Genome-based classification of Halonovum salinarum gen. nov., sp. nov., and Halonovum rutilum sp. nov., two novel halophilic archaea isolated from a solar saltern

Abstract

Background

Haloarchaea, belonging to the class Halobacteria, currently include more than 400 validly published species. The isolation of novel halophilic archaea is important for understanding their evolutionary, ecological, and taxonomic diversity. Within the family Haloferacaceae, the genus Halorarum comprises two species reclassified from Halobaculum and one newly described species. In this study, we propose a new genus, Halonovum, to accommodate two novel species that are phylogenetically related to the genera Halobaculum and Halorarum.

Results

Two novel halophilic archaeal strains, MBLA0143^T and MBLA0147^T, were isolated from a solar saltern in Republic of Korea. Strain MBLA0143^T and MBLA0147^T showed the highest 16 S rRNA gene similarity (94.5% and 94.3%) to Halorarum salinum NJ-3-1^T, and these two isolates were confirmed to represent different species, with 93.7% 16 S rRNA gene similarity between them. Their growth occurred at 25–55 °C (optimum, 40 °C), 2.5–5.1 M NaCl (optimum, 4.2 M), 0.1–1.0 M MgCl₂ (optimum, 0.1 M), and pH 5.0–9.0 (optimum, 6.0–7.0). The predominant polar lipids detected in these two strains included phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. The genome size and G + C content of strain MBLA0143^T were 3.58 Mbp and 68.3%, while those of strain MBLA0147^T was 4.29 Mbp and 67.6%. The OrthoANI (74.6–76.1% for MBLA0143ᵀ and 73.8–75.3% for MBLA0147ᵀ), AAI (66.9–71.7% for MBLA0143ᵀ and 66.9–67.8% for MBLA0147ᵀ), and isDDH (20.8–21.5% for MBLA0143ᵀ and 20.0–20.7% for MBLA0147ᵀ) values between these two species and currently recognized Halobaculum and Halorarum species support their placement in different genera within the family Haloferacaceae, based on the genus delimitation thresholds. In addition, phylogenomic analyses confirmed that the two novel species are distinct from species of other genera.

Conclusions

These results combined with polyphasic taxonomic analyses support that strains MBLA0143^T and MBLA0147^T represent a novel genus related to the genera Halobaculum and Halorarum. Two novel species are proposed to be classified into a novel genus and species, named Halonovum salinarum gen. nov., sp. nov. and Halonovum rutilum sp. nov. The type strains are MBLA0143^T (= KCTC 4317^T = JCM 36640^T) and MBLA0147^T (= KCTC 4319^T = JCM 36641^T).

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04481-8.

Background

Artificial and natural hypersaline environments, including solar salterns, serve as typical habitats for halophilic archaea belonging to the class Halobacteria [1]. Currently, the class Halobacteria comprises two orders, seven families, and eighty-five genera. The genus Halobaculum (Hbl.), classified under the family Haloferacaceae, order Halobacteriales, and class Halobacteria, was first proposed in 1995 [2] and currently comprises 13 validly published species (as of October 2025), according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN, https://lpsn.dsmz.de/). The type species, Halobaculum gomorrense, was isolated from the Dead Sea. Members of the genus Halobaculum are Gram-stain-negative, red-pigmented, and chemoheterotrophic archaea, distinguished by their major polar lipid composition, which includes phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), and sulfated mannosyl glucosyl diether (S-DGD-1) [3, 4]. Some species in genus Halobaculum such as Hbl. halophilum, Hbl. rubrum, Hbl. saliterrae, and Hbl. salinum contain phosphatidylglycerol sulfate (PGS) [5–7].

Subsequently, the genus Halorarum was proposed based on genome-based analyses of the family Haloferacaceae and was validly published in 2024 with two reclassified species, Hrm. halophilum and Hrm. salinum [8]. Recently, the third species, Hrm. halobium, was also validly published [9]. Members of the genus Halorarum are Gram-stain-negative, aerobic, chemoorganotrophic archaea with variable polar lipid compositions, including PG, PGP-Me, S-DGD-1, phosphatidylglycerol sulfate (PGS), and mannosyl glucosyl diether (DGD-1) [8].

Phylogenomic evidence indicates that certain strains previously affiliated with the genus Halobaculum and Halorarum represent distinct evolutionary lineages. In this study, we collected samples from the Sorae solar saltern, located in the Yellow Sea of the Republic of Korea, and isolated novel species candidates. A polyphasic taxonomic analysis was conducted on strains MBLA0143^T and MBLA0147^T. Genome-based classification further confirmed that these strains are distinct from Halobaculum and Halorarum, supporting the proposal of a novel genus, Halonovum, within the family Haloferacaceae.

Materials and methods

Isolation of pure haloarchaeal cultures

An unpurified surface sample was collected at a solar saltern in Sorae, Republic of Korea (37°24′40″N, 126°44′43″E) in 2018. The isolation procedure of halophilic archaea followed previously described methods [10, 11]. Briefly, 1 g of sample was inoculated into 100 mL of sterile DB Characterization Medium No. 2 (DBCM2). The composition of the medium per liter was as follows: 200 g NaCl, 29 g MgSO₄·7H₂O, 25 g MgCl₂·6H₂O, 5.8 g KCl, 0.25 g peptone (KisanBio, Seoul, Republic of Korea), 0.05 g yeast extract (BD Difco™, Franklin Lakes, NJ, USA), 1.1 g sodium pyruvate, 4.16 ml of 1 M CaCl₂ solution, 1 ml of 4.8 mM FeCl₂ solution, 5 ml of 1 M NH₄Cl, 2 ml of 0.5 M potassium phosphate buffer (pH 7.5), 3 ml of vitamin solution, and 1 ml of trace element solution. The final pH was adjusted to 7.0–7.3 using 1 M Tris-base buffer [12]. The inoculated broth was incubated for approximately 1 month. The culture was then serially diluted with sterile 20% (w/v) NaCl solution, and each diluted sample was spread on DBCM2 supplemented with 2.0% (w/v) agar. Plates were incubated at 37 °C for at least two weeks. Colonies that appeared were selected and sub-cultured on fresh DBCM2 agar more than three times to ensure purity. Among them, two pure isolates, designated MBLA0143^T and MBLA0147^T, were selected for further taxonomic analysis. The strains were preserved at −80 °C in 20% (w/v) glycerol solution containing 20% (w/v) NaCl.

For comparative polyphasic analyses, reference strains Hbl. gomorrense JCM 9908^T, Hbl. saliterrae JCM 32,473^T, Hrm. halophilum JCM 33,550^T, and Hrm. salinum JCM 33,552^T were obtained from the Japan Collection of Microorganisms (JCM).

Phylogenetic analyses

Genomic DNA from strains MBLA0143^T and MBLA0147^T was extracted using the Genomic DNA Mini Kit (Cosmogenetech, Seoul, Republic of Korea). The 16 S rRNA genes of two strains were amplified and cloned following previously described methods [13]. PCR products were purified using the Cosmogenetech PCR Purification Kit and sequenced at Macrogen Co., Ltd. (Seoul, Republic of Korea). The resulting sequences were assembled using SeqMan™ II expert sequence analysis software. The 16 S rRNA gene sequences of strains MBLA0143^T and MBLA0147^T were compared with those of related taxa using the EzBioCloud database (https://www.ezbiocloud.net/). In addition, the RNA polymerase subunit B′ (rpoB′) genes were retrieved from the genome sequences of the strains. rpoB′ gene sequences of related species were obtained from the Genome Analysis Database and the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genome/) [14]. Multiple sequence alignments were performed using the ClustalW algorithm implemented in BioEdit to evaluate sequence similarities between the isolates and reference taxa [15]. Phylogenetic trees based on 16 S rRNA and rpoB′ gene sequences were constructed using the maximum-likelihood (ML) method in MEGA X software, with 1,000 bootstrap replications to assess branch support, applying the Kimura two-parameter substitution model [16–18].

Microscopy, growth conditions, and chemotaxonomy

All experiments were conducted using DBCM2 medium, following the minimal standards protocol for haloarchaea as previously reported [19]. Gram staining was performed using a method specifically designed for haloarchaea [20]. The cell morphology of strains MBLA0143^T and MBLA0147^T were examined using a Field-Emission Scanning Electron Microscope (FESEM) (SIGMA 360; Carl Zeiss, Oberkochen, Germany). Sample preparation for FESEM observation was performed according to previously described methods [21]. Growth conditions, including NaCl concentration, temperature, and pH were determined according to the previously described methods [22]. Nutrient requirements and biochemical activities were assessed in accordance with the minimal standards for haloarchaea [19]. Polar lipids were extracted and separated by one- and two-dimensional thin-layer chromatography (TLC) following established protocols [23].

Genome sequencing, annotation, and analysis

The genome of strains MBLA0143^T and MBLA0147^T were sequenced and assembled using the Pacific Biosciences Sequel platform. De novo genome assembly was performed with Flye assembler v2.8.3, under default parameters within the PacBio SMRT Analysis software v2.3.0 [24]. General genomic features were retrieved from the NCBI Assembly database (https://www.ncbi.nlm.nih.gov/assembly/) and annotated using the Rapid Annotations using Subsystems Technology (RAST) server (https://rast.nmpdr.org/) via the RASTtk pipeline [25, 26]. Gene prediction and functional annotation were carried out with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [27]. Predicted genes were classified into Clusters of Orthologous Groups (COG) using the eggNOG v5.0 database for homology-based functional assignment [28, 29]. Secondary metabolite biosynthetic gene clusters were identified with antiSMASH v7.0 under stringent detection settings and additional modules, including KnownClusterBlast, ClusterBlast, SubClusterBlast, MIBiG comparison, ActiveSiteFinder, and RREFinder (https://antismash.secondarymetabolites.org/) [30].

For genome comparisons, all available genomes of the genera Halobaculum and Halorarum were retrieved from the NCBI Genome Database (https://www.ncbi.nlm.nih.gov/genome/). To evaluate genomic relatedness and delineate species boundaries, Ortho-average nucleotide identity (OrthoANI), average amino acid identity (AAI), and in silico DNA–DNA hybridization (isDDH) analyses were conducted. OrthoANI values between strains MBLA0143ᵀ and MBLA0147^T and other Halobaculum and Halorarum species were calculated using OAT software version 0.93.1 [31]. AAI values were determined using EzAAI version 1.2 [32]. isDDH values were estimated with the Genome-to-Genome Distance Calculator (GGDC 2.1; https://ggdc.dsmz.de/ggdc.php), employing the recommended BLAST + alignment and formula 2 (identities/HSP length) [33]. Intergenomic distances between strains MBLA0143^T and MBLA0147^T and the type strains of Halobaculum and Halorarum were further used to construct a balanced minimum evolution tree with branch support using FASTME 2.1.4, with subtree pruning and regrafting (SPR) postprocessing, via the Type Strain Genome Server (TYGS; https://tygs.dsmz.de/) [34, 35].

For reproducible phylogenomic analysis, core genes were identified with the Up-to-date Bacterial Core Gene (UBCG) pipeline v3.0 [36]. A phylogenomic tree was reconstructed from the concatenated sequences of core genes within the family Haloferacaceae using RAxML as implemented in the UBCG pipeline [37]. Bootstrap support was calculated from 1,000 replicates, and tree visualization was performed using the Interactive Tree of Life (iTOL) tool v7.2 [38].

Pan-genomic analysis

Pan-genome analysis was conducted using the Bacterial Pan-Genome Analysis (BPGA) software [39]. The genomes of Halobaculum and Halorarum species, together with strains MBLA0143^T and MBLA0147^T, were categorized into core genes (conserved across all strains), accessory genes (shared by more than two species), unique genes (strain-specific), and exclusively absent genes (absent strain-specific). Functional and pathway analyses of the pan-genome were performed using the COG database (https://www.ncbi.nlm.nih.gov/research/cog/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/), based on representative sequences of all orthologous gene families [40]. Comparative functional analyses were conducted for core, accessory, and unique pan-genome orthologous groups (POGs). Unique genes identified in strains MBLA0143^T and MBLA0147^T were further annotated using KEGG database. Both POGs were clustered with the USEARCH algorithm, applying a 50% sequence identity cut-off. Data visualizations were generated with Gnuplot v4.6.6, and multiple sequence alignments were performed using MUSCLE [41].

Results and discussion

Phylogenetic analysis based on 16 S rRNA and rpoB' genes

Phylogenetic analyses based on the 16 S rRNA and rpoB′ gene sequences were performed to determine the taxonomic positions of strains MBLA0143^T and MBLA0147^T within the related genera. In ML trees constructed from 16 S rRNA gene sequences, the two strains showed the highest similarity to Hrm. salinum NJ-3-1^T (94.5 and 94.3%, respectively) (Fig. 1). The strains clustered together with 100% bootstrap support. However, the 16 S rRNA gene sequence similarity between MBLA0143^T and MBLA0147^T was 93.7%, indicating that they represent distinct species. The strains were also confirmed to be taxonomically distinct from other genera of the family Haloferacaceae, including Haloprofundus, Halogranum, and Salinigranum. Instead, they formed a separate cluster closely related to Halobaculum and Halorarum. This distinction was further supported by phylogenetic trees based on rpoB′ gene sequences, which consistently separated the two strains from the closely related genera within the family Haloferacaceae (Fig. 2). These results support the proposal that strains MBLA0143^T and MBLA0147^T represent a novel genus.

Fig. 1.

Maximum likelihood phylogenetic tree of strains MBLA0143^T, MBLA0147^T and closely related species based on the 16 S rRNA gene sequence. Bootstrap values (%) were based on 1,000 replicates and are shown for branches with more than 70% bootstrap support. Bar, 0.02 accumulated changes per nucleotide

Fig. 2.

Maximum likelihood phylogenetic tree of strains MBLA0143^T, MBLA0147^T and closely related species based on the rpoB′ gene sequence. Bootstrap values (%) were based on 1,000 replicates and are shown for branches with more than 70% bootstrap support. Bar, 0.05 accumulated changes per nucleotide

Cell morphology, growth physiology, and chemotaxonomy

Strains MBLA0143ᵀ and MBLA0147^T were both Gram-stain-negative, motile, and lysed in distilled water. Cells of strain MBLA0143ᵀ were coccoid-shaped, whereas those of strain MBLA0147^T were rod-shaped (Fig. 3). Colonies of both strains grown on DBCM2 agar at 37 °C for 14 days, appeared red, irregular, and flat. Optimal growth for both strains occurred at 4.2 M NaCl (growth range: 2.5–5.1 M), 0.1 M MgCl₂ (growth range: 0–1.0 M), 40 °C (growth range: 25–55 °C), and pH 6.0–7.0 (growth range: 5.0–9.0). Anaerobic growth was not observed in the presence of nitrate, DMSO, or L-arginine, and neither strain reduced nitrate to nitrite. Catalase activity was positive, whereas oxidase activity, H₂S production, and indole formation were negative in both strains. Neither strain hydrolyzed casein, gelatin, starch, or Tween 80. With respect to substrate utilization, both strains metabolized D-glucose, D-ribose, and pyruvate. Strain MBLA0143^T additionally utilized sucrose, lactose, L-malate, and L-lysine, whereas MBLA0147^T utilized D-mannose, D-galactose, D-fructose, glycerol, D-sorbitol, acetate, citrate, glycine, and L-glutamate. Neither strain grew on L-sorbose, D-xylose, maltose, starch, D-mannitol, DL-lactate, succinate, fumarate, L-alanine, L-arginine, L-aspartate, and L-ornithine. A summary of phenotypic characteristics differentiating these two strains from related species of the genera Halobaculum and Halorarum is presented in Table 1.

Fig. 3.

Scanning electron micrograph. A Strain MBLA0143^T. B Strain MBLA0147^T

Table 1.

Differential characteristics among strains MBLA0143^T, MBLA0147^T, and the current species of the genus Halobaculum. Strains: 1, MBLA0143^T; 2, MBLA0147^T; 3, Hbl. Saliterrae JCM 32,473^T; 4, Hrm. Halophilum JCM 33,550^T; 5, Hrm. Salinum JCM 33,552^T; 6, Hbl. Gomorrense JCM 9908^T. Symbols: +, positive; −, negative. All data obtained from this study

Characteristics	1	2	3	4	5	6
Temperature (°C) (Optimum)	25–55 (40)	25–55 (40)	30–55 (45)	30–55 (40)	25–55 (40)	25–55 (40)
NaCl (M) (Optimum)	2.5–5.1 (4.2)	2.5–5.1 (4.2)	0.9–5.1 (2.6–3.4)	1.7–5.1 (3.4)	0.9–5.1 (3.4)	1.7–5.1 (2.6–3.4)
pH (Optimum)	5.0–9.0 (6.0–7.0)	5.0–9.0 (6.0–7.0)	5.0–9.0 (7.0)	5.0–9.0 (7.0)	5.0–9.0 (7.0)	5.0–9.0 (7.0)
Anaerobic growth with nitrate	−	−	−	+	+	−
Anaerobic growth with L-arginine	−	−	−	+	+	−
Utilization of
D-mannose	−	+	−	−	−	−
D-galactose	−	+	−	−	+	+
D-fructose	−	+	+	+	+	+
Maltose	−	−	+	−	−	+
Sucrose	+	−	−	−	−	+
Lactose	+	−	−	−	−	−
Glycerol	−	+	+	+	+	+
D-sorbitol	−	−	−	−	−	−
Acetate	−	+	−	−	−	−
L-malate	+	−	−	−	−	+
Fumarate	−	−	+	−	−	−
Citrate	−	+	−	−	−	+
Glycine	−	+	+	−	−	+
L-alanine	−	−	−	+	−	+
L-glutamate	−	+	+	−	−	+
L-lysine	+	−	−	−	−	−
L-ornithine	−	−	−	+	−	+
Presence of PGS	−	−	−	+	+	−

TLC analysis of strains MBLA0143^T and MBLA0147^T revealed that phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-ME) were major polar lipid components (Fig. S1). Strain MBLA0143^T contained three polar lipids (PLs), six unidentified lipids (Ls), and four glycolipids (GLs), and a comparable lipid profile was observed in strain MBLA0147^T. A key chemotaxonomic distinction between the two strains was that MBLA0143^T possessed an amino phosphoglycolipid (APGL), whereas MBLA0147^T contained an amino phospholipid (AGL). This difference supports their separation at the species level. One-dimensional TLC showed slight differences between MBLA0143^T and MBLA0147^T and the reference species (Fig. S2). Glycolipid profiles also exhibited minor variations among all compared strains. Regarding to the polar lipids, phosphatidylglycerol sulfate (PGS) was detected in some strains, but it was absent in both MBLA0143^T and MBLA0147^T.

Genome features, annotation, and comparative genomic analyses

The genomes of strains MBLA0143^T and MBLA0147^T were assembled into two and six contigs, respectively. The genome of MBLA0143^T had a total length of 3,580,614 bp with a G + C content of 68.3%, whereas the genome of MBLA0147^T had a total length of 4,292,040 bp with a G + C content of 67.6%. Genome annotation of strain MBLA0143^T predicted 3,610 genes, including 3,559 protein-coding sequences and 51 RNA genes (46 tRNAs, 3 rRNAs, and 2 ncRNAs). Genome annotation of strain MBLA0147^T predicted 4,024 genes, including 3,970 protein-coding sequences and 54 RNA genes (49 tRNAs, 3 rRNAs, and 2 ncRNAs). General genomic features of the two strains are summarized in Table 2.

Table 2.

General characteristics of the genome of strains MBLA0143^T and MBLA0147^T in this study

Attribute	MBLA0143T	MBLA0147T
Sequencing platforms	PacBio Sequel	PacBio Sequel
Assembler	FLYE v. 2.8.3	FLYE v. 2.8.3
Genome coverage	1,093.1x	1471.0x
Assembly size (bp)	3,580,614	4,292,040
Total contig	2	6
Contig length (bp)
- contig 1	2,991,388	3,004,410
- contig 2	589,226	451,027
- contig 3	-	307,905
- contig 4	-	226,510
- contig 5	-	220,336
- contig 6	-	81,852
G + C contents (mol%)
- contig 1	68.51	69.03
- contig 2	67.27	66.39
- contig 3	-	69.17
- contig 4	-	58.81
- contig 5	-	60.13
- contig 6	-	59.13
Total genes	3,610	4,024
Total CDS	3,559	3,970
Total coding genes	3,504	3,919
RNAs	51	54
- rRNAs (5 S, 16 S, 23 S)	3 (1, 1, 1)	3 (1, 1, 1)
- tRNAs	46	49
- ncRNAs	2	2

According to COG functional classification, the most abundant categories in MBLA0143^T and MBLA0147^T were amino acid transport and metabolism (E; 260 genes, 11.36%; 265 genes, 9.81%) and transcription (K; 184 genes, 8.04%; 244 genes, 9.03%) (Table S1). In MBLA0143^T, 161 genes (7.03%) were classified under translation, ribosomal structure, and biogenesis (J), and 156 genes (6.82%) under replication, recombination, and repair (L). In contrast, MBLA0147^T showed the reverse trend, with 185 genes (6.85%) in category L and 163 genes (6.03%) in category J. These distributions were similar to the average COG distribution profiles of the genera Halobaculum and Halorarum, in which most species exhibited a high abundance of genes in categories E, K, J, and C, in descending order (Fig. S3). Notably, strain MBLA0147^T exhibited 5.48% of its genes in the category of carbohydrate transport and metabolism (G), which was higher than the average of 4.09% observed in other Halobaculum and Halorarum species.

The antiSMASH analysis revealed that strains MBLA0143^T and MBLA0147^T harbor terpene and lanthipeptide biosynthetic gene clusters (BGCs) (Table S2). Strain MBLA0143^T possessed two terpene and two lanthipeptide BGCs, predicted to encode closoxazole, archalan α, lichenicidin, and choline. Strain MBLA0147^T contained four BGCs, including three terpene and one lanthipeptide BGCs, associated with the predicted production of zeaxanthin, stlassin, archalan α, and kanamycin. These results highlight the diverse secondary metabolite biosynthetic potential of the two strains, underscoring their value as promising resources for biotechnological applications. In particular, the genomes of strains MBLA0143^T and MBLA0147^T harbor genes associated with carotenoid biosynthesis, including phytoene synthase (crtB), phytoene desaturase (crtD), prenyltransferase (lyeJ), and bisanhydrobacterioruberin hydratase (cruF) (Fig. S4A and Table S3). These gene clusters are predicted to produce bacterioruberin (BR), a compound of increasing interest in bioindustrial applications [42]. Several haloarchaeal genera, including Haloferax, Halorubrum, Haloarcula, Halococcus, Halobacterium, Halorhabdus, and Haloterrigena, have been reported to produce bacterioruberin and to exhibit superior antioxidant activity [43, 44]. Comparative analysis showed that strains MBLA0143^T, MBLA0147^T, and all species of the genera Halobaculum and Halorarum shared more than 60% amino acid sequence similarities in bacterioruberin biosynthetic genes (Fig. S4B). MBLA0143^T and MBLA0147^T exhibited amino acid sequence identities of 83.5% (CrtB), 88.1% (CrtD), 81.9% (LyeJ), and 72.2% (CruF) in their respective carotenoid biosynthetic genes. In comparison, the corresponding sequence similarities across other strains were 66.7–76.1% (CrtB), 73.2–78.9% (CrtD), 61.4–62.4% (LyeJ), and 61.8–64.4% (CruF). These results suggest that the newly isolated strains are capable of producing bacterioruberin, and that carotenoid biosynthesis-related genes are evolutionarily well conserved within the proposed novel genus.

Genes associated with polyhydroxyalkanoate (PHA) biosynthesis were identified in strinas MBLA0143^T and MBLA0147^T, including acetyl-CoA acetyltransferase (phaA), two 3-hydroxybutyryl-CoA dehydrogenases (phaB), two enoyl-CoA hydratases (fadB), and 3-hydroxyacyl-CoA dehydrogenase (fadE) (Table S4). The presence of these genes indicates that two novel strains possess the potential to synthesize PHA, a valuable biopolymer with diverse industrial applications. PHA-associated genes were also found in MBLA0143^T, MBLA0147^T, and all species of the genera Halobaculum and Halorarum, with amino acid sequences similarities ranging from 32.3 to 88.9% (Fig. S5). However, the key enzymes involved in PHA biosynthesis, the PHA synthases phaC, was not been identified in either genome. To date, phaC has only been reported in some halophilic archaeal genera, including Haloferax and Haloarcula [45, 46]. Because most of the haloarchaeal genomes are neither fully sequenced nor fully annotated [47], the absence of phaC may reflect incomplete annotations or the presence of an as-yet-uncharacterized class of PHA synthases. Although phaC was not identified, we confirmed that phaA and phaB, which are essential for PHA biosynthesis, are conserved within the genera Halobaculum and Halorarum, showing high amino acid sequence similarities of 76.4–87.3% and 79.0–84.7%, respectively (Fig. S5). These results suggest that PHA synthases in Halobaculum and Halorarum may have been overlooked due to limited research, or that alternative classes of synthases exist [48]. Continued discovery of novel halophilic archaea will be important for elucidating the diversity of genes encoding functional biomolecules.

Comparative genomic indices are represented in Table 3. The OrthoANI, AAI, and isDDH values between the two novel strains were 79.2%, 74.3%, and 24.1%, respectively, indicating that they represent distinct species. OrthoANI values calculated among strains MBLA0143^T, MBLA0147^T, and closely related species of the genus Halobaculum and Halorarum ranged from 73.8% to 94.6%. MBLA0143^T and MBLA0147^T exhibited OrthoANI values of less than 75.3% and 76.1%, respectively, when compared to previously reported Halobaculum and Halorarum species. Across all species analyzed in this study, AAI values ranged from 66.9% to 95.2%. MBLA0143^T shared an AAI of less than 67.8%, showing the highest similarity to Hrm. salinum NJ-3-1^T. MBLA0147^T exhibited an AAI of 71.7% with Hbl. gomorrense DSM 9297^T, representing the highest identity observed for this strain. The isDDH values of strains MBLA0143^T and MBLA0147^T did not exceed 20.7% and 21.5%, repectively, in comparison with previously reported species. These values fall well below the accepted thresholds for species delineation (95–96% for OrthoANI and AAI, and 70% for isDDH), clearly indicating that MBLA0143^T and MBLA0147^T represent distinct species within the genera Halobaculum and Halorarum [31, 33, 49]. Importantly, the consistently low OrthoANI and AAI values also indicate that MBLB0143^T and MBLA0147^T likely represent a novel genus within the family Haloferacaceae [8]. Furthermore, phylogenomic analysis based on genome-scale intergenomic relatedness revealed that MBLA0143^T and MBLA0147^T, together with 13 other species of the genus Halobaculum and Halorarum, did not cluster within any recognized species or subspecies (within the same species or related species) groups (Fig. S6), providing additional support for its taxonomic novelty [35].

Table 3.

Overall genome-related indices compared strains MBLA0143^T and MBLA0147^T with current species of the genera Halobaculum and Halorarum

Taxon	Hvm. salinarum MBLA0143T				Hvm. rutilum MBLA0147T
	OrthoANI (%)	AAI (%)	isDDH (%)	OrthoANI (%)		AAI (%)	isDDH (%)
Hvm. rutilum MBLA0147T	79.2	74.3	24.1	-		-	-
Hrm. halophilum Gai3-2T	75.6	67.5	20.6	75.0		67.4	21.4
Hrm. salinum NJ-3-1T	76.0	67.8	20.5	75.2		67.6	21.5
Hrm. halobium XH14T	75.1	67.4	20.6	75.5		67.2	21.4
Hbl. gomorrense DSM 9297T	75.4	67.0	20.3	74.9		71.7	21.1
Hbl. halobium SYNS20T	75.2	67.0	20.2	74.7		67.3	21.3
Hbl. limi YSMS11T	74.6	66.9	20.0	73.8		66.9	20.8
Hbl. lipolyticum DT31T	75.9	67.2	20.4	75.3		67.4	21.1
Hbl. litoerum DT92T	76.1	67.4	20.4	75.3		67.7	21.4
Hbl. magnesiiphilum MGY-184T	75.7	66.9	20.7	75.2		67.1	21.3
Hbl. marinum DT55T	75.4	67.0	20.4	74.8		67.0	21.4
Hbl. roseum D90T	75.8	67.1	20.4	75.3		67.3	21.5
Hbl. rubrum C46T	75.6	67.1	20.4	74.8		67.4	21.3
Hbl. saliterrae WSA2T	75.5	67.1	20.6	75.1		67.0	21.1

The phylogenomic tree reconstructed using the UBCG tool revealed that strains MBLA0143^T and MBLA0147^T formed an independent clade within the major cluster containing the genera Halobaculum and Halorarum (Fig. 4). MBLA0143^T and MBLA0147^T formed a well-supported branch with strong bootstrap values, positioned as a sister clade to the cluster comprising all species of Halobaculum and Halorarum, thereby suggesting their taxonomic novelty. UBCG-based gene support indices (GSI) analysis indicated that the clade comprising the two novel species and the clade containing Hbl. gomorrense DSM 9297^T and Hrm. salinum NJ-3-1^T showed high GSI support values (25/27) (Fig. S7). In contrast, all members of Halobaculum and Halorarum, along with Halolamina pelagica CGMCC 1.10329ᵀ (the type species of Halolamina), formed a separate cluster in the UBCG tree, which showed a GSI of 22 and supported their classification as distinct genera. Our two novel species formed a distinct cluster separate from the genera Halobaculum and Halorarum. The two strains clustered within the same node with a GSI of 27, but with the genera Halobaculum and Halorarum, exhibited with a GSI of 25. These results support the conclusion that MBLA0143^T and MBLA0147^T represent a novel genus distinct from previously described taxa.

Fig. 4.

Phylogenomic tree of strains MBLA0143^T, MBLA0147^T, and closely related species reconstructed based on concatenated nucleotide sequences using UBCG pipeline. Bootstrap values (%) were based on 1,000 replicates and are shown for branches with more than 70% bootstrap support. Bar, 0.1 accumulated changes per nucleotide

Pan-genomic analysis with closely related species

Pan-genomic analysis revealed an open-type pangenome for the genera Halobaculum and Halorarum, characterized by continuous expansion of gene clusters and a gradual stabilization of the core genome. The combined pan-genome expanded to 14,768 genes with 1,188 core genes (Fig. S8A). COG annotations showed that most gene clusters were associated with metabolism, cellular processes and signaling, and information storage processing, although a large portion remained poorly characterized (Fig. S8B). Strain-specific gene counts varied among species, with Hrm. halophilum Gai3-2^T and Hrm. salinum NJ-3-1^T showing relatively high numbers of unique genes (1,021 and 1,121, respectively) (Table S5). Strains MBLA0143^T and MBLA0147^T also showed high numbers of unique genes counts compared to other species (1,138 and 1,405, respectively), suggesting their unique genomic features and novelty (Fig. S8C). The high number of strain-specific genes in the two novel strains, combined with their placement in sister clades within the pan-genome phylogenetic tree (Fig. S8D), indicates evolutionary divergence from other species of the genera Halobaculum and Halorarum. This pattern of phylogenetic distinctness was consistently supported by trees constructed from 16 S rRNA, rpoB′, and conserved core genes.

Conclusions

Based on phenotypic, phylogenetic, phylogenomic, and comparative genomic analyses, strains MBLA0143ᵀ (= KCTC 4317ᵀ = JCM 36640^T) and MBLA0147ᵀ (= KCTC 4319ᵀ = JCM 36641ᵀ) are proposed to form a novel genus Halonovum gen. nov., with two species named Halonovum salinarum sp. nov. and Halonovum rutilum sp. nov.

Description of Halonovum gen. nov

Halonovum (Ha.lo.no’vum. Gr. masc. n. hals halos, salt; L. neut. adj. novum, new; N.L. neut. n. Halonovum, a new organism inhabiting salt environments).

Cells are Gram-stain-negative, motile, showing different shapes (cocci or rods) under optimal growth conditions. Cells lyse in distilled water. Colonies on agar plates are red, irregular, and flat. Growth occurs at 25–55 °C, 2.5–5.1 M of NaCl, 0–1.0 M MgCl₂, and pH 5.0–9.0. Chemoorganotrophic and aerobic. Sugars and amino acids can be utilized for growth. The major polar lipids are PG and PGP-Me. Minor unidentified glycolipids may be presented. The type species is Halonovum salinarum. Recommended three-letter abbreviation: Hvm.

Description of Halonovum salinarum sp. nov

Halonovum salinarum (sa.li.na’rum. L. gen. fem. pl. n. salinarum, of salt works).

Cells are Gram-stain-negative, motile, and coccoid-shaped under the optimal growth conditions, and lyse in distilled water. The colonies grown on DBCM2 agar for 14 days under 37 °C are red, irregular, and flat. Growth occurs optimally at 4.2 M NaCl (2.5–5.1 M), 0.1 M MgCl₂ (0–1.0 M), 40 °C (25–55 °C), and pH 6.0–7.0 (5.0–9.0). Anaerobic growth does not observe in nitrate, DMSO, and L-arginine. Reduction of nitrate to nitrite do not observe. Catalase activity is positive while oxidase activity is negative. H₂S and indole formation are negative. Casein, gelatin, starch, or Tween 80 cannot be hydrolyzed. D-Glucose, D-ribose, sucrose, lactose, pyruvate, L-malate, and L-lysine can support growth, respectively. No growth occurred on D-mannose, D-galactose, D-fructose, L-sorbose, D-xylose, maltose, starch, glycerol, D-mannitol, D-sorbitol, acetate, DL-lactate, succinate, fumarate, citrate, glycine, L-alanine, L-arginine, L-aspartate, L-glutamate, and L-ornithine. The major polar lipids are PG and PGP-Me. Genome size and G + C content are 3.58 Mbp and 68.3%.

The type strain MBLA0143^T (KCTC 4317^T = JCM 36640^T) was isolated from a solar saltern, Republic of Korea.

Description of Halonovum rutilum sp. nov

Halonovum rutilum (ru.ti’lum. L. neut. adj. rutilum, red-colored, pertaining to the colony pigmentation of the type strain).

Cells were Gram-stain-negative, motile, and rod-shaped under the optimal growth conditions, and lyse in distilled water. The colonies grown on DBCM2 agar for 14 days under 37 °C are red, irregular, and flat. Growth occurs optimally at 4.2 M NaCl (2.5–5.1 M), 0.1 M MgCl₂ (0–1.0 M), 40 °C (25–55 °C), and pH 6.0–7.0 (5.0–9.0). Anaerobic growth does not observe in nitrate, DMSO, and L-arginine. Reduction of nitrate to nitrite do not observe. Catalase activity is positive while oxidase activity is negative. H₂S and indole formation are negative. Casein, gelatin, starch, or Tween 80 cannot be hydrolyzed. D-Glucose, D-mannose, D-galactose, D-fructose, D-ribose, glycerol, D-sorbitol, acetate, pyruvate, citrate, glycine, and L-glutamate can support growth, respectively. No growth occurred on L-sorbose, D-xylose, maltose, sucrose, lactose, D-mannitol, DL-lactate, succinate, L-malate, fumarate, L-alanine, L-arginine, L-aspartate, L-lysine, and L-ornithine. The major polar lipids are PG and PGP-Me. Genome sizer and G + C content are 4.29 Mbp and 67.6%.

The type strain MBLA0147^T (KCTC 4319^T = JCM 36641^T) was isolated from a solar saltern, Republic of Korea.

Abbreviations

AAI

Average Amino Acid Identity

AGL

Amino Phospholipid

BGCs

Biosynthetic Gene Clusters

BPGA

Bacterial Pan-Genome Analysis

BR

Bacterioruberin

COG

Clusters of Orthologous Groups

DBCM2

DB Characterization Medium No. 2

FESEM

Field-Emission Scanning Electron Microscope

GGDC

Genome-to-Genome Distance Calculator

GL

Glycolipids

GSI

Gene Support Indices

iTOL

Interactive Tree of Life

isDDH

In silico DNA-DNA Hybridization

JCM

Japan Collection of Microorganisms

KEGG

Kyoto Encyclopedia of Genes and Genomes

KCTC

Korean Collection for Type Cultures

L

Unidentified Lipids

ML

Maximum Likelihood

NCBI

National Center for Biotechnology Information

OrthoANI

Ortho-Average Nucleotide Identity

PG

Phosphatidylglycerol

PGAP

Prokaryotic Genome Annotation Pipeline

PGP-Me

Phosphate Methyl Ester

PGS

Phosphatidylglycerol Sulfate

PL

Polar Lipids

PHA

Polyhydroxyalkanoate

POGs

Pan-Genome Orthologous Groups

rpoB'

RNA Polymerase Subunit B′

S-DGD-1

Sulfated Mannosyl Glucosyl Diether

SPR

Subtree Pruning and Regrafting

TLC

Thin-Layer Chromatography

TYGS

Type Strain Genome Server

UBCG

Up-to-Date Bacterial Core Gene

Authors’ contributions

Chi Young Hwang performed the data acquisition, analysis, interpretation, and drafted the original manuscript. Eui-Sang Cho preformed data analysis. Ki-Eun Lee and Eun-Young Lee reviewed and edited the manuscript. Myung-Ji Seo designed and supervised the study.

Funding

This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the ministry of Environment (MOE) of the Republic of Korea (NIBR202304104). This study was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-00554776).

Data availability

Sequences data have been deposited at NCBI GenBank. The 16 S rRNA gene sequences of strains MBLA0143T and MBLA0147T have been deposited in GenBank under the accession number OR646561 and OR646562, respectively. Whole genome sequencing project of these two strains have been deposited at DDBJ/ENA/GenBank under the accession number JBGKAC000000000 and JBGKAD000000000, respectively.

Declarations

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.