Introduction

Numerous actinomycetes have been isolated from soils, and these have contributed significantly to the discovery of many useful bioactive compounds, such as antibiotics, antitumor agents and immunosuppressive agents.1 Over recent years, it has become more and more difficult to find novel bioactive compounds derived from actinomycete strains isolated from soils, so the focus of attention has switched to find novel actinomycetes from other sources. The majority of actinomycetes isolated from soils belong to the genus Streptomyces,2 although the microbial flora in plants is different and many rare actinomycetes are being isolated from plants.3, 4 We searched for actinomycetes from plants for the purpose of finding new microbial resources to use in routine screening for novel bioactive compounds. We proposed novel genera, Phytohabitans suffuscus K07-0523T and Actinophytocola oryzae GMKU 367T, obtained through our research.5, 6 It is clear that plant roots are a useful source for new actinomycetes. Furthermore, we also discovered new antitrypanosomal compounds, the spoxazomicins, from the cultured broth of actinomycete strain K07-0460T. This research will be reported in the accompanying paper.7 The strain K07-0460T was phylogenetically identified as being a member of the genus Streptosporangium described by Couch.8 At present, the genus Streptosporangium comprises 15 species and 2 subspecies: Streptosporangium roseum,8 S. amethystogenes subsp. amethystogenes, S. album, S. vulgare,9 S. longisporum,10 S. nondiastaticum, S. pseudovulgare,11 S. violaceochromogenes,12 S. fragile,13 S. carneum,14 S. amethystogenes subsp. fukuiense,15 S. claviforme,16 S. subroseum,17 S. purpuratum, S. yunnanense18 and S. canum.19 We describe taxonomy of the strain K07-0460T in this paper.

Materials and methods

Strain K07-0460T was isolated from the root of a plant, a variety of orchid collected in Okinawa prefecture in March 2007. It was cultured on water-proline agar (proline 1.0%, tap water, pH 7.0) using the method previously described by Inahashi et al.5 The strain K07-0460T, S. amethystogenes subsp. amethystogenes DSM 43179T, S. amethystogenes subsp. fukuiense IFO 15365T and S. longisporum DSM 43180T were cultured for 2 weeks at 27 °C to observe cultural characteristics on ISP (International Streptomyces Project) media 2, 3, 4, 5, 6 and 7 (ref. 20), glucose–asparagine agar, sucrose–nitrate agar, glycerol–calcium malate agar, peptone–beef extract agar21 and glucose–nitrate agar (glucose 3.0%, NaNO3 0.2%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, KCl 0.05%, FeSO4·7H2O 0.001%, agar 1.5%, pH 7.0). The color of aerial and substrate mycelia and soluble pigments were determined using the Color Harmony Manual.22 The morphological characteristics were observed by light microscopy and scanning electron microscopy (JEOL JSM-5600, JEOL, Tokyo, Japan), of 28-day-old culture grown on ISP medium 3. The temperature range, the pH range and the NaCl tolerance for growth were determined on nutrient agar (Difco, Detroit, MI, USA). Utilization of carbohydrates as the sole carbon source was tested using ISP medium 9 supplemented with B-vitamins.20, 23, 24 ISP medium 4 for starch hydrolysis, ISP medium 8 for nitrate reduction,20 glucose–peptone–gelatin medium (glucose 2.0%, peptone 0.5%, gelatin 20%, pH 7.0) for gelatin liquefaction, 10% skim milk for coagulation and peptonization of milk, ISP medium 6 for H2S production and skim-milk agar for casein hydrolysis were used.25 Tyrosinase activity was determined using ISP medium 7 and other enzyme activities were determined using the API ZYM system (bioMérieux, Lyon, France), according to the manufacturer's instructions. Biomass for the molecular systematics and chemotaxonomic studies was obtained after cultivation in yeast extract–glucose broth (yeast extract 1.0%, glucose 1.0%, pH 7.0) for 1 week at 27 °C. After extraction of isoprenoid quinones, as described by Collins et al.,26 analysis was done by HPLC (Agilent 1100, Agilent, Santa Clara, CA, USA) and mass spectrometer (JEOL JMS-T 100LP) using a Pegasil ODS column (Senshu, Tokyo, Japan), according to Tamaoka et al.27 Isomers of diaminopimelic acid were determined by TLC using whole-cell hydrolysates.28 The N-acyl types of muramic acid were determined by the method of Uchida and Aida.29 Phospholipids in cells were extracted and identified by the method of Minnikin et al.30 The presence of mycolic acids was examined by TLC following Tomiyasu (1982).31 Whole-cell sugar composition was analyzed according to the methods of Becker et al.28 Methyl esters of cellular fatty acids were prepared by direct transmethylation with methanolic hydrochloride, and analyzed on a GLC system (HP 6890; Hewlett Packard, Palo Alto, CA, USA). Identification and quantification of the fatty-acid methyl esters, as well as the numerical analysis of the fatty acid profiles, were performed according to the instructions for the Microbial Identification System (MIDI, Newark, NJ, USA). Chromosomal DNA was prepared following the procedure of Saito and Miura,32 and the DNA G+C content was determined by HPLC, according to Tamaoka and Komagata.33 DNA–DNA hybridization was performed by the photobiotin-labelling method of Ezaki et al.34 After the 16S ribosomal RNA (rRNA) gene was amplified using PCR, the sequence was analyzed according to the method of Inahashi et al.5 The clustalw2 program was used for multiple alignments with selected sequences for calculating evolutionary distances35 by Sea View version 4.2 (ref. 36). The phylogenetic tree was constructed based on the neighbor-joining method,37 maximum-likelihood method38 and the maximum-parsimony method.39 Data were resampled with 1000 bootstrap replications.40 The values of sequence similarities among the closest strains were determined using the EzTaxon server.41

Results and discussion

Morphological, cultural and physiological characteristics

Strain K07-0460T grew well on ISP medium 2, 3 and other media, but globose sporangia on aerial mycelia were produced only on ISP medium 3 (Figure 1) and sucrose-nitrate agar. Vegetative mycelia were branched but not fragmented. The colony color of strain K07-0460T was red, and red soluble pigment was produced in various agar media (Table 1). The temperature and pH range for growth were 13–36 °C and pH 6–11, with optimum growth at 21–32 °C and pH 7–10. Strain K07-0460T did not grow on 3% NaCl medium. Casein was degraded. Starch was hydrolyzed. Gelatin was liquefied weakly. Milk was not peptonized and coagulated. Nitrate was not reduced to nitrite. H2S and melanin were not produced. The strain K07-0460T utilized D-glucose, D-xylose, D-mannitol and D-fructose, and did not utilize L-arabinose, raffinose, melibiose, L-rhamnose, myo-inositol, sucrose and cellulose. The strain K07-0460T did not produce tyrosinase. Enzyme activities of the API ZYM system were positive for alkaline phosphatase, esterase (C4), leucine allylamidase, valine allylamidase, cystine allylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-mannosidase, weakly positive for esterase lipase (C8), α-chymotrypsin and β-galactosidase and negative for lipase (C14), α-galactosidase, β-glucuronidase and α-fukosidase (Table 2).

Figure 1
figure 1

Scanning electron micrograph of strain K07-0460T grown on International Streptomyces Project (ISP) medium 3 for 4 weeks at 27 °C. Bar=10 μm.

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Table 1 Cultural characteristics of strain K07-0460T and type strains of related species
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Table 2 Comparison of physiological characteristics of strain K07-0460T and type strains of related species
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Chemotaxonomy

Strain K07-0460T contained meso-isomer of diaminopimelic acid as the diagnostic diamino acid. Galactose, glucose, mannose, madurose, ribose and xylose were detected as whole-cell sugars. The N-acyl type of muramic acid was acetyl. Phosphatidylethanolamine and unknown glucosamine-containing phospholipids were detected. Phosphatidylcholine and phosphatidylglycerol were not detected. Phospholipid pattern corresponded to type IV.42 Mycolic acids were not detected. The predominant menaquinones were MK-9 (H2) (50%) and MK-9 (H4) (41%) and the minor menaquinones were MK-9 (H0) (7%) and MK-9 (H6) (2%). The major cellular fatty acids were C17:0 10-methyl (24.6%), C17:1 ω8c (17.0%), C17:0 (10.5%) (Table 3). The G+C content of the genomic DNA was 72 mol%. The chemotaxonomic properties of strain K07-0460T were consistent with those of members of the genus Streptosporangium.

Table 3 Cellular fatty-acid compositions (%) of strain K07-0460T and type strains of related species
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Phylogenetic analysis

The 16S rRNA gene sequence of strain K07-0460T showed a close relationship with members of the genus Streptosporangium, and the similarity values between strain K07-0460T and the type strains in the genus Streptosporangium were 95.3–99.4%. The species showing the highest similarity values to strain K07-0460T were S. amethystogenes subsp. amethystogenes DSM 43179T (99.4%), S. amethystogenes subsp. fukuiense IFO 15365T (99.2%) and S. longisporum DSM 43180T (98.7%). The phylogenetic analysis based on the 16S rRNA gene sequences also indicated that strain K07-0460T formed a cluster with S. amethystogenes subsp. amethystogenes DSM 43179T and S. amethystogenes subsp. fukuiense IFO 15365T (Figure 2). The DDBJ accession number of the 16S rRNA gene sequences of strain K07-0460T is AB594818.

Figure 2
figure 2

Neighbor-joining tree based on 16S ribosomal RNA gene sequences showing relationship between K07-0460T and members of the genus Streptosporangium. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. L, branch also recovered in the maximum-likelihood tree; P, branch also recovered in the maximum-parsimony tree; Bar, 0.01 nucleotide substitutions per site.

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DNA–DNA hybridization

DNA–DNA relatedness values with strain K07-0460T among S. amethystogenes subsp. amethystogenes DSM 43179T, S. amethystogenes subsp. fukuiense IFO 15365T and S. longisporum DSM 43180T were below the value of 70% recommended by Wayne et al.43 for the assignment of strains to the same species (Table 4).

Table 4 DNA–DNA re-association between strain K07-0460T and type strains of related species
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Conclusion

The phylogenetical, morphological and chemotaxonomical properties indicated that strain K07-0460T belongs to the genus Streptosporangium. However, DNA–DNA relatedness values between strain K07-0460T and the related strains were below the value of 70% (Table 4). Furthermore, colonies of strain K07-0460T were red in color and produced red soluble pigment, whereas those of related strains were white to pale pink and did not produce soluble pigment (Table 1). The strain K07-0460T is also distinguished from related strains by differences in the utilization of D-glucose, L-arabinose, D-xylose, raffinose, D-mannitol, D-fructose and myo-inositol, reduction of nitrate, degradation of casein and enzyme activities of esterase (C4), valine allylamidase, cystine allylamidase, trypsin, acid phosphatase and α-mannosidase (Table 2). The above results support that strain K07-0460T represents a novel species in the genus Streptosporangium for which the name Streptosporangium oxazolinicum sp. nov. is proposed.

Description of S. oxazolinicum sp. nov.

Streptosporangium oxazolinicum (o.xa.zo.li’ni.cum. N.L. n. oxazolinum, oxazoline; L. neut. suff. -icum, suffix used with the sense of pertaining to; N.L. neut. adj. oxazolinicum, pertaining to oxazoline, referring to the production of oxazoline compounds).

Aerobic, Gram-positive and mesophilic actinomycete. Colonies are red in color. Vegetative mycelia are branched and not fragmented. Red soluble pigment is produced. Exhibits good growth and forms globose sporangia on aerial mycelia. Diagnostic diamino acid is meso-diaminopimelic acid. Whole-cell sugars are galactose, glucose, mannose, madurose, ribose and xylose. The acyl type of the peptidoglycan is acetyl. Phosphatidylethanolamine and unknown glucosamine-containing phospholipids are detected. Phospholipid pattern corresponds to type IV. Mycolic acids are not detected. The predominant menaquinones are MK-9 (H2) and MK-9 (H4). Major fatty acids are C17:0 10-methyl, C17:1ω8c and C17:0. Growth occurs at 13–36 °C (optimum 21–32 °C) and pH 6–11 (optimum pH 7–10). No growth in the presence of 3% NaCl. Casein is degraded. Starch is hydrolyzed. Gelatin is liquefied weakly. Cellulose is not degraded. Milk is not peptonized or coagulated. Nitrate is not reduced to nitrite. H2S and melanin are not produced. D-Glucose, D-xylose, D-mannitol and D-fructose are utilized, but L-Arabinose, raffinose, melibiose, maltose, L-rhamnose, myo-inositol and sucrose are not utilized. Tyrosinase is not produced. According to the API ZYM system, alkaline phosphatase, esterase (C4), leucine allylamidase, valine allylamidase, cystine allylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-mannosidase are positive, esterase lipase (C8), α-chymotrypsin and β-galactosidase are weakly positive and lipase (C14), α-galactosidase, β-glucuronidase and α-fukosidase are negative. The G+C content of the genomic DNA is 72 mol%. The type strain is S. oxazolinicum K07-0460T (=JCM 17388T).