Introduction

The genus Spirosoma in the family Cytophagaceae of phylum Bacteroidetes was first proposed by Larkin and Borrall [22]. At the time of writing, the genus Spirosoma comprised seventeen species with validly published names, including four recently described species, Spirosoma knui [23], Spirosoma lacussanchae [25], Spirosoma luteolum [24, 28], and Spirosoma swuense [12]. The type strains of Spirosoma species have been isolated from various natural sources including air [14, 16], fresh water [2, 23], soil [6, 12, 33], glacier till [4], dust [13], and Zn/Cd-accumulating Salix caprea [8]. Members of the genus Spirosoma were Gram-stain-negative, yellow or orange in colony color, catalase-positive, non-motile or motile, morphologies of rods, filaments and coils, strictly aerobic or facultatively anaerobic bacteria [1] that characterized chemotaxonomically by MK-7 as the predominant respiratory quinone [10], and phosphatidylethanolamine as the major polar lipid. The major fatty acids were summed feature 3 (composed of C16:1 ω7c/C16:1 ω6c) and C16:1 ω5c, [2, 33], and DNA G+C content ranged from 47.2 to 57.0 mol% [14]. To determine its exact taxonomic position, strain S7-3-3T was investigated in detail following 16S rRNA gene sequence analysis and polyphasic taxonomic approach that including phenotypic, chemotaxonomic, and genotypic analyses. All these data suggested that strain S7-3-3T represents a novel species of Spirosoma species.

Materials and Methods

Isolation of Bacterial Strain and Culture Condition

Strain S7-3-3T was isolated from the apple orchard soil in Gyeongsangnam-do province, South Korea (35°35ʹ20″N, 127°51ʹ29ʺE). One gram of soil was suspended in 10 ml saline [0.85% (w/v) NaCl] and serially diluted. One hundred microliters of each dilution was spread onto R2A agar plates (Difco, USA) and incubated at 25 °C for 1 week. On the 107-diluted plate, 30–40 colonies appeared, of which one yellow colony, designated S7-3-3T, was purified by transferring it onto fresh plate and incubating again under the same conditions. Strain S7-3-3T was routinely cultured on R2A agar at 25 °C and was maintained as a glycerol suspension (20%, w/v) at − 70 °C. The isolate was deposited in the Korean Collection for Type Cultures (KCTC) and the Japan Collection of Microorganisms (JCM). Spirosoma pulveris KCTC 42550T, Spirosoma rigui KACC 13387T, and Spirosoma linguale KACC 121565T were obtained from KCTC and the Korean Agricultural Culture Collection (KACC), respectively, and used as reference strains.

Phenotypic and Biochemical Characteristics

The cell morphology and motility of strain S7-3-3T were observed under a light microscope (BX50, Olympus, Japan; 1000X) and a transmission electron microscope (HT7700, Hitachi, Japan), with cells grown for 3 days at 25 °C on R2A agar. Gram test was operated using Gram staining [31]. Catalase and oxidase tests were performed according to the procedures outlined by Cappuccino and Sherman [3]. Growth was assessed on R2A agar (Difco), Luria–Bertani agar (LB; Difco), nutrient agar (NA; Difco), and trypticase soy agar (TSA; Difco). The pH range for growth (pH 4.0–10.0) was assessed in R2A broth (MB Cell, Seoul, Korea) medium using three different buffers: sodium acetate buffer (for pH 4.0–6.0), potassium phosphate buffer (for pH 7.0–8.0), and Tris buffer (for pH 9.0–10.0). Growth at 4, 10, 15, 20, 25, 30, 37, and 42 °C was tested on R2A agar after 7 days of incubation. Salt tolerance was determined by amending R2A broth with NaCl to final concentrations of 0.5, 1, 2, 3, 4, 5, and 10% (w/v) NaCl, and the growth was checked after 7 days of incubation. Enzyme activities, assimilation of carbon sources, acid production from substrates, and other physiological characteristics were determined by using API ZYM, API 20 NE, API ID 32 GN, and API 50CH strips according to the manufacturer’s instructions (bioMérieux).

16S rRNA Gene Sequencing and Phylogenetic Analysis

For the phylogenetic analysis, genomic DNA was extracted and purified using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and a PowerClean DNA Clean-Up Kit (MO BIO Laboratories, Carlsbad, CA, USA) as described previously [15, 20]. The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primers 27F and 1492R [36], and the purified PCR products were sequenced by Genotech (Daejeon, South Korea). The obtained partial 16S rRNA sequence was assembled using SeMan software (DNASTAR, Madison, WI, USA). Phylogenetic neighbors were identified, and pairwise 16S rRNA gene sequence similarities were calculated using EzBioCloud server [38] and NCBI BLAST search program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of 30 related taxa were obtained from the GenBank. The recovered sequences were aligned with the sequence of strain S7-3-3T using the program Clustal X [34]. Gaps and 5′ and 3′ ends of the alignment were edited manually using the BioEdit program [9]. Evolutionary distance matrices for the neighbor-joining algorithm were calculated using Kimura’s two-parameter model [17]. Tree topologies were inferred by the neighbor-joining (NJ) [29], maximum-likelihood (ML) [5], and maximum-parsimony (MP) [7] methods using the program MEGA7 [19]. A bootstrap analysis with 1000 replicate datasets was performed to assess the support of clusters.

Chemotaxonomic Analyses

The fatty acid profiles of strain S7-3-3T and three reference strains see above were analyzed using cells grown on R2A agar for 3 days at 25 °C. Two or three loops of fresh cells were harvested, and then the fatty acid were saponified, extracted, and methylated according to a Sherlock Microbial Identification System (MIDI) protocol [30]. Fatty acid methyl esters were analyzed by gas chromatography using the Microbial Identification software package (TSBA, version 6.0) [30]. Polar lipids were extracted using the procedure described by Minnikin et al. [27] and examined by two-dimensional thin layer chromatography (TLC), followed by spraying with appropriate detection reagents [18]. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), evaporated under a vacuum, and re-extracted in n-hexane/water (1:1, v/v). The extract was purified using Sep-Pak Silica Vac Cartridges (Waters) and then analyzed by high-performance liquid chromatography (HPLC) as described previously [11].

DNA G+C Content

To determine the DNA G+C content, the genomic DNA of strain S7-3-3T was extracted according to the standard cetyltrimethylammonium bromide/NaCl protocol [37]. Individual nucleosides were obtained by digesting the genomic DNA using nuclease P1 and alkaline phosphatase. Single-stranded DNA from salmon testes (D7656; DNA G+C content, 41.2 mol%, Sigma-Aldrich, St. Louis, MO, USA) was used as a standard. The nucleosides were analyzed by reverse-phase high-performance liquid chromatography as described previously [26].

DPD TaxonNumber and Nucleotide Sequence Accession Numbers

The Digital Protologue database TaxonNumber for strain S7-3-3T is TA00286. The 16S rRNA gene sequence of strain S7-3-3T in this study was deposited in NCBI GenBank/EMBL/DDBJ under the accession number LC269320. The accession numbers of the reference strains that closely related to strain S7-3-3T are indicated in Fig. 1.

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the phylogenetic position of strain S7-3-3T among related strains in the genus Spirosoma and representatives of other members of the family Cytophagaceae. Bootstrap values (based on 1000 replications) greater than 50% are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the neighbor-joining and maximum-parsimony algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the neighbor-joining algorithm. The tree was rooted using Bacteroides fragilis ATCC 25285T (X83935) as an outgroup. Bar, 0.05 substitutions per nucleotide position

Full size image

Results and Discussion

Phylogenetic Analysis

A nearly complete 16S rRNA gene sequence of strain S7-3-3T (1420 bps) was obtained. The sequence similarity search in the EzBioCloud database revealed that the isolate had the highest similarity with Spirosoma rigui WPCB118T (94.3%), followed by Spirosoma pulveris JSH5-14T (93.9%) and Spirosoma linguale DSM 74T (93.7%). Sequence similarities to other genera, including Fibrisoma, Huanghella, Nibrella, Larkinella, Rudanella, and Fibrella, were less than 90.0%. The phylogenetic position of the new isolate, determined using various tree-making algorithms (ML, MP, and NJ), revealed that strain S7-3-3T appeared within the genus Spirosoma (Fig. 1). As mentioned above, the level of 16S rRNA gene sequence similarity between strain S7-3-3T and three closest Spirosoma species is lower than the threshold generally employed for the delineation of novel species (i.e., 97% similarity or below) [32, 35]. Thus, strain S7-3-3T could not be assigned to any recognized species within the genus Spirosoma and should be considered to represent a novel species of the genus Spirosoma.

Morphological and Phenotypic Characteristics

The morphological, physiological, and biochemical properties of strain S7-3-3T are given in the species description and its negative characteristics in API 20 NE, API 32 GN, API ZYM, and API 50CH tests are listed in Supplementary Table S1. Comparison of differential characteristics with closely related Spirosoma species is shown in Table 1. In particular, strain S7-3-3T could be differentiated from three reference strains based on its abilities hydrolyze gelatin, to produce β-glucuronidase, and to utilize gluconate, d-mannitol, l-proline, l-serine, and d-ribose, and by its inability to produce acid production from amygdalin and starch. There are several other phenotypic features, which could be used to distinguish the novel isolate from its closest phylogenetic neighbor Spirosoma rigui and the type strains of other closely related members of the genus Spirosoma.

Table 1 Differential phenotypic characteristics between strain S7-3-3T and its phylogenetically closest relatives in the genus Spirosoma
Full size table

Chemotaxonomic Characteristics

The cellular fatty acid profile of strain S7-3-3T is characteristic of members of the genus Spirosoma [2, 13], supporting an affiliation of the isolate with the genus Spirosoma (Table 2). However, some qualitative and quantitative differences in fatty acid content could be observed between strain S7-3-3T and its closest neighbors. In particular, strain S7-3-3T could be differentiated from its above-mentioned phylogenetically closest relatives by the absence of summed feature 9 (C17:1 iso ω9c/C16:0 10-methyl) and by the presence of C17:1 iso ω5c. The major polar lipids of strain S7-3-3T were phosphatidylethanolamine (PE), which was detected in other Spirosoma species as the main component [4], an unidentified aminophospholipid (APL1), and an unidentified polar lipid (L2). In addition, the polar lipid profile of the isolate included moderate amount of an unidentified polar lipid (L3) and minor amounts of an unidentified aminophospholipid (APL2), an unidentified aminolipid (AL), an unidentified phospholipid (PL), and an unidentified polar lipid (L1) (Supplementary Fig. S1). The predominant isoprenoid quinone of stain S7-3-3T was MK-7, which is the major respiratory quinone found in other members of the genus Spirosoma [13, 23].

Table 2 Cellular fatty acid profiles of strain S7-3-3T and phylogenetically related members of the genus Spirosoma
Full size table

DNA G+C Content

The genomic DNA G+C content of strain S7-3-3T was 49.6 mol%, which lies within the range of those reported for other Spirosoma species (47.2–57.0 mol%) [1, 8].

Taxonomic Conclusion

All of the characteristics determined for strain S7-3-3T are in accordance with those of the genus Spirosoma. However, there are several phenotypic differences between strain S7-3-3T and its phylogenetically closest relatives (Table 1). The phylogenetic distinctiveness of strain S7-3-3T confirmed that this isolate is distinct from recognized Spirosoma species. Therefore, on the basis of the data presented, strain S7-3-3T should be classified as a novel species of the genus Spirosoma, for which the name Spirosoma agri sp. nov. is proposed.

Description of Spirosoma agri sp. nov.

Spirosoma agri (a’gri. L. gen. n. agri of a field). Cells are Gram-negative, non-motile, rod-shaped, aerobic, 0.8–1.2 µm wide, and 1.2–5.3 µm long. After 2 days of incubation at 25 °C on R2A agar, colonies are convex, smooth, circular, yellow, and slimy. Cells grow on R2A, NA, and TSA (weak growth) agar, but not on LB agar. Growth occurs at 10–30 °C and pH 6–8, with an optimal temperature of 25 °C and pH 7. Cells tolerate NaCl at a concentration of 1% but not 2%. Catalase and oxidase activities are positive. In API 20 NE tests, positive for β-glucosidase, β-galactosidase, gelatin hydrolysis, but negative for arginine dihydrolase, glucose fermentation, indole production, nitrate reduction, or urease activity. Utilizes for growth N-acetyl-d-glucosamine, l-arabinose (weakly, w), d-glucose, gluconate, d-maltose, d-mannose, d-melibiose, l-proline, d-ribose, salicin, l-serine (w), and d-sucrose, but other substrates in API 32 GN and API 20 NE systems are not utilized. In the API ZYM tests, positive for N-acetyl-β-glucosaminidase, acid phosphatase, alkaline phosphatase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase (C8), α-galactosidase, β-galactosidase, β-glucuronidase (w), α-glucosidase, β-glucosidase, leucine arylamidase, α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase. Acid is produced from N-acetyl-glucosamine (w), d-arabinose (w), l-arabinose (w), arbutin, d-cellobiose, esculin, d-fructose, l-fucose (w), d-galactose, gentiobiose, d-glucose, inulin (w), 5-ketogluconate, d-lactose, d-lyxose, d-maltose, d-mannose, d-melezitose, d-melibiose, methyl-α-d-glucopyranoside (w), methyl-α-d-mannopyranoside (w), methyl-β-d-xylopyranoside (w), d-raffinose, d-ribose (w), d-sucrose, salicin, d-tagatose (w), d-trehalose, d-turanose (w), d-xylose, and l-xylose (w), but not from other substrates tested in the API 50CH system. The major fatty acids are summed feature 3 (C16:1 ω7c/C16:1 ω6c) and C16:1 ω5c. The predominant menaquinone is MK-7. Phosphatidylethanolamine, an unidentified aminophospholipid, and an unidentified lipid are the major polar lipids. The DNA G+C content is 49.6 mol%. The type strain S7-3-3T (= KCTC 52727T = JCM 32199T) was isolated from apple orchard soil in Gyeongsangnam-do province (35°35ʹ20″N, 127°51ʹ29″E), South Korea.