Taxonomic Characterization of Streptomyces Strain CH54-4 Isolated from Mangrove Sediment
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Ann Microbiol (2010) 60:299–305 DOI 10.1007/s13213-010-0041-4 ORIGINAL ARTICLE Taxonomic characterization of Streptomyces strain CH54-4 isolated from mangrove sediment Rattanaporn Srivibool & Kanpicha Jaidee & Morakot Sukchotiratana & Shinji Tokuyama & Wasu Pathom-aree Received: 19 January 2010 /Accepted: 9 March 2010 /Published online: 15 April 2010 # Springer-Verlag and the University of Milan 2010 Abstract An actinobacterium, designated as strain CH54-4, wall chemotype I with no characteristic sugar, and type II was isolated from mangrove sediment on the east coast of the polar lipids that typically contain diphosphatidyl glycerol, Gulf of Thailand using starch casein agar. This isolate was phosphatidylinositol, phosphatidylethanolamine, and phos- found to contain chemical markers typical of members of the phatidylinositol mannoside. Members of the genus Strepto- genus Streptomyces: This strain possessed a broad spectrum myces are widely distributed in soils and played important of antimicrobial activity against Gram-positive, Gram- role in soil ecology (Goodfellow and Williams 1983). They negative bacteria and fungi. In addition, this strain also are prolific sources of secondary metabolites, notably showed strong activity against breast cancer cells with an antibiotics (Lazzarini et al. 2000). −1 IC50 value of 2.91 µg ml . Phylogenetic analysis of a 16S The search and discovery of novel microbes for new rRNA gene sequence showed that strain CH54-4 forms a secondary metabolites is significant in the fight against distinct clade within the Streptomyces 16S rRNA gene tree antibiotic resistant pathogens (Bernan et al. 2004) and and closely related to Streptomyces thermocarboxydus. emerging diseases (Taylor et al. 2001). One strategy is to isolate novel actinomycetes from poorly studied habitats to Keywords Mangrove sediment . Streptomyces . Taxonomy uncover novel sources of new bioactive compounds. Marine environments emerge as one of the most fascinating sources to search for novel taxa of actinomycetes (Pathom- Introduction aree et al. 2006a, b; Bull and Stach 2007). It is now accepted that actinomycetes are widespread in various The genus Streptomyces was proposed by Waksman and marine habitats from seashore (Bredholdt et al. 2007)to Henrici in 1943 for bacteria which resemble fungi in their the deepest ocean (Pathom-aree et al. 2006b). In the present branching filamentous structure. They are characterized by study, an actinomycete strain, CH54-4, was isolated from a the formation of chains of spores on the aerial mycelium, sediment sample taken from mangrove area on the east coast of Thailand. Taxonomic characterizations were : carried out based on 16S rDNA sequence analysis in R. Srivibool K. Jaidee combination with biochemical, chemotaxonomic and mor- Institute of Marine Science, Burapha University, Chonburi 20131, Thailand phological data. The ability to produce antimicrobial and anticancer compounds was also investigated. M. Sukchotiratana : W. Pathom-aree (*) Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Materials and methods e-mail: [email protected] Selective isolation of strain CH54-4 S. Tokuyama Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Strain CH54-4 was isolated from a sediment sample Shizuoka, Japan collected from tropical mangrove area on the east coast of 300 Ann Microbiol (2010) 60:299–305 Thailand by dilution plate technique on starch casein agar HotStar Taq Master Mix Kit protocol in a final volume of plates (Küster and Williams 1964) supplemented with 50 µl containing 10 µl of 10x PCR buffer, 25 mM MgCl2, 50 µg ml−1 actidione to inhibit fungal growth. The sediment 2 µl dNTP (10 mM), 2 µl 9F (5′-GAGTTTGATCCTGGCT sample was pre-treated at 55°C for 15 min and at 100°C for CAG-3′) , 2 µl 1510R (5′-GGCTACCTTGTTACGA-3′), 60 min. All of the inoculated plates were incubated at 30°C 5 µl template DNA, 0.5 µl HotStar Taq DNA polymerase, for 7–10 days. and distilled water made up to 50 µl and direct sequencing of the purified PCR product was carried out with auto- Morphological and physiological characteristics sequencer (ABI PRISM model 377) and 4 primers: 9F, 399F (5′-CTCCTACGGGAGGCAGCAG-3′), 785F (5′- Cultural characteristics of strain CH54-4 were examined by GGATTAGATACCCTGGTAGTC-3′) and 1099F (5′- eyes of 14-day-old culture grown on various International GCAACGAGCGCAACCC-3′). The resultant 16S rRNA Streptomyces Project (ISP) media (Shirling and Gottlieb gene sequences were aligned manually with corresponding 1966) and Actinomycete isolation agar (Mertz and Yao almost complete sequences from Streptomyces strains 1993). Micromorphology and sporulation were observed retrieved from the GenBank database as described previ- under light microscope by inclined cover slip technique ously. Evolutionary distances were calculated (Jukes and (Williams et al. 1989) on starch casein agar after incubating Cantor, 1969) and a phylogenetic tree constructed using the at 30°C for 7 days. Colors of aerial and substrate mycelia neighbor-joining method (Saitou and Nei 1987) with the were determined and recorded using National Bureau of TREECON program (Van de Peer and De Wachter 1994). Standards (NBS) Colour Name Charts (Kelly 1958). The The resultant tree topology was evaluated by a bootstrap spore chain morphology and spore surface ornamentation analysis (Felsenstein 1985) based on 1,000 resampled were examined by scanning electron microscopy (JEOL) of datasets. 14-day-old cultures grown on oatmeal agar. Physiological characteristics were examined by conventional methodolo- Preliminary determination of antimicrobial and anticancer gy and API 20 NE system. Resistance to some antibiotics activities was detected by disc diffusion method. Antimicrobial activity was determined by the cross-streak Chemotaxonomic analysis method on Mueller Hinton agar for bacteria and Potato Dextrose agar for fungi, incubated at 30°C for 3 days after The isomeric forms of diaminopimelic acid (DAP) and inoculation of actinomycete. Test organisms were Bacillus diagnostic sugar in whole-cell hydrolysates were deter- subtilis TISTR008, methicillin-resistant Staphylococcus mined by thin layer chromatography as described by aureus (MRSA) 815, Micrococcus luteus TISTR784, Staneck and Roberts (1974). Menaquinones and phospho- Pseudomonas aeruginosa NBRC 13736, Aspergillus niger lipids were extracted using established methodology NRIC1221, Candida albicans IFO1594, Debaryomyces (Minnikin et al. 1984). The menaquinone extracts were hansenii NRIC1303, Mucor racemosus NBRC 4581, purified using one-dimensional TLC (10 × 20 cm plastic- Schizosaccharomyces pombe NRIC1434, Pichia dispora backed silica gel plates; Kieselgel 60F254 Merck No. 5735; NRIC1348 and Penicillium chrysogenum NRIC11271. Merck, Darmstadt, Germany) prior to analysis by high Seven-day culture medium was extracted by ethyl acetate, performance liquid chromatography (HPLC; Octadodecyl evaporated under vacuum, and the crude extract was dried silica, C18, reverse phase, methanol-I-propanol 2:1 at up under nitrogen gas. Crude extract was dissolved in 270 nm). Polar lipids were detected by two-dimensional DMSO before use. KB (Human epidermoid carcinoma of TLC (aluminium-backed silica gel sheets; Kieselgel 60 cavity, ATCC CCL-17) and BC (breast cancer cell line) F254; Merck No. 1.05554; Merck). Lipid types were were determined by calorimetric cytotoxicity assay (Skehan determined with ethanolic molybdophosphoric acid, et al. 1990). Elliptine and doxorubicin were used as positive Dittmer and Lester Molypdenum blue, ninhydrin and control. DMSO was used as negative control. The OD was α-naphthol-sulphuric acid spray reagents. read in microtiter plate reader at the wavelength of 510 nm. Molecular identification Results Chromosomal DNA of strain CH54-4 was prepared from cells grown in trypticase soy broth (TSB) for 1–2 days by Strain CH54-4 showed extensively branched substrate DNA Trap kit (Cat No. 100-10009; BIOTEC, Thailand). mycelia and aerial hyphae under light microscopy. The PCR amplification of 16S rRNA gene was performed by scanning electron micrograph of strain CH54-4 revealed Ann Microbiol (2010) 60:299–305 301 Table 1 Cultural characteristics of Streptomyces CH 54-4 Medium Growth Aerial mycelium Substrate mycelium Yeast extract–malt extract agar Good Red Dark reddish-brown Oatmeal agar Good Whitish-pink Pinkish-orange Inorganic salt–starch agar Moderate Purple Purple Glycerol asparagine agar Good Greyish-pink Yellowish-pink Tyrosine agar Good No aerial mycelium Buff Actinomycete isolation agar Good Whitish-pink Purple Starch casein agar Good No aerial mycelium Orange Table 2 Phenotypic properties T of strain CH 54-4 Characteristics CH 54-4 S. thermocarboxydus AT37 Catalase - ND Spore surface Hairy Warty Spore mass Red, purple Grey Spore chain Spirales/retinaculiaperti Retinaculiaperti Red or purple red pigment + - Melanin from tyrosine agar - - Diffusible pigment - - Nitrate reduction + + Glucose fermentation + + Arginine dihydrolase - ND Urease - - Degradation of aesculin - + Gelatin + ND Resistance to antibiotics (μgml−1) Lincomycin (2) S ND Tobramycin (10) S S Rifampicin (50) S S Penicillin G (10 i.u.) R R Carbenicillin (100) S ND Vancomycin (30) S S Streptomycin (10) S S Growth temperature 37°C + + 45°C + + 50°C - + Growth with (%w/v) NaCl 3 % + ND NaCl 7% - ND Phenol 0.1% + - Utilization of Glucose + + Arabinose - ND L-Rhamnose + ND Meso-Inositol + + 302 Ann Microbiol (2010) 60:299–305 Table