Abstract
Three Gram-negative, aerobic and non-motile bacterial strains, BT552T, BT553T and KR1UV-12T, were isolated from soil samples in Gwangju-si and Gangneung-si, the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequence showed that strains BT552T, BT553T and KR1UV-12T clustered to a distinct clade within the family Sphingomonadaceae (order Sphingomonadales, class Alphaproteobacteria). The strains exhibited the highest genetic similarity with representatives of the genus Sphingomonas; moreover, strains BT552T and BT553T tightly clustered with Sphingomonas melonis DAPP-PG 224T (98.2 and 98.1 %) and Sphingomonas aquatilis JSS-7T (98.1 and 98.0 %), while strain KR1UV-12T clustered with S. melonis DAPP-PG 224T (97.9%) and Sphingomonas rubra BH3T (97.8%), respectively. The major cellular fatty acids of all three strains were summed feature 8 (C18:1 ω7c/C18:1 ω6c), comprising 44.7, 46.4 and 48.5%. Additionally, their respiratory quinone is Q-10, and polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids, sphingolipid and phosphatidylcholine. They all grow well at an optimum temperature of 25 °C, at pH 7. The draft genomes of strains BT552T, BT553T and KR1UV-12T measures 4 035 561 bp, 3 941 714 bp and 3 418 792 bp, respectively, comprising 3 804 3648 and 3236 coding sequences and 50, 48 and 45 RNA genes. The average nucleotide identity analysis and digital DNA–DNA hybridization values between BT552T, BT553T and KR1UV-12T and closely related Sphingomonas species range from 72.7 to 80.2% and 19.4 to 24.3%, respectively. Based on phenotypic, genotypic and chemotaxonomic data, these three strains BT552T, BT553T and KR1UV-12T represent three novel bacterial species within the genus Sphingomonas for which the names Sphingomonas longa sp. nov. (type strain BT552T= KCTC 82094T =NBRC 114993T), Sphingomonas mollis sp. nov. (type strain BT553T =KCTC 82095T =NBRC 114994T) and Sphingomonas aurea sp. nov. (type strain KR1UV-12T = KCTC 92959T = TBRC 18506T) are proposed.
Keywords: 16S rRNA gene sequencing, Sphingomonadaceae, Sphingomonas, taxonomy, whole-genome sequencing
Introduction
The genus Sphingomonas is a recognized taxonomic group within the Alphaproteobacteria class, specifically belonging to Sphingomonadaceae family. The taxonomic classification of Sphingomonas was first defined by Yabuuchi et al. (1990), with Sphingomonas paucimobilis as the designated type species. By June 2024, the genus included 163 validly published species as per List of Prokaryotic names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/sphingomonas) and have been isolated from diverse environments such as soil, water, plants and fruits [1,7]. The genus Sphingomonas is Gram-negative, aerobic and rod-shaped and forms yellow or orange colonies [8]. The chemotaxonomic characteristics of the Sphingomonas genus include sphingoglycolipid, phosphatidyl dimethylethanolamine, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine as the predominant polar lipids. The predominant respiratory quinone in the genus Sphingomonas is Q-10. The major cellular fatty acids are summed feature 3 (C16:1 ω7c/C16:1 ω6c), summed feature 8 (C18:1 ω7c/C18:1 ω6c) and C14:0 2OH [9]. Most species of the genus Sphingomonas take sym-homospermidine as the major polyamine [10].
Sphingomonas exhibit remarkable capabilities that make them invaluable in various environmental and agricultural contexts. Their proficiency in degrading organic pollutants is particularly noteworthy, regarding their contribution to eliminating contaminated soils, water bodies and other environments. Moreover, in its involvement in processes like nitrogen fixation and denitrification, Sphingomonas species help ensure the availability of essential nutrients for plant growth, thereby contributing to higher agricultural productivity. Their biocontrol and stress tolerance abilities further highlight their importance in sustaining plant health and stability in the soil environment [8,11]. The numerous functional characteristics of Sphingomonas emphasize its potential as a significant participant in studies of the environment and biotechnological applications. This ability helps restore ecosystems and mitigates potential harm to human health and other organisms in the affected areas.
Additionally, the biocontrol mechanisms exhibited by Sphingomonas against pathogens and their tolerance to environmental stressors further enhance their utility in agriculture. By protecting plants from diseases and environmental stresses, these bacteria maintain crop health and stability, ultimately improving agricultural yields and resilience. The multifaceted functional characteristics of Sphingomonas make them essential subjects of scientific research and potential candidates for various biotechnological applications. Whether in environmental remediation, agriculture or beyond, their versatility and efficacy make them valuable assets in addressing contemporary challenges related to pollution, food security and sustainability [8,9, 11].
This work focuses on genomic analysis, taxonomic categorization and characterization of the recently obtained strains BT552T, BT553T and KR1UV-12T, representing distinctive Sphingomonas features. Our research, especially the genomic insights, offers a deeper understanding of how these strains fulfil the key functional roles of Sphingomonas in soil environments. By analysing their genetic makeup, we clarify their capabilities in biodegradation, nutrient cycling and plant interaction, highlighting the importance of these strains in microbial taxonomy and biotechnological applications.
Methods
Organism and culture conditions
Strains BT552T and BT553T were isolated from soil in Gwangju-si (35° 9′ 34.363″ N and 126° 51′ 9.364″ E), whereas strain KR1UV-12T was isolated from soil in Gangneung-si (37° 45′ 6.84″ N and 128° 52′ 41.7″ E), the Republic of Korea. One gram of soil was suspended in 10 ml of sterile normal saline, incubated at 37 °C for 1 h and then serially diluted. A 100 µl aliquot of the diluent was spread onto Reasoner’s 2A (R2A) (BD Difco) agar plates and incubated at 25 °C. After 3 days of incubation, several colonies were examined and selected for further purification. Among the isolated strains, BT552T, BT553T and KR1UV-12T were sub-cultured routinely and preserved at −80 °C in R2A broth containing 20% (v/v) glycerol. The closely related strains were obtained from the Korean Agricultural Culture Collection, Republic of Korea.
Phylogenetic analysis and genome sequencing
The genomic DNA of strains BT552T, BT553T and KR1UV-12T was extracted using a Qiagen DNA extraction kit (Qiagen, Germany), following the manufacturer’s instructions. The 16S rRNA gene was amplified via standard PCR with the bacterial primer pairs 27F and 1492R described by Weisburg et al. [12]. The purified PCR products were sequenced by Bionics (Republic of Korea). The taxonomic classification of the strains was determined by comparing their 16S rRNA gene sequences on the EzBioCloud database (https://www.ezbiocloud.net). Sequences of phylogenetically related taxa were retrieved from the EzBioCloud database. Additionally, NCBI blast (basic local alignment search tool) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to obtain updated information unavailable on the EzBioCloud. The 16S rRNA gene sequences of BT552T, BT553T and KR1UV-12T and the closely related strains were aligned using the EzEditor2 program. Phylogenetic trees were constructed using mega 11 software [13], employing three algorithms: neighbour joining (NJ) by Saitou and Nei [14], maximum likelihood (ML) by Felsenstein [15] and maximum parsimony (MP) by Fitch [16]. Bootstrap analysis with 1000 replicates, as introduced by Felsenstein [17], was performed to assess the reliability of the trees.
Genomic DNA from strains BT552T, BT553T and KR1UV-12T was extracted using the Solgent genomic DNA extraction kit (Solgent, South Korea). After extraction, the concentration of DNA was measured, and Illumina’s Nextera DNA Flex Library Prep Kit was used to construct and generate DNA libraries. Whole-genome sequencing was conducted on the iSeq 100 platform, producing 150 bp paired-end reads. The sequences were assembled using SPAdes (v.3.13.0), developed by the Algorithmic Biology Lab at the St. Petersburg Academic University of the Russian Academy of Sciences [18].
The genome sequences of strains BT552T, BT553T and KR1UV-12T have been submitted to the GenBank database (www.ncbi.nlm.nih.gov/) and annotated using the Prokaryotic Genome Annotation Pipeline provided by the National Center for Biotechnology Information (NCBI). The genomic distance between the strains was calculated by the average nucleotide identity (ANI) using the EzBioCloud web tool (https://www.ezbiocloud.net/tools/ani) [19]. The digital DNA–DNA hybridization (dDDH) values were calculated using the Genome-to-Genome Distance Calculator, explicitly utilizing formula 2 to enhance precision in the calculation [20].
For the taxonomic classification of the bacterial genomes of novel bacteria, the Genome Taxonomy Database Toolkit (GTDB-Tk) (v.2.3.0) was used to generate a GTDB tree and approximate the taxonomy of the novel bacteria (https://github.com/Ecogenomics/GTDBTk). This method involved a concatenated multiple-sequence alignment of 120 marker genes. The resulting tree was visualized using iTOL version 4 (https://itol.embl.de).
Whole-genome assemblies for novel strains and their closely related type strains, as identified in the GTDB tree, were used to reconstruct a whole-genome-based phylogenetic tree using the up-to-date bacterial core gene (UBCG) set pipeline. This approach, detailed by Na et al. [21], leverages a concatenated sequence dataset of 92 single-copy bacterial core genes (www.ezbiocloud.net/tools/ubcg) for phylogenetic reconstruction. Furthermore, functional genes were analysed and categorized to evaluate these strains’ metabolic characteristics and ecological roles using the Rapid Annotation using Subsystem Technology (RAST) server [22,23].
Morphological, physiological and biochemical analysis
The cell morphology was examined using transmission electron microscopy (JEOL, JEM1010) after incubating for 3 days on R2A agar plates at 25 °C. The Gram staining of strains BT552T, BT553T and KR1UV-12T was performed using a standard Gram reaction kit by bioMérieux. Growth conditions were tested at various temperatures: 4, 10, 15, 25, 30, 35 and 40 °C. The pH tolerance range was determined using nutrient agar (NA), with pH values from 5.0 to 9.0 in 0.5 unit increments, all maintained at 25 °C. pH-dependent growth was assessed with two different buffers at a final concentration of 100 mM: acetate buffer for pH 5.0–6.5 and phosphate buffer for pH 7.0–9.0. NaCl tolerance concentrations ranged from 1 to 10% (w/v at 1% intervals). Oxidase activity was assessed using a 1 % (w/v) tetramethyl-p-phenylenediamine solution [24], and catalase activity was evaluated by observing bubble production after applying a 3% (v/v) hydrogen peroxide solution [25]. Growth on various culture media was observed on R2A agar, NA (BD Difco), tryptic soy agar (TSA) (BD Difco), MacConkey agar (BD Difco) and lysogeny broth (LB) (BD Difco). Carbon source utilization and fermentation were determined using the API 20NE test kit, and enzymatic activities were assessed using the API ZYM test kit (bioMérieux) according to the manufacturer’s guidelines.
Chemotaxonomic characteristics
Polar lipids from strains BT552T, BT553T and KR1UV-12T were extracted following the method described by Minnikin et al. [26] and analysed using two-dimensional TLC. The separated polar lipids were identified with a series of reagents, using a mixture of chloroform, methanol and water in ratios of 9:10:3 v/v/v for the first dimension and 5:10:4 v/v/v for the second (Komagata and Suzuki 1987) [27]. For identifying total lipids, glycolipids, sphingolipids, glycosphingolipids, phosphatidylcholine, amino groups and phosphorus-containing lipids, the TLC plate was treated with ethanolic molybdatophosphoric acid (Merck), α-naphthol in sulfuric acid reagent (Wako), Dragendorff’s reagent (Merck), a ninhydrin spray solution (Merck) and molybdenum blue reagent (Sigma). Quinones of strains BT552T, BT553T and KR1UV-12T were extracted using Sep-Pak Vac cartridges (Waters) and analysed by HPLC [28].
For cellular fatty acid and polyamine analysis, strains BT552T, BT553T and KR1UV-12T were cultured on NA for 3 days at 25 °C. Cellular fatty acids were purified via saponification, methylation and extraction procedures, according to Sasser [29]. The resulting fatty acid methyl esters were identified using the Sherlock Microbial Identification System V6.01 (MIS, database TSBA6; MIDI Inc., Newark, DE). Polyamines were extracted and analysed according to the methods described by Busse and Auling [30] and Busse et al. [31].
Results and discussion
Morphological, physiological and biochemical analyses
The strains BT552T, BT553T and KR1UV-12T were Gram-negative, non-motile and rod-shaped (Fig. 1). The colonies of strains BT552T and BT553T exhibited a yellow colour, convex and round after 72 h of incubation on NA at 30 °C. Similarly, strain KR1UV-12T exhibited an orange-yellow colour with rounded, slightly convex-shaped colonies after 72 h of incubation on NA at 30 °C. BT552T and KR1UV-12T strains grew at 20–30 °C (optimum at 25 °C) without NaCl. However, strain BT553T grew within a temperature range of 10–30 °C (optimum at 25 °C) in the absence of NaCl. Although BT552T and BT553T have similar colony morphology, BT552T shows positive reactions for gelatin hydrolysis and α-chymotrypsin activity, while BT553T shows negative reactions, indicating differences in biochemical characteristics between these strains. Table 1 lists the distinct physical characteristics that distinguish strains BT552T, BT553T and KR1UV-12T and their closest neighbours in the Sphingomonas genus.
Fig. 1. Transmission electron micrographs of strains BT552T (a), BT553T (b) and KR1UV-12T (c).
Table 1. Differential characteristics of strains BT552T, BT553T and KR1UV-12T and closely related species.
Taxa: 1, strain BT552T; 2, strain BT553T; 3, strain KR1UV-12T; 4, S. melonis DAPP-PG 224T; 5, S. aquatilis JSS-7T; 6, S. rubra BH3T; 7, S. liriopis RP10T; 8, S. cynarae SPC-1T; 9, S. metalli 9O-1T. Data of strains BT552T and BT553T and reference strains were obtained in this study unless indicated otherwise. +, Positive; −, negative; w, weak positive; nd, no data. All strains had positive results for aesculin hydrolysis. All strains were negative for indole production and acid production from glucose and urease.
| Characteristic | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|---|
| Characteristic morphology | Convex, smooth | Convex, smooth | Slightly convex, glistening | Domed, smooth | Low-convex, smooth, opaque |
Elevated, circular | Convex, smooth | Convex, smooth | Opaque, convex |
| Colony colour | Yellow | Yellow | Orange yellow | Deep yellow | Yellow | Red | Light yellow | Bright orange | Orange |
| Nitrate reduction to NO2 | − | − | − | − | − | nd | + | − | − |
| Nitrate reduction to N2 | − | − | − | nd | − | nd | + | − | − |
| Enzyme activity | |||||||||
| Arginine dihydrolase | − | − | − | − | − | − | + | − | − |
| Gelatin hydrolysis | + | − | + | − | − | − | − | + | + |
| N-Acetyl-β-glucosaminidase | w | + | + | + | + | − | − | nd | nd |
| Trypsin | w | − | w | + | + | − | + | nd | nd |
| α-Chymotrypsin | w | − | + | − | − | + | − | nd | nd |
| β-Galactosidase (ONPG) | + | + | + | + | + | − | − | nd | nd |
| Assimilation | |||||||||
| Adipate | − | − | − | − | nd | − | − | − | − |
| Caprate | − | − | − | − | + | − | − | − | − |
| Citrate | − | − | − | − | − | nd | − | − | − |
| d-Glucose | − | − | − | + | + | w | + | + | + |
| d-Maltose | − | − | − | + | + | nd | + | + | + |
| d-Mannitol | − | − | − | − | nd | nd | − | + | − |
| d-Mannose | − | − | − | + | − | nd | − | + | + |
| Gluconate | − | − | − | − | + | − | − | + | + |
| l-Arabinose | − | − | − | + | + | nd | − | + | + |
| l-Malate | − | − | − | nd | − | nd | + | − | + |
| N-Acetyl-d-glucosamine | − | − | − | + | + | − | − | − | − |
| Phenyl acetate | − | − | − | + | − | − | − | − | − |
| G+C content | 66.8 | 65.9 | 68.3 | 67.0* | 67.1 | 68.8† | 68.2 | 66.6‡ | 68.6§ |
Phylogenetic analysis and genome sequencing
In the context of phylogenetic classification, the comparative study of strains BT552T, BT553T and KR1UV-12T utilizing 16S rRNA gene sequencing and comprehensive genome analysis has produced significant results. The analysis classified these strains as members of the Sphingomonadaceae family. The 16S rRNA gene sequence comparisons indicate that these strains belong to the genus Sphingomonas. This alignment is further corroborated by genome-based phylogenetic analysis, providing a cohesive view of their taxonomic placement. These findings affirm the placement of strains BT552T, BT553T and KR1UV-12T within the genus Sphingomonas, contributing to the taxonomic clarity of the Sphingomonadaceae family. The 16S rRNA gene sequence of strain BT552T showed high similarity to Sphingomonas melonis DAPP-PG 224T (98.2%), Sphingomonas aquatilis JSS-7T (98.1%), Sphingomonas kyungheensis THG-B283T (97.7%) and Sphingomonas liriopis RP10T (97.6%). Strain BT553T showed the highest similarity to S. melonis DAPP-PG 224T (98.1%), S. aquatilis JSS-7T (98.0%), S. liriopis RP10T (97.8%) and Sphingomonas taxi ATCC 55669T (97.6%). Strain KR1UV-12T showed the highest similarity to S. melonis DAPP-PG 224T (97.9%), Sphingomonas rubra BH3T (97.8%), S. liriopis RP10T (97.8%) and S. aquatilis JSS-7T (97.8%). The strains used are listed in Table S1 (available in the online Supplementary Material). Phylogenetic trees were constructed using the NJ method (Fig. 2), ML method (Fig. S1) and MP method (Fig. S2).
Fig. 2. NJ phylogenetic tree based on 16S rRNA gene sequences showing the position of strains BT552T, BT553T and KR1UV-12T and other relative species of the genus Sphingomonas. Numbers at nodes are bootstrap percentages (> 70%) based on the NJ algorithms. Rhizorhabdus argentea SP1T was used as an outgroup. [34] Bar, 0.01 substitutions per nucleotide position.
Genomic sequencing revealed that the genome of strain BT552T spans 4 035 561 base pairs, with a sequencing coverage of 54.1×. This genome comprises 3804 coding sequences (CDSs). The calculated DNA G+C content is 66.8%, which falls within the typical range for the genus Sphingomonas (58.7 –71.0 %), as referenced in the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome/browse). In a parallel analysis, the genome of strain BT553T was determined to encompass 3 941 714 base pairs, with a genome coverage of 55.3×. This genome is characterized by 3648 CDSs and a DNA G+C content of 65.9%, aligning with the known range for Sphingomonas species. Lastly, the genome of strain KR1UV-12T was determined to encompass 3 418 792 base pairs, with a genome coverage of 59.3×. This genome is characterized by 3236 CDSs and a DNA G+C content of 68.3%, also within the known range for Sphingomonas species. The genome sequences of strains BT552T, BT553T and KR1UV-12T have been deposited in the DDBJ/EMBL/GenBank databases under the accession numbers JAFEMC000000000, JAELXS000000000 and JAUUDS000000000, respectively. Genome sequences of closely related species were sourced from NCBI for comparative purposes. The details of these genomes and their assembly statistics are comprehensively listed in Tables S2 and S3.
The genomic comparisons between strain BT552T and other members of the Sphingomonas genus yielded ANI values ranging from 72.8 to 77.6% and dDDH values between 19.9 and 22.1%. For strain BT553T, the ANI values with other Sphingomonas species were between 72.8 and 79.9 %, with dDDH values ranging from 19.4 to 23.7%. For strain KR1UV-12T, the ANI values with other Sphingomonas species were between 73.2 and 80.2%, with dDDH values ranging from 20.1 to 24.3%. As part of our internal analysis, when comparing distance, ANI and dDDH among the newly identified strains BT552T, BT553T and KR1UV-12T, the distance between BT552T and BT553T was calculated to be 0.2017, with an ANI value of 77.3% and a dDDH value of 21.7%. Similarly, the distance between BT552T and KR1UV-12T was 0.1978, with an ANI value of 77.8% and a dDDH value of 22.2%. The distance between BT553T and KR1UV-12T was 0.2010, with an ANI value of 77.9% and a dDDH value of 21.8%. Notably, BT552T, BT553T and KR1UV-12T displayed ANI values significantly below the conventional species delineation threshold of 95–96%. Similarly, the dDDH values for these strains were well below the established criterion for species differentiation in prokaryotes, which is ≥70% in in silico DDH. The comprehensive genome comparisons of strains BT552T, BT553T and KR1UV-12T are detailed in Tables S4, S5 and S6. These findings are critical in establishing the taxonomic uniqueness of strains BT552T, BT553T and KR1UV-12T within the genus Sphingomonas.
The results of phylogenetic analysis based on the 16S rRNA gene and whole-genome sequences revealed relationships for these strains. When analysing the phylogenetic relationships based on the 16S rRNA gene, strains BT552T and BT553T showed the closest phylogenetic affinity to S. melonis and S. aquatilis. In contrast, strain KR1UV-12T showed a closer relationship to S. rubra and Sphingomonas criticola (Figs 2, S1 and S2). Further, in the reconstructed phylogenetic analysis using the genome-based UBCG tool, strains BT552T and BT553T were closely aligned with Sphingomonas cynarae and S. rubra. However, the phylogenetic positioning of BT552T and BT553T is sufficiently unique to support their classification as new species within the genus Sphingomonas. In contrast, the UBCG phylogenetic tree places strain KR1UV-12T closest to Sphingomonas metalli (Fig. S3). Despite the variations in the closest species determined by genome-based phylogenetic analyses, KR1UV-12T showed enough phylogenetic distinction to be proposed as a new species of the Sphingomonas genus. These genomic results support the proposal to classify strains BT552T, BT553T and KR1UV-12T as novel species in the Sphingomonas genus.
The RAST server comprehensively annotated the genomes of strains BT552T, BT553T and KR1UV-12T. For strain BT552T, the RAST analysis identified a total of 275 subsystems (Fig. S4). Similarly, the genome of strain BT553T contained 291 subsystems (Fig. S5), indicating a broad repertoire of biological functions. For strain KR1UV-12T, the RAST analysis identified a total of 268 subsystems (Fig. S6), suggesting a complex genomic architecture with diverse functional capabilities. These annotations provide insights into the novel strains’ metabolic diversity. The RAST annotation details are accessible at https://rast.nmpdr.org/. Using the blastp algorithm, we compared strains BT552T, BT553T and KR1UV-12T to related Sphingomonas species, focusing on genes for auxin biosynthesis and ammonia assimilation. Strain BT552T showed 71.4–93.1 % similarity in auxin biosynthesis genes (Table S7), and BT553T had 73.0–93.6% similarity (Table S8). KR1UV-12T exhibited 72.9–93.2% similarity, suggesting conserved pathways with unique strain-specific features (Table S9). For ammonia assimilation genes, BT552T showed 0–97.3% similarity (Table S10), BT553T ranged from 0 to 99.1% (Table S11) and KR1UV-12T had 0–99.1% similarity (Table S12), indicating diverse nitrogen metabolism. These sequence differences and phylogenetic analyses support the classification of strains BT552T, BT553T and KR1UV-12T as novel species within the Sphingomonas genus.
Chemotaxonomic characterization
The chemotaxonomic analyses were conducted to determine the cellular fatty acid compositions of strains BT552T, BT553T and KR1UV-12T, alongside comparisons with their nearest phylogenetic neighbours (Table 2). For BT552T, the predominant fatty acids were summed feature 8 (comprising C18:1 ω7c and C18:1 ω6c), summed feature 3 (comprising C16:1 ω7c and C16:1 ω6c) and C14:0 2OH, constituting 77.84% of the fatty acid profile. This strain was distinguishable from closely related types by an increased presence of summed feature 3. Similarly, BT553T and KR1UV-12T were characterized by substantial amounts of summed feature 8, summed feature 3 and a significant proportion of C14:0 2OH. The polar lipid assays revealed that all three strains possess lipids, diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phospholipids (PL) and sphingolipid (SL) with BT552T exhibiting additional complex lipids, including aminolipid and aminophospholipid (Figs S7, S8 and S9). The presence of sphingolipids in all strains is a chemotaxonomic marker consistent with the Sphingomonas genus. The respiratory quinone profile, dominated by ubiquinone Q-10 in all three isolates, corroborates their placement within the genus. The major polyamine of BT552T, BT553T and KR1UV-12T are homospermidine. BT552T and KR1UV-12T strains contain putrescine as their minor polyamine, constituting 6.48 and 14.66% of the total polyamines. In contrast, BT553T differs by having spermidine as its minor polyamine, accounting for 1.93% (Fig. S10). These chemotaxonomic signatures and the phylogenetic data support the classification of strains BT552T, BT553T and KR1UV-12T as novel entities within the Sphingomonas genus.
Table 2. Cellular fatty acid profiles of strains BT552T, BT553T and KR1UV-12T and closely related species.
Taxa: 1, strain BT552T; 2, strain BT553T; 3, strain KR1UV-12T; 4, S. melonis DAPP-PG 224T; 5, S. aquatilis JSS-7T; 6, S. rubra BH3T; 7, S. liriopis RP10T; 8, S. metalli 9O-1T; 9, S.cynarae SPC-1T. Data of strains BT552T, BT553T and KR1UV-12T and reference strains were obtained in this study. tr, trace (< 1 %); nd, not detected.
| Fatty acid | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|---|
| Saturated | |||||||||
| C14:0 | tr | 4.86 | 1.23 | 2 | tr | tr | tr | nd | tr |
| C16:0 | 8.54 | 8.41 | 8.13 | 19.8 | 20.58 | 11.2 | 15.6 | 4.8 | 18.4 |
| C18:0 | tr | tr | tr | 4.8 | 1.04 | tr | 3.4 | nd | 8.1 |
| Unsaturated | |||||||||
| C16:1 ω5c | 6.99 | 1.18 | 1.73 | nd | tr | tr | tr | 1.8 | nd |
| C17:1 ω6c | tr | 1.5 | 2.96 | 8.2 | nd | 2.2 | 1.8 | nd | nd |
| C17:1 ω7c | nd | nd | nd | tr | nd | nd | 1.0 | nd | nd |
| C18:1 ω5c | tr | tr | 1.07 | nd | 1.51 | nd | nd | 1.4 | nd |
| C18:1 ω7c | nd | nd | nd | nd | nd | nd | nd | 63.1 | 40.2 |
| C18:3 ω6, 9, 12c | tr | nd | nd | nd | nd | nd | 4.6 | nd | nd |
| Branched-chain fatty acid | |||||||||
| C18:1 ω7c 11-methyl | 4.49 | 3.15 | 3.24 | 5.9 | nd | 3.9 | 3.8 | 3.1 | nd |
| Hydroxy fatty acids | |||||||||
| C13:0 2OH | nd | nd | tr | 1.6 | nd | nd | 1.9 | nd | nd |
| C14:0 2OH | 12.5 | 11.69 | 14.95 | 6.3 | 10.86 | 20.6 | 9.7 | 11.3 | 5.4 |
| C15:0 2OH | tr | tr | 0.43 | nd | nd | tr | nd | nd | nd |
| C18:1 2OH | tr | tr | tr | 1.5 | nd | nd | nd | nd | nd |
| CYCLO | |||||||||
| C17:0 cyclo | nd | nd | nd | nd | nd | nd | 1.1 | nd | 2.0 |
| C19:0 cyclo ω8c | nd | tr | nd | nd | nd | nd | 1.2 | nd | nd |
| Summed feature | |||||||||
| 3; C16:1 ω7c/C16:1 ω6c | 17.67 | 19.77 | 16.21 | 5.5 | 4.61 | 18.9 | nd | nd | nd |
| 8; C18:1 ω7c/C18:1 ω6c | 44.69 | 46.38 | 47.48 | 35.6 | 59.89 | 39.2 | 32.8 | nd | nd |
| 9; C17:1 iso ω9c/C16:0 10-methyl | nd | tr | nd | tr | nd | nd | 2.1 | nd | nd |
The results from the phylogenetic analysis distinctly indicated that strains BT552T, BT553T and KR1UV-12T represent new species within the genus Sphingomonas. Based on phenotypic, genotypic and biochemical characteristics, strains BT552T, BT553T and KR1UV-12T represent three novel species within the genus Sphingomonas. We propose the names Sphingomonas longa, Sphingomonas mollis and Sphingomonas aurea for these novel species.
Description of Sphingomonas longa sp. nov.
Sphingomonas longa (lon’ga. L. fem. adj. longa, long, extended, referring to the length of cells of the type strain).
Cells are Gram-negative, non-motile and rod-shaped. Colonies grown on NA have a convex, circular and smooth appearance with a yellow colour after 72 h of incubation at 30 °C. The cells are ~0.6–1.5 µm in width and 1.5–5.8 µm in length. They grow on NA agar at 25–30 °C (optimum at 25 °C) and tolerate NaCl concentrations of 0–2.5 % (optimal at 0%). The strain grows on NA, TSA, LB and R2A agar but not on MAC agar. Both oxidase and catalase activities are positive. In the API 20NE test, positive reactions for β-glucosidase (aesculin hydrolysis), protease (gelatin hydrolysis) and β-galactosidase (PNPG). It tests negative for nitrate reduction, indole production and acid production from glucose, arginine dihydrolase, urease, d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-d-glucosamine, d-maltose, potassium gluconate, caprate, adipate, l-malate, citrate and phenylacetate. In the API ZYM test, strain BT552T is positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase and β-galactosidase (ONPG). It exhibits weakly positive reactions for esterase (C4), esterase (C8), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase (starch hydrolysis), β-glucosidase and N-acetyl-β-glucosaminidase. It tests negative for β-glucuronidase, α-mannosidase and α-fucosidase.
The major respiratory quinone is Q-10. Dominant cellular fatty acids include summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polyamine is homospermidine (93.5%), with putrescine (6.5%) as the minor polyamine. The major polar lipids in this strain include DPG, PG, PE, PC, PL and SL.
The type strain for Sphingomonas longa, BT552T (=KCTC 82094T = NBRC 114993T), was isolated from soil in the Republic of Korea. The genome sequence of strain BT552T has been deposited in GenBank/DDBJ/EMBL under the accession number JAFEMC000000000. The GenBank accession number for the 16S rRNA gene sequence of strain BT552T is MT893356.
Description of Sphingomonas mollis sp. nov.
Sphingomonas mollis (mol’lis. L. fem. adj. mollis, soft).
Cells are Gram-negative, non-motile and rod-shaped. Colonies grown on NA have a convex, circular and smooth appearance with a yellow colour after 72 h of incubation at 30 °C. The cells are ~1.5 µm in width and 5.8 µm in length. They grow on NA at temperatures ranging from 10 to 30 °C (optimum at 25 °C) and tolerate NaCl concentrations of 0–2.5 % (optimal at 0%). The strain grows on NA, TSA, LB and R2A agar but not MAC agar. Oxidase and catalase activities are positive. In the API 20NE test, positive reactions for β-glucosidase (aesculin hydrolysis) and β-galactosidase (PNPG). It tests negative for nitrate reduction, indole production, acid production from glucose, arginine dihydrolase, urease, protease (gelatin hydrolysis), d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-d-glucosamine, d-maltose, potassium gluconate, caprate, adipate, l-malate, citrate and phenylacetate. In the API ZYM test, strain BT553T is positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, β-galactosidase (ONPG) and N-acetyl-β-glucosaminidase. It exhibits weakly positive reactions for esterase (C4), esterase (C8), cystine arylamidase, α-galactosidase, β-glucuronidase, α-glucosidase (starch hydrolysis), β-glucosidase and α-fucosidase. It tests negative for lipase (C14), trypsin, α-chymotrypsin and α-mannosidase.
The major respiratory quinone is Q-10. Dominant cellular fatty acids include summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polyamine is homospermidine (98.07%), with spermidine (1.93%) as the minor polyamine. The major polar lipids in this strain include DPG, PG, PE, PC and SL.
The type strain for Sphingomonas mollis, BT553T (=KCTC 82095T = NBRC 114994T), was isolated from soil in the Republic of Korea. The genome sequence of strain BT553T has been deposited in GenBank/DDBJ/EMBL under the accession number JAELXS000000000. The GenBank accession number for the 16S rRNA gene sequence of strain BT553T is MT893357.
Description of Sphingomonas aurea sp. nov.
Sphingomonas aurea (au’re.a. L. fem. adj. aurea, golden).
Cells are Gram-stain-negative, non-motile and rod-shaped. Colonies grown on NA agar have a slightly convex, circular and glistening appearance with orange-yellow colouration after 72 h of incubation at 30 °C. The cells range in size from ~0.6–0.8 µm in width and 1.4–1.8 µm in length. The strain grows on NA agar at temperatures between 25–30 °C (optimum at 25 °C) and tolerates NaCl concentrations of 0–2.5 % (optimal at 0%). It exhibits growth on NA, TSA, LB and R2A agar but not MAC agar. Oxidase activity is positive, but catalase activity is negative. In API 20NE testing, positive reactions for β-glucosidase (aesculin hydrolysis) and β-galactosidase (PNPG). It tests negative for arginine dihydrolase, nitrate reduction, indole production and acid production from glucose, urease, protease (gelatin hydrolysis), d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-d-glucosamine, d-maltose, gluconate, caprate, adipate, l-malate, citrate and phenylacetate. In API ZYM testing, it tests positive for alkaline phosphatase, leucine arylamidase, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase (ONPG) and N-acetyl-β-glucosaminidase, with weakly positive reactions for esterase (C4), esterase (C8), valine arylamidase, cystine arylamidase, trypsin, α-glucosidase (starch hydrolysis) and β-glucosidase. It tests negative for lipase (C14), β-glucuronidase, α-mannosidase and α-fucosidase.
The major respiratory quinone is Q-10. Dominant cellular fatty acids include summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polyamine is homospermidine (85.34%), with putrescine (14.66%) as the minor polyamine. The major polar lipids in this strain include DPG, PG, PE, PC, SL and glycosphingolipid.
The type strain for Sphingomonas aurea, KR1UV-12T (=KCTC 92959T = TBRC 18506T), was isolated from soil in the Republic of Korea. The genome sequence of strain KR1UV-12T has been deposited in GenBank/DDBJ/EMBL under the accession number JAUUDS000000000. The GenBank accession number for the 16S rRNA gene sequence of strain KR1UV-12T is OR342729.
Supplementary material
Abbreviations
- ANI
average nucleotide identity
- blast
basic local alignment search tool
- CDSs
coding sequences
- dDDH
digital DNA–DNA hybridization
- DPG
diphosphatidylglycerol
- GTDB
Genome Taxonomy Database
- LB
lysogeny broth
- ML
maximum likelihood
- MP
maximum parsimony
- NA
nutrient agar
- NCBI
National Center for Biotechnology Information
- NJ
neighbour joining
- PC
phosphatidylcholine
- PE
phosphatidylethanolamine
- PG
phosphatidylglycerol
- PL
phospholipids
- R2A
Reasoner’s 2A
- RAST
Rapid Annotation using Subsystem Technology
- SL
sphingolipid
- TSA
tryptic soy agar
- UBCG
up-to-date bacterial core gene
Footnotes
Funding: This research was supported by a research grant from Seoul Women’s University (2024) and by the grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202002203, NIBR202304204).
GenBank accession no.: The GenBank/EMBL/DDBJ/PIR accession numbers for the 16S rRNA gene and whole-genome sequences of strains BT552T, BT553T and KR1UV-12T are MT893356 (JAFEMC000000000), MT893357 (JAELXS000000000) and OR342729 (JAUUDS000000000), respectively.
Author contributions: All authors equally contributed in this work.
Contributor Information
Eo Jin Kim, Email: djwls5442@swu.ac.kr.
Soo Hyun Maeng, Email: sarahmaeng@daum.net.
Myung Kyum Kim, Email: biotech@swu.ac.kr.
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