Ann Microbiol (2010) 60:299–305 DOI 10.1007/s13213-010-0041-4

ORIGINAL ARTICLE

Taxonomic characterization of 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 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 . 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 2 (continued) Characteristics CH 54-4 S. thermocarboxydus AT37T

D-Mannose + ND Mannitol + + Raffinose + - D-Xylose + ND D-Maltose + ND N-acetylglucosamine - ND Potassium gluconate + ND L-Tryptophan + ND Capric acid - ND Adipic acid - ND Malic acid - ND Trisodium citrate - ND Phenylacetic acid - ND

that aerial mycelia were highly branched with compact ethanolamine, diphosphatidylglycerol, phosphatidylinositol spirales and rectiflexibile spore chain. Each spore chain and phosphatidylinositol mannoside. Predominant menaqui- consisted of 40–60 short rod (0.5–0.6×0.7–0.8 μm) hairy none was MK9 (H8). Chemotaxonomic and morphological surface spores. properties of strain CH54-4 are in line with its classification The cultural characteristics are shown in Table 1.Strain in the genus Streptomyces (Williams et al. 1989). CH54-4 grew well on most media except on inorganic salt– The assignment of this strain to the genus Streptomyces starch agar. Typically, the colonies were convex and some was also supported by 16S rRNA gene sequence data. An part of the aerial mycelia and spore chains could be observed almost full sequence of 16S rRNA gene of strain CH54-4 around the colony edge. Aerial mycelia were initially white, (1,501 nucleotides) was obtained and submitted to the becoming pink, orange, reddish orange to purple color GenBank database under an accession number DQ417205. depending on the medium used, and gradually darkened in Comparison of the sequence of strain CH54-4 with the old culture. Likewise, vegetative mycelium colour was corresponding sequences of representative strains of the yellowish-pink to red or purple depending on the medium. genus Streptomyces showed that this organism formed a Melanin and other soluble pigments were not produced. distinct phyletic line with a clade encompassed by The physiological properties are shown in Table 2.Strain Streptomyces thermocarboxydus and S. lusitianus (Fig. 1). CH 54-4 was sensitive to most antibiotics except penicillin G The antimicrobial activity of strain CH54-4 against and was moderately thermotolerant; the optimum range for selected microorganisms is shown in Table 3. The strain growth was between 30–40°C. It showed positive results for inhibited growth of both Gram-positive and Gram-negative utilization of many carbon sources such as mannitol and bacteria, yeast and fungi suggesting a broad spectrum raffinose. However, some organic acids and citrate could not characteristic of the active compound. Red pigment was be utilized. Growth also occurred in 3% NaCl. also produced on ISP2 medium. This pigment was soluble Chemotaxonomic tests showed that whole-cell hydro- in chloroform and developed an orange band of bacterial lysates of strain CH54-4 were rich in LL-diaminopimelic acid carotenoid on the chromatogram. In addition, the strain

(LL-DAP) with no characteristic sugar indicated a wall showed strong activity to breast cancer cells with the IC50 chemotype I. The diagnostic polar lipids were phosphatidyl- of 2.91 µg ml-1 (data not shown). Ann Microbiol (2010) 60:299–305 303

0.1 substitutions/site

62 Streptomyces massasporeus DSM 40035T (AY999836) 54 Streptomyces hawaiiensis NBRC 12784T (AB184143) Streptomyces indiaensis NBRC 13964T (AB184553) Strain 54-4 (DQ417205) Streptomyces thermocarboxydus DSM 44293T (U94490) 62 Streptomyces lusitanus NBRC 13464T (AB184424) Streptomyces spinoverrucosus NBRC 14228T (AB184578) Streptomyces luteogriseus NBRC 13402T (AB184379) Streptomyces purpurascens NBRC 13077T (AB184859) 59 Streptomyces janthinus ISP 5206T (AJ399478) 99 Streptomyces violaceus ISP 5082T (AY999738) 88 Streptomyces roseoviolaceus NBRC 13081T (AB184298) Streptomyces parvulus NBRC 13193T (AB184326) Streptomyces lomondensis LMG 20088T (AJ781352) Streptomyces coerulescens ISP 5146T (AY999720) 98 66 Streptomyces bellus ISP 5185T (AJ399476) 56 Streptomyces coeruleorubidus ISP 5145T (AY999719) 67 T 95 Streptomyces viridodiastaticus NBRC 13106 (AB184317) Streptomyces humidus subsp. antitumoris NBRC 13976T (AB184556) Streptomyces glaucescens NBRC 12774T (AB184843) 75 Streptomyces matensis NBRC 12889T (AB184221) 72 Streptomyces althioticus NBRC 12740T (AB184112) Streptomyces pseudogriseolus subsp. glucofermentans NBRC 13833T (AB184516) 57 96 Streptomyces griseorubens NBRC 12780T (AB184139) 100 Streptomyces variabilis NBRC 12825T (AB184884) 95 Streptomyces griseoincarnatus NBRC 12871T (AB184207) Streptomyces erythrogriseus LMG 19406T (AJ781328)

100 T 94 Streptomyces rameus KCTC 9767 (AY999821) Streptomyces glomeratus LMG 19903T (AJ781754) Streptomyces thermocarboxydovorans NBRC 16324T (AB249929) 57 T 90 Streptomyces thermoviolaceus subsp. thermoviolaceus NBRC 13905 (AB184545) Streptomyces thermodiastaticus NBRC 100020T (AB249928) Streptomyces bluensis NBRC 13460T (AB184421) Streptomyces vinaceus NBRC 13425T (AB184394)(AB1 100 Streptomyces griseoruber NBRC 12873T (AB184209)

Fig. 1 Neighbor-joining tree based on almost complete 16S rRNA neighbor-joining analysis of 1,000 resampled datasets; only values gene sequence showing relationships between streptomycete isolate above 50% are given. Scale bar indicates 0.1 substitutions per CH54-4 and related members of the genus Streptomyces. The numbers nucleotide position at the nodes indicate the level of bootstrap support based on a 304 Ann Microbiol (2010) 60:299–305

Table 3 Antimicrobial activity of Streptomyces CH 54-4 on Test organisms Diameter of clear zone (mm) ISP2 medium Bacillus subtilis TISTR 008 10 Methicillin-resistant Staphylococcus aureus 815 (clinical isolate) 14 Micrococcus luteus TISTR 784 8 Pseudomonas aeruginosa NBRC 13736 w Candida albicans IFO 1594 21 Schizosaccharomyces pombe NRIC 1434 3 Debaryomyces hansenii NRIC 1303 6 Saccharomyces cerevisiae TISTR 5039 4 Pichia dispora NRIC 1348 - Mucor racemosus NBRC 4581 w Aspergillus niger NRIC 1221 w Penicillium chrysogenum NRIC 1271 10 + Positive, - negative, w weak

Discussion N-acetyl-β-D-glucosamidase and α-L-arabinosidase, but not α-galactosidase in API-ZYM test. Therefore, strain CH54-4 With the morphological characteristics of spirales and may represent a novel species of Streptomyces. retinaculiapperti spore chains, wall chemotype I with no The present findings support the view that marine characteristic sugar, and polar lipids, it was clear that strain associated habitats are precious resources for novel actino- CH54-4 belonged to the genus Streptomyces. The result from mycete taxa and bioprospecting. Streptomycetes from phylogenetic analysis based on almost complete 16S rDNA mangrove sediment were reported to be a producer of sequence confirmed its placement in the genus Streptomyces. novel compounds such as production of caprolactones from Streptomyces strain CH54-4 shared a 16S rDNA simi- Streptomyces sp B6007 isolated from mangrove sediment in larity level of 98.8% with S. thermocarboxydus,an Papua New Guinea (Stritzke et al. 2004) and octalactin C, equivalent to 30 nucleotides differences out of 1,479 novel 8-membered macrolide produced by mangrove- positions. High 16S rDNA similarities and nucleotide derived Streptomyces sp. V5 (Guang-Ying et al. 2007). differences of this value have been recorded for the type strains of many Streptomyces species, some of which are Acknowledgements Wasu Pathom-aree is grateful for support from – Thailand Research Fund. We thank Miss Rawiwan Watanadilok for known to have DNA DNA relatedness value lower than the HPLC technical assistance and the Institute of Marine Science, the 70% cutoff point recommended for the delineation of Bangsaen, for all facility support. This work was under the genomic species (Wayne et al. 1987), for instance, the type collaborative project NRCT-JSPS (National Research Council of strains of Streptomyces thermocarboxydus, S. thermodias- Thailand and Japan Society for the Promotion of Science). taticus and S. thermoviolaceus subsp. thermoviolaceus share high 16S rDNA sequence similarity values with one another (98.7–99%) but can be separated on the basis of References genotypic and phenotypic properties, notably DNA–DNA relatedness data (Kim et al. 1998). Currently, a 16S rRNA Bernan VS, Greenstein M, Carter GT (2004) Mining marine micro- gene sequence similarity of 98.7–99% was proposed to be organisms as a source of new antimicrobials and antifungals. mandatory for testing the genomic uniqueness of a novel Curr Med Chem-Anti-Infective Agents 3:181–195 isolate (Stackebrandt and Ebers 2006). Bull AT, Stach JEM (2007) Marine : new opportunities for natural product search and discovery. Trends Microbiol Although strain CH 54-4 shares high 16S rDNA similarity 15:491–499 values with S. thermocarboxydus, they were different in Bredholdt H, Galatenko OA, Engelhardt K, Fjarvik E, Terekhova LP, numbers of phenotypic characteristics. Strain 54-4 produced Zotchev SB (2007) Rare actinomycete bacteria from the shallow red carotenoid pigment on ISP2 medium (Table 2). Hairy- water sediments of the Trondheim fjord, Norway: isolation, diversity and biological activity. Environ Microbiol 9:2756–2764 surfaced spore are borne on aerial mycelium instead of warty Felsenstein J (1985) Confidence limits on phylogeny: an appropriate spore. The aerial spore mass colour is whitish-pink not grey. use of the bootstrap. Evolution 39:783–791 No distinctive substrate mycelium color was observed in S. Goodfellow M, Williams ST (1983) Ecology of actinomycetes. Annu – thermocarboxydus. Strain CH54-4 also produced α- Rev Microbiol 37:189 216 β α Guang-Ying C, Feng Z, Yong-Cheng L (2007) A novel 8-membered galactosidase but not N-acetyl- -D-glucosamidase and -L- macrolide produced by marine-derived actinomycete Streptomy- arabinosidase. S. thermocarboxydus was positive with ces sp. V5. Chinese. J Org Chem 27:1159–1161 Ann Microbiol (2010) 60:299–305 305

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