Introduction

The genus Dactylosporangium, belongs to the family Micromonosporaceae, was first described by Thiemann et al.1 in 1967 for the aerobic mesophilic filamentous bacteria which forms finger-shaped sporangia and globose bodies on substrate mycelium as well as motile spores.2 In this genus, Dactylosporangium matsuzakiense strains have reported to produce an aminoglycoside antibiotic.3 The members of this genus that validly published and isolated from soils comprise, D. aurantiacum, D. fulvum, D. luridum, D. luteum, D. maewongense, D. matsuzakiense, D. roseum, D. salmoneum, D. siamense, D. thailandense, D. tropicum and D. vinaceum2, 3, 4, 5, 6, 7, 8, 9, 10 whereas D. darangshiense was isolated from rock soil.11 In Thailand, D. thailandense,1, 2 D. maewongense,7 D. tropicum9 and D. siamense10 have been reported.

In the course of our investigation for diversity of actinomycetes in peat swamp forest soil of Thailand, the strain RY35-23T was isolated. In this study, we described the taxonomic position of the strain which exhibited the morphology similar to the members of the genus Dactylosporangium using polyphasic approach.

Materials and methods

A peat swamp soil sample was collected from a tropical moist forest at Nong Jum Rung area in Rayong province, Thailand. The sample was air dried at room temperature for 7 days and was grinded using mortar. One gram of sample was added to basic lauryl sulfate solution (0.1 g sodium lauryl sulfate, 1.75 g KH2PO4, 3.5 g K2HPO4, 1000 ml distilled water, pH 7.0) and prepared a serial dilution to 10−4. 100 μl of each resultant suspension was added on humic acid–vitamin agar12 supplemented (l−1) with 25 mg of nalidixic acid and 50 mg of cycloheximide. The plates were incubated at 30 °C for 21 days. The colony of isolate RY35-23T was selected by observing the morphology of colony and sporangia and then transferred to International Streptomyces project media 2 (ISP 2 medium)13 for working culture.

The cultural characteristics of isolate RY35-23T was observed on various media recommended by Shirling and Gottlieb.13 The color of colony, reverse side and soluble pigment were determined using the Color Harmony Manual.14 The morphology of sporangia and globose bodies was observed using a scanning electron microscope (JSM-7610F and JSM-5410LV, Japan) after cultivation on sucrose nitrate agar and ISP 4 agar at 30 °C for 4 weeks.

Phenotypic properties were determined using the standard methods.15, 16 The utilization and acid production of various carbon sources were determined as described by Shirling and Gottlieb13 and Gordon et al.,17 respectively. The effect of pH, temperature and NaCl tolerance for growth was observed on ISP 2 medium at 30 °C for 14–21 days.

All chemotaxonomic studies were analyzed using freeze-dried cells obtained from the culture grown in ISP2 broth at 30 °C for 6 days. Isomers of diaminopimelic acid in cell wall peptidoglycan were determined using TLC based on the method of Staneck and Roberts.18 Whole-cell hydrolysate sugars were analyzed using HPLC following the method of Mikami and Ishida.19 The N-acyl type of muramic acid was analyzed using the method of Uchida and Aida.20 Phospholipids were extracted and analyzed using the procedure of Minnikin et al.21 The isoprenoid quinones were extracted according to the method of Collins et al.22 and were analyzed by HPLC. Fatty acid methyl esters were prepared according to the method described by the manufacturer's instruction (Sherlock Microbial Identification System MIDI, Inc., Newark, DE, USA)23, 24 and analyzed using gas chromatography and a HP-computer with MIDI data base (Hewlett Packard, Palo Alto, CA, USA). The presence of mycolic acids was determined using TLC based on the method of Tomiyasu.25

Genomic DNA was extracted from freeze-dried cells as described by Raeder and Broda.26 The G+C content of DNA was determined using HPLC according to the procedure of Tamaoka and Komagata.27 The amplification of 16S ribosomal RNA gene was carried out as described by Suriyachadkun et al.,28 and the PCR products were sequenced (Macrogen Inc., Seoul, Korea) using the universal primers.29 The sequence was determined using BLAST analysis on the EzTaxon-e database30 and was aligned, Bioedit software (Ibis Biosciences, Carlsbad, CA, USA), against the member of the genus Dactylosporangium. The phylogenetic trees were constructed using MEGA5.0 software31 based on neighbor-joining,32 maximum-parsimony33 and maximum-likelihood34 methods. The confidence values of nodes were evaluated using the bootstrap resampling method with 1000 replications.35 The DNA–DNA hybridization was performed as described by Ezaki et al.36

Results and Discussion

Taxonomic properties of strain RY35-23T

Phenotypic characteristics

Strain RY35-23T produced branch-substrate mycelia, while aerial mycelia were not observed on agar culture. Substrate mycelia were not fragmented. The strain produced finger-shaped sporangia on short sporangiophores. The irregular rugose sporangia were 0.6–1 by 3.2–4.3 μm in size (Figure 1a). Formation of globose bodies was observed on various ISP media, nutrient agar and sucrose nitrate agar. Globose bodies were spherical to oval in shape (1–1.6 μm in size) and emerged directly from substrate mycelia (Figure 1b). The motile spores were observed after 30 min when the agar culture was flooded with yeast dextrose broth. The colony of the strain showed amber to light melon yellow on various ISP media (Table 1). The temperature for growth was 20–37 °C with the optimal temperature at 25–37 °C. No growth was observed at 15 and 45 °C. The pH range for growth was 4–9 with the optimal pH at 6–7. The phenotypic properties of strain RY35-23T are showed in Table 2.

Figure 1
figure 1

Scanning electron micrograph of sporangia (a) and globose bodies (b) of strain RY35-23T grown on sucrose nitrate agar and ISP 4 at 30 °C for 4 weeks, respectively. ISP, International Streptomyces project.

Full size image
Table 1 Cultural characteristics of strain RY35-23T and closely related type strains
Full size table
Table 2 Differential characteristics between strain RY35-23T and closely related type strains
Full size table

Chemotaxonomic characteristics

Cell wall peptidoglycan of strain RY35-23T contained 3-hydroxy-diaminopimelic acid and meso-diaminopimelic acid as the major and minor diaminopimelic acids, respectively. The N-acyl muramic acid was glycolyl type. Whole-cell hydrolysate contained rhamnose, ribose, galactose, mannose, glucose, arabinose and xylose. The two latter sugars, arabinose and xylose, are diagnostic sugars that are classified as whole-cell sugar type D according to the classification of Lechevalier and Lechevalier.37 The major phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides (Figure 2). This polar lipid pattern corresponds to polar lipid type II, which showed one nitrogenous phospholipid, phosphatidylethanolamine, as diagnostic polar lipid.38 The major isoprenoid quinones were MK-9(H8; 75%) and MK-9(H6; 25%), the same as D. darangshiense, the closest species.11 The major cellular fatty acids were C17:0, C18:0, C18:1ω9c, anteiso-C15:0, iso-C15:0, iso-C16:0, iso-C17:0 and anteiso-C17:0 as shown in Table 3. This fatty acid profile of strain RY35-23T was similar to the other closely related Dactylosporangium type strains but was different in the percentage and some minor fatty acids. The mycolic acid was absent. The G+C content was 72.5 mol%. These chemical analysis results showed that the strain RY35-23T exhibited typical chemotaxonomic characteristics of the genus Dactylosporangium.39

Figure 2
figure 2

Polar lipid profiles of strain RY35-23T on a two-dimensional thin-layer chromatogram that were detected with 5% phosphomolybdic acid in ethanol as spraying reagent (for total lipids). DPG, diphosphatidylglycerol; L, unknown lipids; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIMs, phosphatidylinositol mannosides; PL, unknown phospholipid. A full color version of this figure is available at The Journal of Antibiotics journal online.

Full size image
Table 3 Cellular fatty acid compositions (%) of strain RY35-23T and closely related type strains
Full size table

Phylogenetic analysis

BLAST analysis revealed that the strain RY35-23T showed 16S ribosomal RNA gene similar to D. fulvum JCM 5631T (98.94%), D. roseum JCM 3364T (98.87%) and D. darangshiense JCM 17441T (98.86%). The phylogenetic tree based on neighbor-joining analysis revealed that strain RY35-23T shared monophyletic clade with those three closest Dactylosporangium species as showed in Figure 3.

Figure 3
figure 3

Phylogenetic relationships based on neighbor-joining analysis (Saitou and Nei, 1987) of 16S ribosomal RNA gene sequences of strain RY35-23T and all members in the genus Dactylosporangium. Verrucosispora gifhornensis DSM 44337T was used as an out group. Asterisk (*, #) indicated that the branches were recovered in the maximum-likelihood tree and maximum-parsimony tree, respectively. The number at branch nodes indicates bootstrap percentages derived from 1000 replications (only value >50% are shown). Bar=0.01 substitutions per nucleotide position.

Full size image

DNA–DNA hybridization

The levels of DNA–DNA relatedness among strain RY35-23T, D. fulvum JCM 5631T (27.4±3.1 to 36.1±2.9%), D. roseum JCM 3364T (37.1±3.7 to 39.5±1.4%) and D. darangshiense JCM 17441T (25.0±2.9 to 32.9±3.5%) were lower than 70% (Table 4), the cutoff level for assigning strains to the same species.40 This can be indicated that strain RY35-23T represents a genomic distinct from those Dactylosporangium species.

Table 4 DNA–DNA relatedness of strain RY35-23T and closely related type strains
Full size table

Conclusion

Based on the results of polyphasic approach, the strain RY35-23T showed typical morphology and chemical characteristics similar to the members of genus Dactylosporangium. The taxonomic position of strain RY35-23T was confirmed by phylogenetic tree analysis, which revealed that our strain belonged to the genus Dactylosporangium (Figure 2). The strain RY35-23T was compared with its closest Dactylosporangium species and could be distinguished using phenotypic properties, especially the cultural characteristics on ISP 2 medium, whose strain RY35-23T showed the amber colony and light maize pigment, whereas D. fulvum JCM 5631T, D. roseum JCM 3364T and D. darangshiense JCM 17441T showed maple, coral and light maize colony, and tan pigment, chili pigment and no pigment, respectively. Moreover, other phenotypic properties can also use for distinguishing between species as showed in Table 2. Therefore, on the basis of phenotypic properties, chemotypic properties, 16S ribosomal RNA gene analysis and DNA–DNA relatedness, strain RY35-23T represents the novel species of the genus Dactylosporangium, for which the name D. sucinum (type strain RY35-23T=JCM 19831T=TISTR 2212T= PCU 333T) is proposed.

Description of Dactylosporangium sucinum sp. nov.

Dactylosporangium sucinum (su.ci’ num. L. neut. adj. sucinum amber gold color)

Gram-positive, mesophilic, aerobic actinomycete. Amber vegetative mycelia are formed on ISP 2, ISP 3 and ISP 6 media. Finger-shaped sporangia can be observed on ISP 4 and ISP 5 media. Globose bodies are formed on ISP 2, 3, 4, 5, 6 and 7 media. Light wheat pigment is produced on ISP 2, ISP 4 and ISP 7 media. The strain shows good growth on ISP 2 medium, moderate growth on ISP 3, 4, 6 media and nutrient agar, and poor growth on ISP 5 medium. Optimal temperature for growth is 25–37 °C; no growth observed at 45 °C. Growth pH is 4–9 (optimum pH 6–7). The maximum NaCl concentration for growth is 3%. Hydrolysis of esculin and starch is positive. Liquefication of gelatin is negative. Nitrate is not reduced to nitrite. Peptonization of milk is positive but coagulation of milk is negative. Produces acid from salicin, D-melezitose, D-xylose, D-mannitol, D-mannose, L-rhamnose, L-arabinose, D-cellobiose and D-glucose but not myo-inositol and D-sorbitol. Utilizes D-glucose, L-arabinose, D-mannose, D-xylose, D-mannitol, sucrose, D-melezitose, D-cellobiose but not D-melibiose, D-arabitol, D-sorbose and D-raffinose. Cell wall contains 3-hydroxy-diaminopimelic acid as a major diaminopimelic acid and small amount of meso-diaminopimelic acid. The major phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The major isoprenoid quinones were MK-9(H8) and MK-9(H6). The major cellular fatty acids were C17:0, C18:0, C18:1ω9c, anteiso-C15:0, iso-C15:0, iso-C16:0, iso-C17:0 and anteiso-C17:0. The G+C content of type strain is 72.5 mol%.

The type strain RY35-23T (=JCM 19831T= TISTR 2212T= PCU 333T) was isolated from peat swamp forest soil collected from Nong Jum Rung area, Rayong province, Thailand.