitin, laminarin, and starch, and the strain BDSF4-3 T degraded chitin, laminarin, starch, and xylan (Table S5).

The in vitro degradation was supported by the identification of CAZymes genes within the genome. The breakdown of starch was facilitated by the existence of glycoside hydrolase families GH13 and GH16, primarily characterized as α-amylases [72]. All strains degraded chitin and carried GH23 that may involve in the degradation of chitin [73]. The hydrolysis of laminarin and xylan was facilitated by GH16 and GH3 in the genome, respectively (http://www.cazy.org/GH16.html) (Table S5). Oligosaccharides generated from the breakdown of polysaccharides had been studied to exhibit diverse biological activities, rendering them suitable for use in the cosmetic, functional food, and medical industries [74, 75]. The decomposition of laminarin, a highly abundant marine polysaccharide, plays a significant role in the turnover of marine polysaccharides [8]. Furthermore, the degradation of xylan holds significant importance for the biofuel, animal feed, pulp, and paper sectors [76]. Overall, the analyses emphasize the significance of these strains in their capacity to degrade complex polysaccharides, underscoring their importance in the carbon cycle and in various biotechnological applications.

Determination of the minimal inhibitory concentration (MIC) of heavy metals for the isolated strains

All three newly isolated strains were examined for their tolerance to various heavy metals. The MIC values of the heavy metals, including Cu2+, Co2+, Ni2+, Zn2+, Mn2+, and Hg2+ were different for the three isolates. Strain ABR2-2 tolerated high concentrations of various heavy metals compared to other strains. Strain ABR2-2 exhibited tolerance to heavy metals with minimum inhibitory concentrations of Co2+ (6 mM), Ni2+ (5 mM), Zn2+ (4 mM), Mn2+ (50 mM), and Hg2+ (0.4 mM). Strain BKB1-2 T had MIC to Co2+ (6 mM), Ni2+ (5 mM), Zn2+ (2 mM), Mn2+ (50 mM), and Hg2+ (0.2 mM), while strain BDSF4-3 T had MICs of Co2+ (3 mM), Ni2+ (3 mM), Zn2+ (2 mM), Mn2+ (30 mM), and Hg2+ (0.4 mM) (Table 7). Strains capable of growing in the presence of 0.5 mM of Co2+, Cu2+, Ni2+, Zn2+, and 0.05 mM of Hg2+ were classified as tolerant strains [52, 53]. The results are significant and comparable to those of known metal-resistant strains isolated from the deep sea. For example, Dietzia maris and Pseudoalteromonas shioyasakiensis strains, isolated from deep-sea sediments, demonstrated resistance to a range of heavy metals. Dietzia maris showed MICs of Cu2+ (2.8–5.7 mM), Co2+ (0.6 mM), Ni2+ (3.0 mM), Zn2+ (2.2 mM), and Mn2+ (14.3 mM) [77]. On the other hand, Pseudoalteromonas shioyasakiensis, exhibited MICs of Cu2+ (5.7 mM), Co2+ (0.3–0.6 mM), Ni2+ (3.0–3.6 mM), and Zn2+ (0.8–8.5 mM) [77]. Furthermore, the well-known metal-resistant bacteria species Cupriavidus metallidurans MSR33 showed elevated MIC values for Cu2+ (3.8 mM), Co2+ (20 mM), Ni2+ (6.0 mM), Zn2+ (17 mM), and Hg2+ (0.1 mM) [78, 79] (Table 7).

Table 7 Minimum inhibitory concentration of heavy metals for newly isolated strains
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These results were further supported by the detection of heavy metal transporter genes in the genomes of Marivirga strains. All the strains carried a CorA transporter protein involved in the transport of (Co2+) and nickel (Ni2+). Additionally, the CopA genes are prevalent in contaminated environments and participate not only in the metabolism of Cu2+ but also in handling other heavy metal, including Ni2+, Hg2+, and CrO42− [52]. These analyses highlight the importance of Marivirga strains in terms of heavy metal remediation in marine environments.

Description of Marivirga arenosa sp. nov.

Arenosa (a.re.no’sa. L. fem. adj. arenosa, sandy) referring to the source of isolation sea sand.

The cells are Gram negative bacilli, strictly aerobic, and have gliding motility. Produces a dark orange, circular, smooth colony on MA. The optimum growth occurs at temperatures 25–30 °C, pH 6.5–7.5, and NaCl 2–4% (w/v). The strain is positive for the activities of acid phosphatase, alkaline phosphatase, cystine arylamidase, α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase. The strain shows a positive reaction in Biolog GEN III plate for acetic acid, L-arginine, α-D-glucose, D-galactose, L-glutamic acid, D-maltose, D-mannose, D-malic Acid, L-malic Acid, sodium butyrate, and D-trehalose. Moreover, the strain can hydrolyze casein, cellulose, chitin, laminarin, starch, Tween 20, and Tween 40. The major fatty acids are iso-C15:0 and summed feature 3 (C16:1 ω7c/C16:1 ω6c), while the main polar lipids and the respiratory quinone are phosphatidylethanolamine and menaquinone-7. The genomic DNA G + C content of the type strain is 33.7%.

The type strain, BKB1-2 T (= KCTC 82989 T = InaCC B1618T) was isolated from the sea sand collected from the West Sea, Republic of Korea.

Description of Marivirga salinae sp. nov.

Salinae. (L. Sal. is derived from the Latin word sal meaning salt), referring to to the source of isolation at a salt farm.

The cells are Gram negative bacilli, strictly aerobic, and have gliding motility. Produces a light orange, circular, smooth colony on MA. The optimum growth occurs at temperatures 25–30 °C, pH 6.5–7.5, and NaCl 2–4% (w/v). The strain is positive for the activities of acid phosphatase, alkaline phosphatase, cystine arylamidase, α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase. The strain shows a positive reaction in Biolog GEN III plate for acetic acid, acetoacetic acid, L-aspartic acid, D-cellobiose, gentiobiose, α-D-glucose, D-maltose, D-mannose, propionic acid, sodium butyrate, D-salicin, and D-trehalose. The strain can hydrolyze casein, chitin, laminarin, starch, Tween 20, Tween 40, and xylan. The predominant fatty acids are iso-C15:0 and iso-C15:1 G while the main polar lipids and the respiratory quinone are phosphatidylethanolamine and menaquinone-7, respectively. The genomic DNA G + C content of the type strain is 34.4%.

The type strain, BDSF4-3 T (= KCTC 82973 T = InaCC B1619T), was isolated from microbial mat collected from the West Sea, Republic of Korea.

Conclusion

In conclusion, three strains, BKB1-2 T, ABR2-2, and BDSF4-3 T, were isolated from sea sand, green alga, and microbial mats collected at the West Sea, Korea. Using a polyphasic taxonomy approach, it was found that the three isolates were affiliated with the genus Marivirga within the phylum Bacteroidota. Notably, two of them, BKB1-2 T and BDSF4-3 T, were considered to represent two novel species in the genus Marivirga. Currently, the family Marinivrgacease consists of only one genus: Marivirga. The comparative genome analysis of the Marivirgaceae family highlighted the significance of all strains in terms of complex polysaccharide degradation, heavy metal resistance, the synthesis of various amino acids and vitamins. The isolates possessed important nitrogen metabolic pathways, including DNRA, nitrite, nitric oxide, and nitrous oxide reduction, which collectively contribute to the nitrogen cycle within ecosystems. Furthermore, the in vitro analysis revealed that the newly isolated strains are capable of degrading complex polysaccharides, including chitin, cellulose, laminarin, starch, and xylan, which is crucial for the carbon cycle. Additionally, these strains displayed tolerance to heavy metals, including Co2+, Ni2+, Zn2+, Mn2+, and Hg2+. Our analysis highlights that strains within the genus Marivirga hold potential for future utilization in various environmental and industrial sectors.

Availability of data and materials

The datasets generated and analyzed during the current study are available at the National Center for Biotechnology Information (NCBI). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains BKB1-2T, ABR2-2, and BDSF4-3T are OR233632, OR233633, and OR233634, respectively, and the genome sequences are CP129968, CP129970, and CP129971, respectively.

Abbreviations

ANI:

Average Nucleotide Identity

dDDH:

Digital DNA–DNA hybridization

MA:

Marine agar

CAZymes:

Carbohydrate-active enzymes

DNRA:

Dissimilatory nitrate reduction to ammonium

DNS:

Dinitrosalicylic acid

References

  1. Fernández-Gómez B, Richter M, Schüler M, Pinhassi J, Acinas SG, González JM, et al. May.Ecology of marine Bacteroidetes: a comparative genomics approach. Isme J. 2013;1026–37. https://doi.org/10.1038/ismej.2012.169.

  2. Muhammad N, Avila F, Lee Y-J, Han HL, Kim K-H, Kim S-G. Chondrinema litorale gen. nov., sp. nov., of the phylum Bacteroidota, carrying multiple megaplasmids isolated from a tidal flat in the West Sea, Korea. Front Mar Sci. 2023;10:1186809.

  3. Köpke B, Wilms R, Engelen B, Cypionka H, Sass H. Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients. Appl Environ Microbiol. 2005;7819–30. https://doi.org/10.1128/aem.71.12.7819-7830.2005.

  4. Zhu XY, Li Y, Xue CX, Lidbury I, Todd JD, Lea-Smith DJ, et al. Deep-sea Bacteroidetes from the Mariana Trench specialize in hemicellulose and pectin degradation typically associated with terrestrial systems. Microbiome. 2023;175. https://doi.org/10.1186/s40168-023-01618-7.

  5. He T, Zhang X. Characterization of Bacterial Communities in Deep-Sea Hydrothermal Vents from Three Oceanic Regions. Mar Biotechnol (NY). 2016;232–41. https://doi.org/10.1007/s10126-015-9683-3.

  6. Gómez-Pereira PR, Fuchs BM, Alonso C, Oliver MJ, van Beusekom JE, Amann R. Distinct flavobacterial communities in contrasting water masses of the north Atlantic Ocean. Isme J. 2010;472–87. https://doi.org/10.1038/ismej.2009.142.

  7. Pinhassi J, Sala MM, Havskum H, Peters F, Guadayol O, Malits A, et al. Changes in bacterioplankton composition under different phytoplankton regimens. Appl Environ Microbiol. 2004;6753–66. https://doi.org/10.1128/aem.70.11.6753-6766.2004.

  8. Bäumgen M, Dutschei T, Bornscheuer UT. Marine Polysaccharides: Occurrence, Enzymatic Degradation and Utilization. Chembiochem. 2021;2247–56. https://doi.org/10.1002/cbic.202100078.

  9. Thomas F, Hehemann J-H, Rebuffet E, Czjzek M, Michel G. Environmental and gut bacteroidetes: the food connection. Front Microbiol. 2011;93. https://doi.org/10.3389/fmicb.2011.00093.

  10. Reintjes G, Arnosti C, Fuchs B, Amann R. Selfish, sharing and scavenging bacteria in the Atlantic Ocean: a biogeographical study of bacterial substrate utilisation. Isme J. 2019;10:1119–32. https://doi.org/10.1038/s41396-018-0326-3.

  11. Bauer M, Kube M, Teeling H, Richter M, Lombardot T, Allers E, et al. Whole genome analysis of the marine Bacteroidetes'Gramella forsetii' reveals adaptations to degradation of polymeric organic matter. Environ Microbiol. 2006;2201–13. https://doi.org/10.1111/j.1462-2920.2006.01152.x.

  12. Brinkmann S, Kurz M, Patras MA, Hartwig C, Marner M, Leis B, et al. Genomic and Chemical Decryption of the Bacteroidetes Phylum for Its Potential to Biosynthesize Natural Products. Microbiol Spectr. 2022;0247921. https://doi.org/10.1128/spectrum.02479-21.

  13. Lewin RA, Lounsbery DM. Isolation, cultivation and characterization of flexibacteria. J Gen Microbiol. 1969;145–70. https://doi.org/10.1099/00221287-58-2-145.

  14. Nedashkovskaya OI, Vancanneyt M, Kim SB, Bae KS. Reclassification of Flexibacter tractuosus (Lewin 1969) Leadbetter 1974 and 'Microscilla sericea' Lewin 1969 in the genus Marivirga gen. nov. as Marivirga tractuosa comb. nov. and Marivirga sericea nom. rev., comb. nov. Int J Syst Evol Microbiol. 2010;1858–63. https://doi.org/10.1099/ijs.0.016121-0.

  15. Xu Y, Zhang R, Li Q, Liu K, Jiao N. Marivirga lumbricoides sp. nov., a marine bacterium isolated from the South China Sea. Int J Syst Evol Microbiol. 2015;452–6. https://doi.org/10.1099/ijs.0.066027-0.

  16. Muramatsu Y, Kamakura Y, Takahashi M, Nakagawa Y. Reclassification of Flexibacter tractuosus NBRC 15981T as Marivirga harenae sp. nov. in the family Flammeovirgaceae. Int J Syst Evol Microbiol. 2017;1937–42. https://doi.org/10.1099/ijsem.0.001890.

  17. Lin CY, Zhang XY, Liu A, Liu C, Song XY, Su HN, et al. Marivirga atlantica sp. nov., isolated from seawater and emended description of the genus Marivirga. Int J Syst Evol Microbiol. 2015;1515–9. https://doi.org/10.1099/ijs.0.000126.

  18. Zhang M, Zhang Y, Yao Q, Yang F, Zhu H. Marivirga aurantiaca sp. nov., a halophilic nitrite-reducing bacterium, isolated from intertidal surface sediments. Int J Syst Evol Microbiol. 2023;6195. https://doi.org/10.1099/ijsem.0.006195.

  19. Nguyen TTH, Vuong TQ, Han HL, Li Z, Lee YJ, Ko J, et al. Three marine species of the genus Fulvivirga, rich sources of carbohydrate-active enzymes degrading alginate, chitin, laminarin, starch, and xylan. Sci Rep. 2023;6301. https://doi.org/10.1038/s41598-023-33408-4.

  20. Pheng S, Han HL, Park DS, Chung CH, Kim SG. Lactococcus kimchii sp. nov., a new lactic acid bacterium isolated from kimchi. Int J Syst Evol Microbiol. 2020;505–10. https://doi.org/10.1099/ijsem.0.003782.

  21. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017;1613–7. https://doi.org/10.1099/ijsem.0.001755.

  22. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;1547–9. https://doi.org/10.1093/molbev/msy096.

  23. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;406–25. https://doi.org/10.1093/oxfordjournals.molbev.a040454.

  24. Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 1981;368–76. https://doi.org/10.1007/bf01734359.

  25. Fitch WM. Toward defining the course of evolution: minimum change for a specific tree topology. Syst Biol. 1971;20:406–16.

  26. Jeon D, Jiang L, Peng Y, Seo J, Li Z, Park SH, et al. Sphingomonas cannabina sp. nov., isolated from Cannabis sativa L. 'Cheungsam'. Int J Syst Evol Microbiol. 2022. https://doi.org/10.1099/ijsem.0.005566.

  27. Tittsler RP, Sandholzer LA. The Use of Semi-solid Agar for the Detection of Bacterial Motility. J Bacteriol. 1936;575–80. https://doi.org/10.1128/jb.31.6.575-580.1936.

  28. Muhammad N, Le Han H, Lee YJ, Ko J, Nguyen TTH, Kim SG. Flavobacterium litorale sp. nov., isolated from red alga. Int J Syst Evol Microbiol. 2022;5458. https://doi.org/10.1099/ijsem.0.005458.

  29. Le Han H, Jiang L, Thu Tran TN, Muhammad N, Kim SG, Tran Pham VP, et al. Whole-genome analysis and secondary metabolites production of a new strain Brevibacillus halotolerans 7WMA2: A potential biocontrol agent against fungal pathogens. Chemosphere. 2022; 136004. https://doi.org/10.1016/j.chemosphere.2022.136004.

  30. Cowan ST. Cowan and Steel's manual for the identification of medical bacteria. Cambridge: Cambridge university press; 1993.

  31. Vila E, Hornero-Méndez D, Azziz G, Lareo C, Saravia V. Carotenoids from heterotrophic bacteria isolated from Fildes Peninsula, King George Island, Antarctica. Biotechnol Rep (Amst). 2019;e00306. https://doi.org/10.1016/j.btre.2019.e00306.

  32. Muhammad N, Nguyen TTH, Lee YJ, Ko J, Avila F, Kim SG. Vibrio ostreae sp. nov., a novel gut bacterium isolated from a Yellow Sea oyster. Int J Syst Evol Microbiol. 2022;5586. https://doi.org/10.1099/ijsem.0.005586.

  33. Heo H, Kwon M, Song B, Yoon S. Involvement of NO3− in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method. Appl Environ Microbiol. 2020;1054–20. https://doi.org/10.1128/AEM.01054-20.

  34. Zhang M, Li A, Yao Q, Wu Q, Zhu H. Nitrogen removal characteristics of a versatile heterotrophic nitrifying-aerobic denitrifying bacterium, Pseudomonas bauzanensis DN13–1, isolated from deep-sea sediment. Bioresour Technol. 2019;122626. https://doi.org/10.1016/j.biortech.2019.122626.

  35. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI technical note 101. Newark, DE: MIDI inc; 1990.

    Google Scholar 

  36. Komagata K, Suzuki K. Lipid and Cell-Wall Analysis in Bacterial Systematics. Method in Microbiol. 1988;161–207. https://doi.org/10.1016/S0580-9517(08)70410-0.

  37. Wick RR, Judd LM, Holt KE. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol. 2019;129. https://doi.org/10.1186/s13059-019-1727-y.

  38. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019;540–6. https://doi.org/10.1038/s41587-019-0072-8.

  39. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;1043–55. https://doi.org/10.1101/gr.186072.114.

  40. Manni M, Berkeley MR, Seppey M, Zdobnov EM. BUSCO: Assessing Genomic Data Quality and Beyond. Curr Protoc. 2021;e323. https://doi.org/10.1002/cpz1.323.

  41. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;60. https://doi.org/10.1186/1471-2105-14-60.

  42. Na SI, Kim YO, Yoon SH, Ha SM, Baek I, Chun J. UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction. J Microbiol. 2018;280–5. https://doi.org/10.1007/s12275-018-8014-6.

  43. Li W, O'Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, et al. RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Res. 2021;1020–8. https://doi.org/10.1093/nar/gkaa1105.

  44. Graham ED, Heidelberg JF, Tully BJ. Potential for primary productivity in a globally-distributed bacterial phototroph. Isme J. 2018;1861–6. https://doi.org/10.1038/s41396-018-0091-3.

  45. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008;75. https://doi.org/10.1186/1471-2164-9-75.

  46. Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, et al. AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 2019;81–7. https://doi.org/10.1093/nar/gkz310.

  47. Ausland C, Zheng J, Yi H, Yang B, Li T, Feng X, et al. dbCAN-PUL: a database of experimentally characterized CAZyme gene clusters and their substrates. Nucleic Acids Res. 2021;523–8. https://doi.org/10.1093/nar/gkaa742.

  48. Gao B, Jin M, Li L, Qu W, Zeng R. Genome Sequencing Reveals the Complex Polysaccharide-Degrading Ability of Novel Deep-Sea Bacterium Flammeovirga pacifica WPAGA1. Front Microbiol. 2017;600. https://doi.org/10.3389/fmicb.2017.00600.

  49. Muhammad N, Avila F, Nedashkovskaya OI, Kim SG. Three novel marine species of the genus Reichenbachiella exhibiting degradation of complex polysaccharides. Front Microbiol. 2023;1265676. https://doi.org/10.3389/fmicb.2023.1265676.

  50. Deshavath NN, Mukherjee G, Goud VV, Veeranki VD, Sastri CV. Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing sugars: Interference of furfural and 5-hydroxymethylfurfural. Int J Biol Macromol. 2020;180–5. https://doi.org/10.1016/j.ijbiomac.2020.04.045.

  51. Noisangiam R, Nuntagij A, Pongsilp N, Boonkerd N, Denduangboripant J, Ronson C, et al. Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand. Syst Appl Microbiol. 2010;374–82. https://doi.org/10.1016/j.syapm.2010.06.002.

  52. Altimira F, Yáñez C, Bravo G, González M, Rojas LA, Seeger M. Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile. BMC Microbiol. 2012;193. https://doi.org/10.1186/1471-2180-12-193.

  53. Dekker L, Osborne TH, Santini JM. Isolation and identification of cobalt- and caesium-resistant bacteria from a nuclear fuel storage pond. FEMS Microbiol Lett. 2014;81–4. https://doi.org/10.1111/1574-6968.12562.

  54. Muhammad N, Avila F, Lee Y-J, Han HL, Kim K-H, Kim S-G. Chondrinema litorale gen. nov., sp. nov., of the phylum Bacteroidota, carrying multiple megaplasmids isolated from a tidal flat in the West Sea, Korea. Frontiers in Marine Science. 2023;1186809. https://doi.org/10.3389/fmars.2023.1186809.

  55. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer KH, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol. 2014;635–45. https://doi.org/10.1038/nrmicro3330.

  56. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017;1281–6. https://doi.org/10.1007/s10482-017-0844-4.

  57. Hii SL, Tan JS, Ling TC, Ariff AB. Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res. 2012;921362. https://doi.org/10.1155/2012/921362.

  58. Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A, et al. Applications of Microbial Enzymes in Food Industry. Food Technol Biotechnol. 2018;16–30. https://doi.org/10.17113/ftb.56.01.18.5491.

  59. Vachher M, Sen A, Kapila R, Nigam A. .Microbial therapeutic enzymes: A promising area of biopharmaceuticals. Curr Res Biotechnol.2021;195–208. https://doi.org/10.1016/j.crbiot.2021.05.006.

  60. Liu Y, Zhang Y, Huang Y, Niu J, Huang J, Peng X, et al. Spatial and temporal conversion of nitrogen using Arthrobacter sp. 24S4–2, a strain obtained from Antarctica. Front Microbiol. 2023;1040201. https://doi.org/10.3389/fmicb.2023.1040201.

  61. Liu S, Dai J, Wei H, Li S, Wang P, Zhu T, et al. Dissimilatory Nitrate Reduction to Ammonium (DNRA) and Denitrification Pathways Are Leveraged by Cyclic AMP Receptor Protein (CRP) Paralogues Based on Electron Donor/Acceptor Limitation in Shewanella loihica PV-4. Appl Environ Microbiol. 2021;1964–20. https://doi.org/10.1128/aem.01964-20.

  62. Ye F, Duan L, Sun Y, Yang F, Liu R, Gao F, et al. Nitrogen removal in freshwater sediments of riparian zone: N-loss pathways and environmental controls. Front Microbiol. 2023;1239055. https://doi.org/10.3389/fmicb.2023.1239055.

  63. Hochmuth T, Piel J. Polyketide synthases of bacterial symbionts in sponges-evolution-based applications in natural products research. Phytochemistry. 2009;1841–9. https://doi.org/10.1016/j.phytochem.2009.04.010.

  64. Barbosa J, Caetano T, Mendo S. Class I and Class II Lanthipeptides Produced by Bacillus sp. J Nat Prod. 2015;2850–66. https://doi.org/10.1021/np500424y.

  65. Thomas F, Bordron P, Eveillard D, Michel G. Gene Expression Analysis of Zobellia galactanivorans during the Degradation of Algal Polysaccharides Reveals both Substrate-Specific and Shared Transcriptome-Wide Responses. Front Microbiol. 2017;1808. https://doi.org/10.3389/fmicb.2017.01808.

  66. Chernysheva N, Bystritskaya E, Stenkova A, Golovkin I, Nedashkovskaya O, Isaeva M. Comparative Genomics and CAZyme Genome Repertoires of Marine Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T. Mar Drugs. 2019;661. https://doi.org/10.3390/md17120661.

  67. Chettri D, Verma AK. Biological significance of carbohydrate active enzymes and searching their inhibitors for therapeutic applications. Carbohydr Res. 2023;108853. https://doi.org/10.1016/j.carres.2023.108853.

  68. Li W, Wang L, Li X, Zheng X, Cohen MF, Liu YX. Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functioning CopA in Environmental Microbiomes. Genomics Proteomics Bioinformatics. 2022;101–2. https://doi.org/10.1016/j.gpb.2022.08.006.

  69. Zhang Y, Rodionov DA, Gelfand MS, Gladyshev VN. Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC Genomics. 2009;78. https://doi.org/10.1186/1471-2164-10-78.

  70. Hantke K. Is the bacterial ferrous iron transporter FeoB a living fossil? Trends Microbiol. 2003;192–5. https://doi.org/10.1016/s0966-842x(03)00100-8.

  71. Lau CK, Krewulak KD, Vogel HJ. Bacterial ferrous iron transport: the Feo system. FEMS Microbiol Rev. 2016;273–98. https://doi.org/10.1093/femsre/fuv049.

  72. Janeček Š, Svensson B, MacGregor EA. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci. 2014;1149–70. https://doi.org/10.1007/s00018-013-1388-z.

  73. Wang YJ, Jiang WX, Zhang YS, Cao HY, Zhang Y, Chen XL, et al. Structural Insight Into Chitin Degradation and Thermostability of a Novel Endochitinase From the Glycoside Hydrolase Family 18. Front Microbiol. 2019;2457. https://doi.org/10.3389/fmicb.2019.02457.

  74. de Jesus Raposo MF, de Morais AM, de Morais RM. Marine polysaccharides from algae with potential biomedical applications. Mar Drugs. 2015;2967–3028. https://doi.org/10.3390/md13052967.

  75. Ruocco N, Costantini S, Guariniello S, Costantini M. Polysaccharides from the Marine Environment with Pharmacological, Cosmeceutical and Nutraceutical Potential. Molecules. 2016;551. https://doi.org/10.3390/molecules21050551.

  76. Schröder SP, de Boer C, McGregor NGS, Rowland RJ, Moroz O, Blagova E, et al. Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes. ACS Cent Sci. 2019;1067–78. https://doi.org/10.1021/acscentsci.9b00221.

  77. Gillard B, Chatzievangelou D, Thomsen L, Ullrich MS. Heavy-metal-resistant microorganisms in deep-sea sediments disturbed by mining activity: An application toward the development of experimental in vitro systems. Front in Mari Sci. 2019;462. https://doi.org/10.3389/fmars.2019.00462.

  78. Trajanovska S, Britz ML, Bhave M. Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site. Biodegradation. 1997;113–24. https://doi.org/10.1023/a:1008212614677.

  79. Rojas LA, Yáñez C, González M, Lobos S, Smalla K, Seeger M. Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation. PLoS One. 2011;17555. https://doi.org/10.1371/journal.pone.0017555.

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Acknowledgements

The sampling of the bacterial strains was made possible by the support and collaboration of the National Science Museum of Korea through the Joint Academic Survey of the Federation of National Biodiversity Organizations in 2021.

Funding

This research was supported the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5232423) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2021M3H9A1030164).

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  1. Biological Resource Center/Korean Collection for Type Cultures (KCTC), Korea ResearchInstitute of Bioscience and Biotechnology, Jeongeup, Jeonbuk, 56212, the Republic of Korea

    Neak Muhammad, Forbes Avila & Song-Gun Kim

  2. Department of Biotechnology, KRIBB School, University of Science and Technology (UST), Daejeon, 34113, the Republic of Korea

    Neak Muhammad, Forbes Avila & Song-Gun Kim

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NM and S-GK designed the study. NM performed the isolation and the phenotypical and genomic characterizations. FA performed bioinformatics analyses. NM and S-GK wrote the manuscript. All authors contributed to the article and approved the submitted manuscript.

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Correspondence to Song-Gun Kim.

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Muhammad, N., Avila, F. & Kim, SG. Comparative genome analysis of the genus Marivirga and proposal of two novel marine species: Marivirga arenosa sp. nov., and Marivirga salinae sp. nov.. BMC Microbiol 24, 245 (2024). https://doi.org/10.1186/s12866-024-03393-3

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