Introduction

The genus Streptomyces belonging to the family Streptomycetaceae was proposed by Waksman and Henrici1 to accommodate aerobic, Gram-positive and spore-forming actinomycetes. The Streptomyces strains represent a group of actinomycetes that are widely distributed in nature. At present, the genus comprises more than 550 recognized species with validly published names, and recently some novel species, including S. cocklensis,2 S. gramineus,3 S. nanhaiensis,4 S. panacagri,5 S. pharmamarensis,6 S. qinglanensis7 and S. staurosporininus,8 have been described. Strains of the genus Streptomyces are superior to other actinomycete strains in their ability to produce various bioactive metabolites, especially antibiotics. Well-known antibiotics derived from Streptomyces strains include tetracycline, streptomycin, chloramphenicol, neomycin, nystatin, amphotericin, kanamycin and cycloheximide. Streptomyces strains are still a rich source of commercially significant compounds, such as antibiotics, enzymes, enzyme inhibitors and other pharmacologically active agents.9 Therefore, new species in the genus Streptomyces remains a focus of efficient research for the discovery of new bioactive compounds. In this paper, we report the taxonomic status of Streptomyces strains KC-038T, KC-031 and KC-106T, which were isolated from soils in the south of Thailand.

Materials and methods

Strains KC-038T and KC-031 were isolated from soil samples collected from the Krung Ching Waterfall, Khao Luang National Park, Nakhon Si Thammarat Province, Thailand, and strain KC-106T was isolated from the Similan Island National Park (8°39′09′′N 97°38′27′′E), Phanga Province, Thailand. The soil samples were serially diluted with distilled water, heated at 55 °C for 5 min and plated onto potato starch-glycerol agar10 and starch casein nitrate agar11 containing nystatin (25 mg l−1) and tetracycline (10 mg l−1). The resulting pure isolates were maintained on SYM agar (starch 1.0%, NZ amine 0.3%, yeast extract 0.1%, meat extract 0.1%, CaCO3 0.3%, agar 1.2%, pH 7.0). Genomic DNA of each isolate was obtained by sonication of a suspension of cells12 grown in YD broth (yeast extract 1.0%, dextrose 1.0%, pH 7.0). The 16S rRNA gene was amplified using the primers described by Takahashi et al.13 The PCR products were sequenced on a DNA sequencer (model 3130 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer’s instructions. The closest phylogenetic neighbors were identified by BLAST searches using the EzTaxon-e server.14 The clustalw2 program was used for multiple alignments with selected sequences for calculating evolutionary distances15 with SeaView version 4.2.16 Phylogenetic trees were constructed using the neighbour-joining,17 maximum-likelihood18 and maximum-parsimony19 methods. Data were resampled with 1000 bootstrap replications.20 Values for sequence similarity among the closely related strains were determined using the EzTaxon-e server.14

Strains KC-038T, KC-031 and KC-106T were cultivated at 27 °C for 2 weeks on ISP (International Streptomyces Project) 2, 3, 4, 5, 6 and 7 media,21 YS agar (yeast extract 2.0%, starch 1.0%, agar 1.5%, pH 7.0) and nutrient agar. The Color Harmony Manual22 was used to determine the color of aerial and substrate mycelia and soluble pigment. The features of the substrate and aerial mycelia and spores were observed by light microscopy (Nikon; model Labophoto-2, Tokyo, Japan) and scanning electron microscopy (model JSM-5600, JEOL, Tokyo, Japan) after cultivation on agar media at 27 °C for 3 weeks. For scanning electron microscopy investigation, the cultures were fixed with 4% osmium tetraoxide vapor in situ for 16 h at room temperature, and then dried at room temperature.23 Physiological characteristics, NaCl tolerance, and the temperature and pH ranges required for growth were determined on ISP 2 medium. Utilization of various carbohydrates as the sole carbon source was tested using ISP 9 medium.24 Starch hydrolysis was examined using ISP 4 medium, while nitrate medium (beef extract 0.3%, peptone 0.5%, KNO3 0.1%, pH 7.0) was used to assess nitrate reduction, and glucose–peptone–gelatin medium (glucose 2.0%, peptone 0.5%, gelatin 20%, pH 7.0) was used to examine gelatin liquefaction. Skim milk (10%) was used to assess coagulation and peptonization of milk, and skim milk agar was used to examine casein hydrolysis. Enzyme activities were determined using the API ZYM system (bioMérieux, Lyon, France), according to the manufacturer’s instructions. Biomass for the genotypic and chemotaxonomic studies was obtained after cultivation in YD broth on a rotary shaker at 27 °C for 3 days. Diaminopimelic acid isomers in whole cells were determined by TLC using whole-cell hydrolysates.25 Whole-cell sugar composition was analyzed according to the methods of Becker et al.25 Isoprenoid quinones were extracted according to the method of Collins et al.26, and were analyzed by LC/MS (JMS-T 100LP, JEOL) using a CAPCELL PAK C18 UG120 column (Shiseido, Tokyo, Japan) with methanol/2-propanol (7:3). The N-acyl types of muramic acid were determined by using the method of Uchida and Aida.27 Phospholipids were extracted and identified by using the method of Minnikin et al.28 The presence of mycolic acids was examined by TLC following the protocol of Tomiyasu.29 Cellular fatty acid composition was determined by gas liquid chromatography, according to the Microbial Identification System (MIDI) Sherlock version 6.0 using the RTSBA6 MIDI database as described by Sasser.30 For DNA base composition analysis, chromosomal DNA was prepared following the procedure of Saito and Miura,31 and the DNA G+C content was determined by HPLC according to the method of Tamaoka and Komagata.32 DNA–DNA hybridization was performed using the photobiotin-labeling method of Ezaki et al.33

Results and Discussion

Chemotaxonomic characteristics

Strains KC-038T, KC-031 and KC-106T exhibited typical characteristics of the genus Streptomyces. LL-diaminopimelic acid was detected in whole-cell hydrolysates. The menaquinones detected were as follows: MK-9 (H6) (62%), MK-9 (H4) (23%) and MK-9 (H8) (15%) for KC-038T, and MK-9 (H8) (70%), MK-9 (H6) (21%) and MK-9 (H4) (10%) for KC-106T. The N-acyl type of muramic acid was acetyl. Strains KC-038T and KC-106T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and unknown phospholipids as phospholipid composition. Strains KC-038T and KC-031 contained C16:0 (23.5, 19.7%), iso-C16:0 (18.4, 22.3%) and anteiso-C15:0 (17.7, 16.6%) (Table 1), whereas strain KC-106T contained anteiso-C15:0 (25.0%), iso-C16:0 (23.2%) and anteiso-C17:0(10.3%) as major cellular fatty acids (Table 2). The cellular fatty acid profiles of strains KC-038T, KC-031 and KC-106Twere almost the same as those of the type strains, but the amount of some fatty acids was different, as shown in Tables 1 and 2. The DNA G+C content was 72 mol% for strains KC-038T and KC-031, and 73 mol% for strain KC-106T.

Table 1 Cellular fatty acid compositions (%) of strains KC-038T, KC-031 and closely related type strains
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Table 2 Cellular fatty acid compositions (%) of strain KC-106T and closely related type strains
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Phylogenetic analysis

The 16S rRNA gene sequence similarity value between strains KC-038T and KC-031 was 99.9%, and they showed the highest sequence similarities to S. olivochromogenes NBRC 3178T (98.1%) and S. psammoticus NBRC 13971T (98.1%) and clustered with them (Figure 1).

Figure 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strains KC-038T, KC-031 and closely related type strains of the genus Streptomyces. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. (●), branches were also recovered in the maximum-parsimony tree; (*), branches were also recovered in the maximum-likelihood tree; Bar, 0.005 nucleotide substitutions per site.

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The 16S rRNA gene sequence of strain KC-106T was most similar to those of S. seoulensis NBRC 16668T (98.9%), S. recifensis NBRC 12813T (98.9%), S. chartreusis NBRC 12753T (98.7%) and S. griseoluteus NBRC 13375T (98.4%). The phylogenetic tree showed that strain KC-106T forms a cluster with three of the above species, with the exception of S. chartreusis (Figure 2).

Figure 2
figure 2

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain KC-106T and closely related type strains of the genus Streptomyces. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. (●), branches were also recovered in the maximum-parsimony tree; (*), branches were also recovered in the maximum-likelihood tree; Bar, 0.005 nucleotide substitutions per site.

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Phenotypic characteristics

The cultural characteristics of strains KC-038T and KC-031, along with those of the type strains of the closest related species, S. olivochromogenes NBRC 3178T34 and S. psammoticus NBRC 13971T,35 are shown in Table 3. Strains KC-038T and KC-031 grew well and formed extensively branched substrate mycelia on the various agar media tested. Aerial mycelia of white to gray color were produced on ISP 2–5 and 7 media, YS agar and nutrient agar, while the related type strains produced white aerial mycelia. The aerial mycelia consisted of long spiral chains with a smooth surface and the spores were rod shaped (Figures 3 and 4). Soluble pigment was produced on ISP 6 medium. The phenotypic and differential characteristics of strains KC-038T and KC-031 are listed in the species description below and in Table 4. Strains KC-038T and KC-031 were highly similar to each other but were differentiated from the closest related type strains with respect to carbon utilization.

Table 3 Cultural characteristics of strains KC-038T, KC-031 and closely related type strains
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Figure 3
figure 3

Scanning electron micrograph of strain KC-038T grown on YS agar for 3 weeks at 27 °C.

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Figure 4
figure 4

Scanning electron micrograph of strain KC-031 grown on ISP 4 medium for 3 weeks at 27 °C.

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Table 4 Differential characteristics of strains KC-038T, KC-031 and closely related type strains
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The cultural characteristics of strain KC-106T and the type strains of the closest related species, S. seoulensis NBRC 16668T,36 S. recifensis NBRC 12813T,37 S. chartreusis NBRC 12753T,38 and S. griseoluteus NBRC 13375T,39 are shown in Table 5. The strain grew well and formed extensively branched substrate and aerial mycelia on all agar media tested. Aerial mycelia of white to brownish gray color were produced. The aerial mycelia consisted of long and spiral spore chains with a hairy surface and oval-shaped spores (Figure 5), which is clearly different from the smooth spores produced by the related strains. The phenotypic and differential characteristics of strain KC-106T are listed in the species description below and in Table 6. Strain KC-106T was also differentiated from the closest related type strains with respect to carbon utilization.

Table 5 Cultural characteristics of strain KC-106T and closely related type strains
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Figure 5
figure 5

Scanning electron micrograph of strain KC-106 T grown on ISP 4 medium for 3 weeks at 27 °C.

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Table 6 Differential characteristics of strain KC-106T and closely related type strains
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DNA–DNA hybridization

The DNA–DNA relatedness value between strains KC-038T and KC-031 was 100%; therefore, these two strains were classified as the same species. The DNA–DNA relatedness values between strain KC-038T and the closest related type strains, S. olivochromogenes NBRC 3178T and S. psammoticus NBRC 13971T, were in the range of 4–36% (Table 7). The DNA–DNA relatedness values between strain KC-106T and the closest type strains, S. seoulensis NBRC 16668T, S. recifensis NBRC 12813T, S. chartreusis NBRC 12753T and S. griseoluteus NBRC 13375T, were in the range of 7–46% (Table 8). These values were below the 70% cutoff point recommended by Wayne et al.40 for assigning strains to the same species, and these results thus confirm that strains KC-038T and KC-106T are distinct from their closely related phylogenetic neighbors. Therefore, strains KC-038T and KC-106T are clearly the two novel species within the genus Streptomyces.

Table 7 DNA–DNA relatedness between KC-038T, KC-031 and closely related type strains
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Table 8 DNA–DNA relatedness between KC-106T and closely related type strains
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Conclusion

Based on phylogenetic, phenotypic and chemotaxonomic characteristics, strains KC-038T, KC-031 and KC-106T are classified within the genus Streptomyces. The cultural, physiological and biochemical features, such as aerial mycelia color and utilization of various carbohydrates as the sole carbon source, indicate that strains KC-038T, KC-031 and KC-106T differ from the closest related species. The results of DNA–DNA relatedness also support their classification of these strains into two novel species. Therefore, the name Streptomyces siamensis sp. nov. is proposed for strains KC-038T and KC-031, and the name Streptomyces similanensis sp. nov. is proposed for strain KC-106T.

Description of S. siamensis sp. nov. (si.am.en’sis N.L. masc. adj. siamensis, belonging to Siam, the old name for Thailand, where the first strain was isolated)

Gram-positive, aerobic actinomycete, forming extensively branched substrate mycelia and aerial hyphae that differentiate into long and spiral chains of smooth-surfaced, rod-shaped spores. The color of aerial mycelia varies from white to gray. Dark brown soluble pigment is produced in ISP6 medium. Growth occurs at 16–38 °C (optimum at 16–30 °C) and at pH 4–11 (optimum at pH 5–9). Hydrolyzes casein and starch but not gelatin. Nitrate is not reduced. Utilizes D-glucose, L-arabinose, D-xylose, raffinose, melibiose, D-mannitol, D-fructose, L-rhamnose and myo-inositol, but not sucrose. Positive for alkaline phosphatase, esterase (C4), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrase, β-glucosidase and N-acetyl-β-glucosaminidase, and weakly positive for valine arylamidase, cystine arylamidase, trypsin, β-galactosidase and α-mannosidase. Negative for esterase lipase (C8), lipase (C14), osidase, α-galactosidase and α-glucosidase (API ZYM system). The menaquinones are MK-9 (H6), MK-9 (H4) and MK-9 (H8). Major cellular fatty acids are C16:0, iso-C16:0 and anteiso-C15:0. The DNA G+C content of the type strain is 72 mol%.

The type strain KC-038T (=NBRC 108799T =PCU 328T =TISTR 2107T) was isolated from the soil collected at Krung Ching Waterfall, Khao Luang National Park, Nakhon Si Thammarat Province, Thailand.

Description of S. similanensis sp. nov. (si.mi.lan. en’ sis N.L. masc. adj. similanensis, belonging to Similan, an island in the southern Thailand, where the first strain was isolated)

Gram-positive, aerobic actinomycete, forms extensively branched substrate mycelia and aerial hyphae that differentiate into long and spiral chains of hairy surfaced and oval spores. Aerial mycelia are white to brownish gray in color. Soluble pigment is not produced. Growth occurs at 12–40 °C (optimum at 18–30 °C) and at pH 4–11 (optimum at pH 4–9). Hydrolyzes casein and starch but not gelatin. Nitrate is not reduced. Utilizes D-glucose, L-arabinose, D-xylose, raffinose, melibiose, D-mannitol and D-fructose; weakly utilizes raffinose and melibiose, but not sucrose, L-rhamnose or myo-inositol. Positive for alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-mannosidase. Negative for esterase lipase (C8), trypsin, α-chymotrypsin and β-galactosidase (API ZYM system). Menaquinones are MK-9 (H8), MK-9 (H6) and MK-9 (H4). Major cellular fatty acids are anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The DNA G+C content is 73 mol%.

The type strain KC-106T (=NBRC 108798T=PCU 329T=TISTR 2104T) was isolated from soil collected at the Similan Island National Park, Phanga Province, Thailand.