The genus Sulfitobacter, first described by Sorokin in 1995, belongs to the family Roseobacteraceae within the class Alphaproteobacteria, with Sulfitobacter pontiacus as the type species (Sorokin, 1995). Members of this genus are sulfur-oxidizing, chemoheterotrophic bacteria, comprising 24 valid species identified from diverse marine and extreme environments, such as seawater, tidal flats, and hypersaline lakes, as well as in association with marine organisms, including algae, seagrass, starfish, and coral (Fukui et al., 2015; Lian et al., 2021; Wang et al., 2021). Sulfitobacter species are Gram-negative, aerobic, catalase- and oxidase-positive, with membranes containing primarily fatty acid C_18:1ω7c, a respiratory quinone type Q-10, and a genomic DNA G + C content ranging from 55.0 to 64 mol% (Song et al., 2019; Yoon, 2019; Park and Yoon, 2021). Their sulfur-oxidizing capabilities are central to their ecological function, significantly contributing to sulfur cycling and biogeochemical processes that influence carbon and nutrient dynamics in marine ecosystems (Prabagaran et al., 2007; Xu et al., 2024). In addition, Sulfitobacter species are often associated with marine algae, such as S. algicola from green algae (Ulva australis), S. pacificus and S. porphyrae from red algae (Pyropia yezoensis), and S. undariae from brown algae (Undaria pinnatifida) reservior (Xu et al., 2024, 2015; Park et al., 2015; Wang et al., 2021). They produce essential nutrients like vitamin B12 and siderophores, supporting algal growth under nutrient-limited conditions (Yang et al., 2021; Beiralas et al., 2023).

Sulfitobacter species are widely distributed across coastal and ocean environments, where they contribute to organic sulfur cycling and play a vital role in maintaining marine ecosystem health. In this study, the taxonomic positions of strains HNIBRBA2951^T and HNIBRBA3233^T, isolated from seawater and tidal flats, were investigated using a polyphasic approach, incorporating phenotypic, phylogenetic, and chemotaxonomic analyses.

Strain isolation and 16S rRNA gene sequence determination

The surface seawater sample was collected from the coast of Geogeumdo, Goheung, Republic of Korea (GPS, 34°28'15.41"N, 127°06'11.06") in April 2022. The tidal flat sample was collected from Wando, Republic of Korea (34°23'43.64"N, 126°39'42.01"E) in May 2022. The tidal flat samples were prepared by mix-dilution of 0.2 g with sterile PBS, then 50 μl of both seawater and tidal flat samples were spread onto marine agar 2216 (MA, Difco) plates, and then incubated at 20°C for seven days. The beige-colored colonies formed on MA were selected and cultured routinely under the same isolation conditions. Two novel bacterial strains were designated with the institutional accession numbers HNIBRBA2951^T and HNIBRBA3233^T and stored at -80°C in 20% (v/v) glycerol. The 16S rRNA gene sequences of the two strains were obtained by commercial sequencing (identification service) of the Macrogen (Korea). The nucleotide sequences were amplified by PCR using the universal primers 27F and 1492R (Lane, 1991) for identification purpose, and then determined using universal primers 907R and 785F. The resultant raw sequences were then checked for accuracy and assembled using the Geneious Prime program. The almost completed 16S rRNA gene sequences were evaluated for similarity to the nearest phylogenetic neighbor using the EzBioCloud’s identification service.

Phylogenetic and genomic analyses

The phylogenetic relationships of the two novel strains and their related type strains were determined through the construction of a phylogenetic tree based on the analysis of 16S rRNA gene sequences. The sequences of phylogenetic neighbors were acquired from the EzBioCloud Database and then aligned using the EzEditor software (Jeon et al., 2014). Phylogenetic trees were generated by MEGA version 10 (Kumar et al., 2018) using neighbor-joining (Saitou and Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms. The neighbor-joining and maximum-likelihood trees were reconstructed with the Juke-Cantor and the Kimura 2-parameter models, respectively. The K2 + G + I model was considered and found to be the best-fit. The maximum-parsimony tree was inferred using the Tree-Bisection-Reconnection (TBR) method with the number of initial trees (random addition) as 10 and partial deletion options. The robustness of the topologies for the neighbor-joining and maximum-likelihood trees was evaluated through bootstrap analysis based on 1000 resamplings of the sequences (Felsenstein, 1985).

Draft genome sequences of strains HNIBRBA2951^T and HNIBRBA3233^T were obtained using the Illumina NovaSeq platform, with paired-end reads of 2 × 151 bp. Genomic DNA extraction and genome sequencing were performed using Macrogen Whole Genome Sequencing Services (Korea). De novo assembly was performed with Unicycler (Wick et al., 2017) in the GALAXY (Blankenberg et al., 2010). The quality of the final assemblies was evaluated using QUAST (Gurevich et al., 2013), the coverage was calculated with BBMap (Bushnell, 2014), the completeness and contamination values of the genomes were estimated by CheckM (Parks et al., 2015), and the genomes were then annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). To construct the phylogenomic tree, the Sulfitobacter species genomes available from the EzBioCloud Database and NCBI genome database were downloaded. A genome-based phylogenetic tree was constructed using UBCGs (up-to-date bacterial core gene sets) (Na et al., 2018). Average nucleotide identity (ANI) values were calculated using OrthoANI analysis (Lee et al., 2016). Digital DNA-DNA hybridization (dDDH) values were estimated using the GGDC website (http://ggdc.dsmz.de/distcalc2.php) (Meier-Kolthoff et al., 2022). Metabolic pathways and orthology assignments were reconstructed based on KEGG using BlastKOALA (Kanehisa et al., 2016). Additionally, potential secondary metabolite biosynthetic gene clusters were identified using the web-based tool antiSMASH version 7.1.0 (Blin et al., 2023).

Morphological, physiological and chemotaxonomic analyses

The morphological characteristics and the presence or absence of flagella in the cells were investigated using transmission electron microscopy (TEM) (H-7650; Hitachi) and scanning electron microscopy (SEM) (JSM-IT700HR; JEOL). For sample preparation for TEM, the samples were negatively stained with a 2% (v/v) uranyl acetate solution (Sigma-Aldrich). For sample preparation for SEM, the cells were fixed with a 2% (v/v) glutaraldehyde and 1% (v/v) osmium tetroxide solution. The fixed samples were sputter-coated with platinum under a vacuum. Colonial morphology, including characteristics such as size, color, form, elevation, and margin, was observed in accordance with the methods described by Alexander and Strete (2001). The bacterial characteristics (Gram-reaction, motility, catalase, and oxidase production) of two isolates were determined according to the methods described by Benson (2002). The optimal growth temperature and growth range were tested at eight different temperatures (4, 10, 15, 20, 25, 30, 37 and 42°C) on MA. Growth was examined in marine broth adjusted to pH 5.0–10.0 at increments of 0.5 pH unit intervals. pH values of 5.0–6.0, 6.5–7.0, 7.5–8.0, 8.5–9.0, and 9.5–10.0 were obtained by MES, MOPS, HEPES, and CHES buffers, respectively. Growth in salinity was examined in the absence of NaCl, in the presence of 0.5–2 (at increments of 0.5% intervals), 2–10% (w/v) NaCl (at increments of 1% intervals) and in modified marine broth. However, modified marine broth excluded NaCl from the Difco formula. The growth of the bacteria under anaerobic conditions was determined on MA at 30°C for a period of five weeks by using the GasPak EZ Anaerobe Pouch System (BD). The physiological and biochemical characterization was conducted through the application of API test systems (API ZYM and API 20NE, bioMérieux), in accordance with the instructions provided by the manufacturer. However, an exception was made in that the cells were suspended in a 2% (w/v) NaCl solution. The analysis of cellular fatty acids was conducted on cells of two novel strains and related type strains, harvested from MA plates after incubation at 20°C until the mid-exponential phase was reached. The fatty acids were saponified, methylated, and extracted in accordance with the standard protocol of the MIDI (Sherlock Microbial Identification System) system. The resulting fatty acid methyl esters were then analyzed by gas chromatography (7890A GC system; Agilent) and identified by using the RTSBA6 database of the Microbial Identification System (Sasser, 1990). The analysis of respiratory quinones and polar lipids was carried out using the analytical services of the Korean Culture Center of Microorganisms (KCCM). Respiratory quinones and polar lipids were extracted from freeze-dried cells according to the method described by Minnikin et al. (1984). The extracted quinones were purified using thin-layer chromatography and subsequently analyzed by high-performance liquid chromatography (HPLC) (Collins, 1985). Polar lipid extracts were separated using two-dimensional thin-layer chromatography (TLC) on silica gel-coated plates (10 × 10 cm, Merck). The first direction used a solvent system of chloroform/methanol/water (65:25:3.8), and the second direction used chloroform/acetic acid/methanol/water (40:7.5:6:1.8). The plates were treated with a solution of molybdophosphoric acid (10% in ethanol, Sigma), ninhydrin (0.2% in ethanol, Sigma), molybdenum blue (1.3% molybdenum oxide in sulfuric acid, Sigma), α-naphthol (0.15% in ethanol-sulfuric acid) and Dragendorff’s reagents (Merck) in order to visualize functional group-containing lipids (Minnikin et al., 1977).

Phylogeny based on the 16S rRNA gene sequence

The strains HNIBRBA2951^T and HNIBRB3233^T, isolated from seawater and tidal flats on Korean islands, have almost complete 16S rRNA gene sequences with lengths of more than 1,350 bp. These sequences were deposited in NCBI with lengths of 1,379 bp (PP854564) and 1,408 bp (PP854565), respectively. The sequence of strain HNIBRBA2951^T showed highest similarities with S . donghicola DSW-25^T (98.6%), S . guttiformis EL-38^T (98.0%) and S . mediterraneus CH-B427^T (98.0%), while strain HNIBRBA3233^T exhibited highest similarities with S . mediterraneus CH-B427^T (97.3%) and S . donghicola DSW-25^T (97.3%). The sequence similarity between strains HNIBRBA2951^T and HNIBRBA3233^T was 97.2%. The phylogenetic tree showed the position of both strains within the genus Sulfitobacter is presented in Fig. 1. This was further supported by the maximum-likelihood and maximum parsimony trees. In the neighbor-joining tree, strain HNIBRBA2951^T formed a clade with S. undariae W-BA2^T, S. guttiformis KCTC 32187^T and S. donghicola KCTC 12864^T. Strain HNIBRBA3233^T clustered with S. algicola 1151^T. The overall topology of the neighbor-joining tree closely resembled that of the maximum-likelihood tree (data not shown). This phylogenetic inference, combined with the 98.7% similarity cut-off (Kim et al., 2014) between strains HNIBRBA2951^T, HNIBRBA3233^T, and related Sulfitobacter species, suggested that strains HNIBRBA2951^T and HNIBRBA3233^T represent two novel species of the genus Sulfitobacter.

Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships of strains HNIBRBA2951^T and HNIBRBA3233^T with representatives of the genus Sulfitobacter. Percentages at nodes are levels of bootstrap support (>70%) based on neighbor-joining analyses of 1,000 re-sampled datasets. Hyphomonas polymorpha PS728^T (ARYM01000020) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.

Whole genome sequence and genome relatedness

The draft genome sequences of both strains HNIBRBA2951^T and HNIBRBA3233^T have been deposited at the GenBank database under accession numbers JBEFLO00000000 and JBEFLP00000000, respectively. The genome sizes of strains HNIBRBA2951^T and HNIBRBA3233^T were 3,844,622 and 3,923,515 bp, which assembled into 35 and 27 contigs with coverages of 908.0 and 764.2 × and N50 values of 290,437 and 2,713,271 bp, respectively. In total 3,700 genes of strain HNIBRBA2951^T, based on the PGAP results, 3,653 genes were predicted, including 3,624 protein-coding genes, 41 tRNA genes, 3 rRNA genes, 3 non-coding RNA genes, and 29 pseudogenes. Strain HNIBRBA2951^T had the genomic DNA G + C content of 59.5 mol%. In total 3,806 genes of strain HNIBRBA3233^T, 3,759 genes were predicted, including 3,729 protein-coding genes, 41 tRNA genes, 3 rRNA genes, 3 noncoding RNA genes, and 30 pseudogenes. Strain HNIBRBA3233^T had the genomic DNA G + C content of 63.5 mol% (Supplementary data Table S1). Genomic analysis of strains HNIBRBA2951^T and HNIBRBA3233^T revealed metabolic and biosynthetic capabilities. Both genomes encoded genes involved in sulfur metabolism (seoABC, cysIK) and assimilatory nitrate reduction (nasABCDE). However, genes associated with the SOX system (soxABCDYZ) were detected only in the strain HNIBRBA2951^T. In contrast, carotenoid biosynthesis genes (crtABCDFI) were found only in the stain HNIBRBA3233^T. Secondary metabolite biosynthetic gene cluster (BGC) analysis using antiSMASH indicated that strain HNIBRBA2951^T contained five BGCs, including those for homoserine lactone (hserlactone), β-lactone-containing protease inhibitor (betalactone), terpene, polyketide synthase type I (T1PKS) and ectoine. Strain HNIBRBA3233^T comprised six BGCs, encompassing terpene (one of the two regions is the carotenoid-associated BCG), β-lactone-containing protease inhibitor (betalactone), polyketide synthase type I (T1PKS) and two regions for homoserine lactone (hserlactone) biosynthesis. The ANI and dDDH values between strain HHNIBRBA2951^T and S . donghicola KCTC 12864^T were 76.2 and 19.3%, and those between strain HNIBRBA2951^T and S. undariae DSM 102234^T were 76.0 and 19.5%, respectively. The ANI and dDDH values between strain HNIBRBA3233^T and S . donghicola KCTC 12864^T were 73.8 and 18.4%, and those between strain HNIBRBA3233^T and S. guttiformis KCTC 32187^T were 73.8 and 18.0%, respectively. The ANI and dDDH values in two novel isolates were 75.6 and 19.1%, respectively (Supplementary data Table S2). These values were below the standard criteria value of ANI (95–96%) and DDH (70%), indicating that strains HNIBRBA2951^T and HNIBRBA3233^T represent two novel species of the genus Sulfitobacter. In the phylogenomic tree, two novel strains were formed clade with three related type strains ( S. donghicola KCTC 12864^T, S. undariae DSM 102234^T and S. guttiformis KCTC 32187^T) (Supplementary data Fig. S1).

Morphological, physiological and biochemical analysis

The cells of strains HNIBRBA2951^T and HNIBRBA3233^T were Gram-staining-negative, aerobic and rod-shaped (Supplementary data Fig. S2). Oxidase- and catalase- were positive. The characteristics that differentiate strains HNIBRBA2951^T, HNIBRBA3233^T, as well as the type strains of the closely related species of Sulfitobacter, are summarized in Table 1. The strains HNIBRBA2951^T and HNIBRBA3233^T could be distinguished from their closest relatives by a number of characteristics, including the temperature, pH and NaCl range for growth, hydrolysis of aesculin and gelatin, and enzyme activities. The fatty acid profiles of the isolates are shown in Table 2. The major cellular fatty acids (>5% of the total fatty acids) of strain HNIBRBA2951^T were summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c) and C_16:0, while those of strain HNIBRBA3233^T were summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c), C_19:0ω8c cyclo, C_18:1ω7c 11-methyl and C_16:0. The fatty acid composition of strains HNIBRBA2951^T and HNIBRBA3233^T exhibited a similarity to that of related Sulfitobacter species, with a predominance of summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c). However, they also showed differences in the proportions of some fatty acid, with strain HNIBRBA2951^T characterized by the absence of C_18:1ω7c 11-methyl and strain HNIBRBA3233^T by a higher proportion of C_18:1ω7c 11-methyl and C_19:0ω8c cyclo. The predominant quinone of both novel strains was Q-10. The polar lipid profile of strain HNIBRBA2951^T contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and one unidentified polar lipid (Supplementary data Fig. S3A). The corresponding profile of strain HNIBRBA3233^T contained phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified polar lipids (Supplementary data Fig. S3B). The presence of the polar lipids, phosphatidylglycerol and phosphatidylcholine is consistent with that observed in other species of the genus Sulfitobacter (Song et al., 2019; Yang et al., 2021). However, there were differences in the diphosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and several unidentified polar lipids.

Table 1

Differential Characteristics of strains HNIBRBA2951^T, HNIBRBA3233^T and four related type strains

Strains: 1, HNIBRBA2951^T; 2, HNIBRBA3233^T; 3, S. donghicola KACC 17293^T; 4, S. guttiformis KACC 13741^T; 5, S. mediterraneus KCTC 32188^T; 6, S. undariae KCTC 42200^T. Data of columns 1-4 obtained from this study; data of column 5 and 6 obtained from Fukui et al. (2015) and Yoon (2019), respectively. All strains were positive for catalase and oxidase activities. All strains were negative for nitrate reduction; indole production; glucose fermentation; α-chymotrypsin, β-glactosidase, N-acetyl-glucosaminidase, β-glucuronidase and α-fucosidase activities; and assimilation of D-mannose and capric acid. +, Positive; –, negative; w, weakly positive reaction; nd, no data.

Characteristics	1	2	3	4	5	6
Growth at/in
37°C	-	+	-	-	-	-
pH 6	-	+	-	-	+	-
pH 8.5	-	+	-	+	+	-
0.5% NaCl (w/v)	-	+	-	-	-	+
4% NaCl (w/v)	-	+	-	+	+	+
Hydrolysis of:
Esculin hydrolysis	+	+	+	+	-	-
Arginine dihydrolase, gelatinase	-	-	-	-	+	-
β-Galactosidase (PNPG)	+	-	-	+	nd	nd
Assimilation of:
D-Glucose	+	-	+	-	+	-
L-Arabinose	-	-	+	-	-	-
D-Mannitol	-	-	+	-	+	-
N-Acetyl-glucosamine	-	-	+	-	+	nd
Potassium gluconate	+	-	+	-	+	nd
Adipic acid	-	-	-	-	+	nd
Malic acid	-	-	+	-	+	nd
Trisodium citrate	-	-	-	-	+	nd
Phenylacetic acid	-	+	+	-	+	nd
Enzyme activities (API ZYM):
Naphthol-AS-BI-phosphohydrolase	+	+	+	+	-	w
Esterase lipase (C8), leucine arylamidase	+	+	-	+	+	+
Alkaline phosphatase	-	+	-	+	+	+
Valine arylamidase	-	+	+	-	w	w
Cystine arylamidase	-	+	+	-	-	-
β-Glucuronidase	-	+	-	-	-	-
Lipase (C14), trypsin, α-galactosidase, α-glucosidase	-	-	+	-	-	-
α-Mannosidase	-	-	-	+	-	-

Table 2

Cellular fatty acid compositions (%) of strains HNIBRBA2951^T, HNIBRBA3233^T and four related type strains

Strains: 1, HNIBRBA2951^T; 2, HNIBRBA3233^T; 3, S. donghicola KACC 17293^T; 4, S. guttiformis KACC 13741^T; 5, S. mediterraneus KCTC 32188^T; 6, S. undariae KCTC 42200^T. Data of columns 1-4 obtained from this study; data of column 5 and 6 obtained from Fukui et al. (2015) and Yoon (2019), respectively. Fatty acids amounting to <1.0% of the total fatty acids in all strains are not shown. -, not detected; nd, no data; tr, traces (<0.5%).

Fatty acid	1	2	3	4	5	6
Straight-chain
C14:0	0.7	0.5	tr	tr	nd	tr
C16:0	6.9	15.4	9.7	6.7	5.5	8.9
C17:0	0.5	0.8	tr	0.6	nd	nd
C18:0	2.1	2.6	3.2	1.4	0.8	1.1
Branched
iso-C18:0	tr	-	4.2	0.6	nd	4.4
Unsaturated
C18:1ω7c 11-methyl	-	15.8	1.5	2.7	11.0	-
C19:0ω8c Cyclo	-	27.3	-	-	nd	-
C20:2ω6,9c	-	0.9	-	-	nd	nd
Hydroxy
C10:0 3-OH	-	3.5	-	2.7	2.5	3.3
Summed features*
3	0.6	1.5	0.6	0.8	nd	1.2
7	2.0	0.8	-	1.4	nd	-
8	86.3	30.4	79.5	82.1	68.5	79.9

*Summed feature represents two or three fatty acids that could not be separated by GC with the Microbial Identification system. Summed feature 3 comprised C_16:1ω7c and/or C_16:1ω6c. Summed feature 7 comprised C_19:1ω7c and/or C_19:1ω6c. Summed feature 8 comprised C_18:1ω7c and/or C_18:1ω6c.

Taxonomic conclusion

The phylogenetic, phenotypic, and genomic analyses indicate that strains HNIBRBA2951^T and HNIBRBA3233^T are distinct from their closest relatives within the genus Sulfitobacter. Both strains exhibit distinctive characteristics in regard to their growth conditions, biochemical properties, and enzyme activities. The fatty acid profiles exhibited similarities with those of other Sulfitobacter species, yet also revealed distinct differences of particular interest. The C_18:1ω7c 11-methyl fatty acid is absent in strain HNIBRBA2951^T, whereas strain HNIBRBA3233^T exhibits higher proportions of this and the C_19:0ω8c cyclo fatty acids. Both strains predominant contain Q-10 as the major respiratory quinone. The polar lipid composition exhibited distinct differences. The strain HNIBRBA2951^T has been found to contain diphosphatidylglycerol and phosphatidylethanolamine, which are absent in the strain HNIBRBA3233^T. In contrast, strain HNIBRBA3233^T contained unidentified aminolipid and unidentified polar lipids. Genomic analysis, including the ANI and dDDH values, confirmed that both strains represent novel species, as their values fell below the established thresholds for species delineation. Additionally, phylogenetic inference based on 16S rRNA gene sequences placed strains HNIBRBA2951^T and HNIBRBA3233^T as distinct species within the genus Sulfitobacter, further supporting their classification as novel species. In addition, combined results lead to the proposal that strains HNIBRBA2951^T and HNIBRBA3233^T be classified as two novel species, named Sulfitobacter aquimarinus sp. nov. and Sulfitobacter marinivivus sp. nov., respectively.

Description of Sulfitobacter aquimarinus sp. nov.

Sulfitobacter aquimarinus (a.qui.ma.ri'nus. L. fem. n. aqua water; L. masc. adj. marinus, of the sea, marine; N.L. masc. adj. aquimarinus, living in seawater).

Cells are Gram-staining-negative, non-motile, obligately aerobic, non-flagellated, short rod-shaped, approximately 0.4–0.6 µm wide and 0.9–1.2 µm long after 5 days at 20°C on MA. Colonies are beige-colored, convex and circular with entire margins on MA, and approximately 1.0 mm in diameter after 5 days at 20°C. Growth occurs at 10–30°C (optimum, 25°C), at pH 5.5–8.0 (optimum, pH 7) and with 1–3% NaCl (optimum 2% NaCl). Oxidase- and catalase- activities are positive. Starch, casein, chitin and CM-cellulose are not hydrolysed. In the API 20NE test, positive for aesculin hydrolysis, β-galactosidase activity (PNPG test), assimilation of D-glucose and potassium gluconate; negative for nitrate reduction, indole production, glucose fermentation, gelatinase, arginine dihydrolase, urease, assimilation of L-arabinose, D-mannose, D-mannitol, N -acetyl-glucosamine, D-maltose, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. In the API ZYM system, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities are present; alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities are absent. The major cellular fatty acids (>5%) are summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c) and C_16:0. The only respiratory quinone is Q-10. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and one unidentified polar lipid.

The type strain is HNIBRBA2951^T (= KCTC 8586^T = MCCC 1K09399^T), isolated from seawater in Geogeumdo, Goheung, Republic of Korea. The DNA G + C content of the type strain is 59.5%. The GenBank accession numbers for the 16S rRNA gene sequence and the whole genome sequence of strain HNIBRBA2951^T are PP854564 and JBEFLO00000000 (BioProject PRJNA1118828; BioSample SAMN41628379), respectively.

Description of Sulfitobacter marinivivus sp. nov.

Sulfitobacter marinivivus (ma.ri.nivi'vus. L. masc. adj. marinus, of the sea, marine; L. masc. adj. vivus, alive; N.L. masc. adj. marinivivus, living in the sea).

Cells are Gram-staining-negative, non-motile, obligately aerobic, non-flagellated, rod-shaped, approximately 0.4–0.6 µm wide and 1.0–1.2 µm long after 5 days at 20°C on MA. Colonies are beige-colored, convex and circular with entire margins on MA, and approximately 1.0 mm in diameter after 5 days at 20°C. Growth occurs at 10–30°C (optimum, 25°C), at pH 6–9 (optimum, pH 7) and with 0–10% NaCl (optimum 2% NaCl). Oxidase- and catalase-activities are positive. Starch, casein, chitin and CM-cellulose are not hydrolysed. In the API 20NE test, positive for aesculin hydrolysis and assimilation of phenylacetic acid; negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, β-galactosidase activities (PNPG test) and assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N -acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid and trisodium citrate. In the API ZYM system, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase and β-glucuronidase activities are present; esterase (C4), lipase (C14), trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N -acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities are absent. The major cellular fatty acids (>5%) are summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c), C_19:0ω8c cyclo, C_18:1ω7c 11-methyl and C_16:0. The only respiratory quinone is Q-10. The polar lipids are phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified polar lipids.

The type strain is HNIBRBA3233^T (= KCTC 8585^T = MCCC 1K09401^T), isolated from tidal flat in Wando, Republic of Korea. The DNA G + C content of the type strain is 63.5%. The GenBank accession numbers for the 16S rRNA gene sequence and the whole genome sequence of strain HNIBRBA3233^T are PP854565 and JBEFLP00000000 (BioProject PRJNA1118829; BioSample SAMN41628380), respectively.

남해 섬 지역의 해양 환경에서 분리된 균주 HNIBRBA2951^T와 HNIBRBA3233^T는 그람 음성, 절대 호기성, 비운동성, 막대 모양의 세포 형태를 가진다. 16S rRNA 유전자의 염기서열을 기반으로 한 계통발생학적 분석 결과, 두 균주는 Sulfitobacter 속에 속하며, 각각 Sulfitobacter donghicola DSW-25^T (98.6% 및 97.3%)와 Sulfitobacter mediterraneus CH-B427^T (98.0% 및 97.3%)와 가장 높은 유사성을 나타냈다. 신종 두 균주 간의 서열 유사성은 97.2%였다. ANI와 dDDH 값은 관련 표준 균주와 비교하여 각각 종 구분 기준치 미만(<78.2% 및 <20.8%)이었다. 두 균주의 주요 퀴논은 ubiquinone-10이었으며, 주요 지방산으로는 summed feature 8 (C_18:1ω7c 및/또는 C_18:1ω6c)을, 주요 극성 지질로는 포스파티딜글리세롤을 함유하고 있었다. 계통발생학적 및 표현형적 특성을 종합적으로 고려하여, 균주 HNIBRBA2951^T와 HNIBRBA3233^T는 Sulfitobacter 속의 새로운 종으로 제안하며, 표준 균주는 각각 Sulfitobacter aquimarinus sp. nov. (HNIBRBA2951^T = KCTC 8586^T = MCCC 1K09399^T)와 Sulfitobacter marinivivus sp. nov. (HNIBRBA3233^T = KCTC 8585^T = MCCC 1K09401^T)이다.

This work was supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (HNIBR202101111).

The authors declare that there is no conflict of interests regarding the publication of this paper.