Saccharopolyspora pathumthaniensis sp. nov., a novel actinomycetes

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Morphological and chemotaxonomic characterization of actinomycete strain S582 isolated from the gut of a termite (Speculitermes sp.) in Pathum Thani Province ...
J. Gen. Appl. Microbiol., 57, 93 100 (2011)

Full Paper Saccharopolyspora pathumthaniensis sp. nov., a novel actinomycetes isolated from termite guts (Speculitermes sp.) Kanokkorn Sinma,1 Yuumi Ishida,3 Tomohiko Tamura,3 Vichien Kitpreechavanich,2 and Shinji Tokuyama1, * 1

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422 8529, Japan 2 Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 3 Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Chiba 292 0812, Japan (Received October 15, 2010; Accepted January 4, 2011)

Morphological and chemotaxonomic characterization of actinomycete strain S582 isolated from the gut of a termite (Speculitermes sp.) in Pathum Thani Province, Thailand, clearly demonstrated that this strain is a member of the genus Saccharopolyspora. 16S rDNA sequence analysis for the strain supported the assignment of the strain to the genus Saccharopolyspora. The similarity value of sequences between this strain and the closely related species Saccharopolyspora endophytica was 99.5%. The DNA G+C content was 70.2 mol%. DNA DNA hybridization results (53.3%) and some physiological and biochemical properties indicated that strain S582T was distinguished from the phylogenetically closest relatives. Based on these genotypic and phenotypic data, strain S582T should be a new species in the genus Saccharopolyspora and the name Saccharopolyspora pathumthaniensis sp. nov. is proposed for the strain. The type strain is S582T (=NBRC 104112T =BCC 28624T). Key Words—actinomycetes; Pseudonocardiaceae; Saccharopolyspora; termite guts

Introduction

 The genus Saccharopolyspora was proposed by Lacey and Goodfellow (1975) for actinomycetes isolated from spontaneously heated sugarcane bagasse. The genus currently encompasses aerobic, non-acidfast organisms which form an extensively branched substrate mycelium that fragments into rod-shaped, non motile elements and an aerial mycelium that differentiates into bead-like chains of spores. Saccharopolyspora is also characterized by the presence of  * Address reprint requests to: Dr. Shinji Tokuyama, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422 8529, Japan.  Tel/Fax: +81 54 238 4879  E-mail: [email protected]

meso-diaminopimelic acid, arabinose and galactose in their wall peptidoglycans (wall chemotype IV sensu Lechevalier and Lechevalier, 1970). The organisms have large amounts of phosphatidylglycerol, phosphatidylcholine, phosphatidyl ethanolamine and phosphatidylmethylethanolamine (phospholipids pattern III sensu Lechevalier et al. (1977)), are rich in iso- and anteiso-branched chain fatty acids, and have a large amount of tetra-hydrogenated menaquinone with nine isoprene units but lack mycolic acids (Embley et al., 1987; Goodfellow et al., 1989). The DNA base compositions of members of the genus fall within the range of 66 77 mol% G+C (Goodfellow et al., 1989).  The genus Saccharopolyspora includes the following validly described species: Saccharopolyspora erythraea (Labeda, 1987), Saccharopolyspora gregorii (Goodfellow et al., 1989), Saccharopolyspora hordei

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(Goodfellow et al., 1989), Saccharopolyspora rectivirgula (Korn-Wendisch et al., 1989), Saccharopolyspora spinosa (Mertz and Yao, 1990), Saccharopolyspora taberi (Korn-Wendisch et al., 1989), Saccharopolyspora hirsuta (Lacey and Goodfellow, 1975), Saccharopolyspora spinosporotrichia (Zhou et al., 1998), Saccharopolyspora flava and Saccharopolyspora thermophila (Lu et al., 2001), Saccharopolyspora antimicrobica (Yuan et al., 2008), Saccharopolyspora cebuensis (Pimento-Elardo et al., 2008), Saccharopolyspora shandongensis (Zhang et al., 2008), Saccharopolyspora endophytica (Qin et al., 2008), Saccharopolyspora halophila (Tang et al., 2009), Saccharopolyspora jiangxiensis (Zhang et al., 2009), Saccharopolyspora qujiaojingensis (Tang et al., 2009), Saccharopolyspora rosea (Yassin, 2009), Saccharopolyspora tripterygii (Li et al., 2009) and Saccharopolyspora patthalungensis (Duangmal et al. 2010). These taxa form a distinct phyletic branch within the evolutionary radiation encompassed by the family Pseudonocardiaceae (Embley et al., 1988; Warwick et al., 1994). Members of the genus Saccharopolyspora are a potentially rich source of natural products, but only erythromycin, produced by Saccharopolyspora erythraea, is currently commercially important (Embley, 1992).  Strain S582T was isolated during a search for lignocellulosic compounds degrading actinomycetes from termite guts and found to produce a xylan-degrading enzyme. The novel strain was isolated from the gut of a grass-feeding termite, Speculitermes sp., collected from an orangery in Pathum Thani Province, Thailand. Strain S582T was found to have morphological properties consistent with its assignment to the genus Saccharopolyspora. In the present investigation, this organism was examined for an array of genotypic and phenotypic properties. This isolate represents a novel species of Saccharopolyspora, for which we propose the name Saccharopolyspora pathumthaniensis. Materials and Methods

 Isolation. Strain S582T was isolated from the gut of a Speculitermes sp. grass-feeding termite on a humic acid vitamin agar (Hayakawa and Nonomura, 1987). The termite sample was collected from an orangery in Pathum Thani Province, Thailand. The strain was maintained on ISP medium No. 2 (Shirling and Gottlieb, 1966).  Morphology. The characteristic of hyphae, notably

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aerial hyphae and spore chain morphology, was observed on ISP media No. 2, 3, 4, 5, 6 and 7 (Shirling and Gottlieb, 1966) using the coverslip technique. Additional morphological properties were observed by examining gold-coated, dehydrated specimens of the organism using a scanning electron microscope (Model JSM6060; JEOL, Ltd., Tokyo, Japan).  Cultural, physiological, and biochemical characteristics. The strain was examined for a range of phenotypic properties following incubation for 14 d at 30 C on various agar media according to procedures of Lacey and Goodfellow (1975) and Zhou et al. (1998). Decomposition of various compounds was examined using the basal medium recommended by Gordon et al. (1974). The temperature for growth and NaCl tolerance were determined on ISP medium No. 2. Carbon utilization was tested by using ISP medium No. 9 supplemented with a final concentration of 1% of tested carbon sources. Nitrogen utilization was examined on the basal medium containing 10 g of glucose, 0.5 g of MgSO4・7H2O, 0.5 g of NaCl, 0.01 g of FeSO4・7H2O, 1 g of K2HPO4 and 12 g of agar in (1 L) distilled water (William et al., 1989). Gelatin liquefaction, peptonization of milk, nitrate reduction, cellulose composition and starch hydrolysis were determined by cultivation on various media described by Hamada (2000). Chitin hydrolysis was detected on colloidal chitin agar (Hsu and Lockwood, 1975). Lipid hydrolysis was tested on Siera s medium (Siera, 1957) supplemented with Tween 80 (1% w/v). The organism was also examined for its ability to grow at 4 45 C.  Chemotaxonomy. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strain in Tryptic Soy Broth (TSB) at 30 C for 5 d. At maximum growth, the broth culture was checked for purity, harvested by centrifugation, washed three times with distilled water, and freeze-dried. Established procedures were used to determine the diagnostic isomers of diaminopimelic acid (DAP), the predominant whole-cell sugars and the major polar lipids (Hasegawa et al., 1983; Lechevalier and Lechevalier, 1980). Fatty acid methyl ester analysis was performed by GLC according to the instructions for the Microbial Identification System (MIDI) (Sasser, 1990). Menaquinones were extracted and purified according to Tamura et al. (1994) and analyzed by LC-MS.  Preparation of DNA, DNA base composition and DNA DNA hybridization. Genomic DNA was isolated from the strain by following the procedure described

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by Hopwood et al. (1985). The DNA base ratio of this strain was determined using the HPLC method of Tamaoka and Komagata (1984). DNA DNA relatedness was measured fluorometrically using the microplate hybridization method devised by Ezaki et al. (1989).  16S rDNA sequencing analysis. 16S rDNA amplification was performed by PCR using KOD plus (TOYOBO, Japan) and universal bacteria 16S rDNA primers. The forward primer, 9F (5 -GAGTTTGATCCTGGCTC AG-3 ), and reverse primer, 1541R (5 -AAGGAGGTG ATCCAGCC-3 ) were used for the amplification. Amplification was carried out using a DNA thermal cycler with the following program: 94 C for 2 min followed by 25 cycles of denaturation (94 C for 15 s), primer annealing (50 C for 30 s) and primer extension (68 C for 1.3 s) at the end of the cycle, with the reaction mixture kept at 4 C. The PCR products were purified using PCR purification kits (MachereyNagel, Germany). The purified product was directly sequenced using a Big Dye® Terminator V3.1 cycle sequencing kit (Applied Biosystems) and the universal primers 9F (5 -GAGTTT GATCCTGGCTCAG-3 ), 785F (5 -GGATTAGATACCCT GGTAGTC-3 ), 802R (5 -TACCAGGGTATCTAATCC-3 ) and 1541R (5 -AAGGAGGTGATCCAGCC-3 ). Sequence gel electrophoresis was carried out and nucleotide sequences were automatically obtained using an Applied Biosystems DNA sequencer (model 373A) and software provided by the manufacturer.  Analysis of sequence data. The 16S rDNA sequence of the strain was multiply aligned using the BioEdit program (version 7.0.5.3, Hall, 1999) against corresponding nucleotide sequences of members of Saccharopolyspora retrieved from the DDBJ (Thomp-

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son et al., 1997) and GenBank (Benson et al., 1997) databases. The alignment was manually verified and adjusted prior to the construction of a phylogenetic tree using MEGA version 4.1 (Kumar et al., 2008) and neighbor joining (Saitou and Nei, 1987). Tree topologies were evaluated by bootstrap analysis based on 1,000 resamplings (Felsenstein, 1985).  Nucleotide sequence accession number. The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain S582T is HM067865. Results and Discussion

 Strain S582T exhibited a range of phenotypic and chemotaxonomic properties consistent with its classification in the genus Saccharopolyspora (Lacey and Goodfellow, 1975). The phenotypic properties of strain S582T were typical of those of the genus Saccharopolyspora. The organism was an aerobic, Grampositive, non-acid alcohol-fast actinomycete that forms extensively branched substrate mycelia. Aerial hyphae differentiate into short chains containing four to six smooth-surfaced and non-motile spores (Fig. 1). The spores are oval and covered by a sheath. The color of the substrate mycelium is pale yellow. Soluble pigment was not produced (Table 1). The temperature for growth is between 28 and 37 C.  The organism contains meso-DAP as the wall diamino acid and contains arabinose and galactose as the major whole-cell sugars (wall chemotype IV pattern). A phospholipid pattern contains phosphatidylcholine and phosphatidylethanolamine corresponding to phospholipid type III (Lechevalier et al., 1977). Major cellular fatty acids were iso-C15:0 (25.7%), iso-C16:0

Fig. 1. Scanning electron micrograph of strain S582T showing short spore chains of smooth surface spores after 2 weeks growth at 30 C on humic acid vitamin agar.

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Table 1. Cultural characteristics of strain S582T. Culture medium

Growth

Color of aerial mycelium

Color of substrate mycelium

Soluble pigment

Yeast extract-malt extract agar (ISP2) Oatmeal agar (ISP3) Inorganic salt-starch agar (ISP4) Glycerol asparagine agar (ISP5) Peptone-yeast extract-iron agar (ISP6) Tyrosine agar (ISP7)

abundant abundant poor moderate moderate moderate

white white white ― white white

yellow light yellow light yellow very pale yellow pale yellow pale/light yellow

― ― ― ― ― ―

 ―: Not produced. Table 2. Comparison of cellular composition of strain S582T with related species of the genus Saccharopolyspora. Composition Major fatty acid

Phospholipid Menaquinone G+C content (mol%)

S582T

S. endophytica YIM 61095T

S. tripterygii YIM 65359T

S. flava AS 4.1520T

iso-C15:0 (25.7) iso-C16:0 (18.3) anteiso-C17:0 (17.9) anteiso-C15:0 (10.2) PC, PE MK-9(H4) 70.2

anteiso-C17:0 (24.9) iso-C16:0 (18.2) iso-C17:0 (12.2) iso-C15:0 (9.4) PC, PE, PG, PI MK-9(H4) 66.2a

iso-C16:0 (27.5) anteiso-C17:0(17.0) iso-C17:0 (12.5) iso-C15:0 (11.5) PG, PE, PC MK-9(H4) 70.5b

anteiso-C17:0 (27.8) iso-C16:0 (14.1) iso-C17:0 (13.1) iso-C15:0 (10.9) PC, PE MK-9(H4) 67

 a Qin et al., 2008, b Li et al., 2009.  Data for strain S582T, S. endophytica YIM 61095T, S. tripterygii YIM 65359T and S. flava AS 4.1520T are from this study. Percentages of fatty acids are shown in parentheses. PC phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol.

(18.3%), anteiso-C17:0 (17.9%) and anteiso-C15:0 (10.2%). Strain S582T contains a tetrahydrogenated menaquinone with nine isoprene units as the predominant isoprenoid quinine. Comparison of the cellular compositions of strain S582T and other type strains of validly described Saccharopolyspora species is shown in Table 2. This chemical profile distinguishes strain S582T from members of wall-chemotype-IV taxa, apart from those classified in the genus Saccharopolyspora (Zhou et al., 1998). In addition, strain S582T was found to have G+C-rich DNA (70.2 mol%).  Almost complete 16S rDNA sequences were generated for strain S582T (1,495 nucleotides). Comparison of this nucleotide sequence with those of members of genus Saccharopolyspora clearly showed that this strain belongs to the genus Saccharopolyspora (Lacey and Goodfellow, 1975) (Fig. 2). The nucleotide sequence of strain S582T shows substantial differences from the corresponding sequences of its nearest neighbors including S. endophytica YIM 61095T, S. tripterygii YIM 65359T and S. flava AS 4.1520T, and shares nucleotide similarity values of 99.5%, 99.0%

and 97.9%, respectively. The close relationship between strain S582T and the type strain of S. endophytica YIM 61095T (Qin et al., 2008) is supported both by treeing algorithms and by a high bootstrap value (Fig. 2).  Thus, despite the high 16S rDNA gene sequence similarity between strain S582 and S. endophytica YIM 61096T (99.5%), morphological and physiological characteristic of strain S582 indicated that it is not a strain of S. endophytica YIM 61096T. Strain S582T produced short chain aerial hyphae containing 4 6 oval spores covered by a sheath while S. endophytica YIM 61096T produced a long chain of smooth surface spores and lacked a spore-covering sheath. Furthermore, S. endophytica YIM 61096T produced pink diffusible pigment when grown on glycerol-asparagine agar (ISP No. 5) (Qin et al., 2008) while strain S582 did not.  In particular, degradation of xylan, uric acid and utilization of D-lactose, sorbitol, acetate, L-histidine, L-valine and L-arginine were effective for discrimination between S582T and related species. Furthermore, the

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Fig. 2. Neighbor-joining tree (Saitou and Nei, 1987) based on almost complete 16S rDNA sequences, showing relationships between strain S582T and representatives of the genus Saccharopolyspora. Streptomyces inusitatus NBRC 13601T (AB184445) is used as an outgroup.  The numbers at the nodes indicate the levels of bootstrap support, based on a neighbor-joining analysis of 1,000 resample datasets; only values >50% are indicated. Bar, 0.01 substitutions per nucleotide position.

levels of DNA DNA relatedness of strain S582T to S. endophytica YIM 61095T, S. tripterygii YIM 65359T and S. flava AS4.1520T were determined to be 53.3%, 44.8% and 39.3%, respectively. The DNA DNA relatedness values between strain S582T and related species was lower than the 70% recommended threshold value for the delineation of genomic species (Stackebrandt and Goebel, 1994). These data supported the finding that strain S582T represents a separated genomic species. Strain S582T can also be distinguished from type strains of the validly described species of Saccharopolyspora by using a combination of phenotypic properties (Table 3). The chemotaxonomic, molecular systematic and phenotypic data showed that strain S582T should be given species status within the genus Saccharopolyspora. It is proposed, therefore, that strain S582T be classified as Saccharopolyspora pathumthaniensis sp. nov.

Description of Saccharopolyspora pathumthaniensis sp. nov.

 Saccharopolyspora pathumthaniensis sp. nov. (pa. thum.tha.ni en.sis. N. L. fem. adj. pathumthaniensis pertaining to the Pathum Thani Province in Thailand where the type strain was collected).  Aerobic, Gram-positive, non-acid-alcohol-fast, nonmotile actinomycete which forms an extensively branched, pale yellow substrate mycelium that fragments into rod-shaped elements after 3 4 d at 30 C. Aerial hyphae are produced upon prolonged cultivation on ISP medium No. 2. Grows well on ISP medium No. 2 (yeast extract-malt extract agar) and ISP medium No. 3 (oatmeal agar). The aerial mycelium carries abundant chains of 4 6 smooth-surfaced spores. Diffusible pigments are not produced in this strain. Adenine, casein, starch, tyrosine and uric acid are degraded. Chitin and cellulose are not degraded. Nitrate is reduced. Uses L-arabinose, D-galactose, D-lactose, Dmaltose, D-raffinose, L-rhamnose, sucrose, fructose, glucose, mannose, melezitose, mannitol, D-xylose, erythritol and glycerol as sole carbon sources for en-

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Table 3. Biochemical properties of strain S582T with the phylogenetically related species of the genus Saccharopolyspora. Microorganisms Characters Degradationa of

Nitrate reduction NaCl range for growth (%, w/v) 0.1% phenol Maximum growth temperature Utilizationb of carbohydrate

Adenine Casein Chitin Starch Tyrosine Cellulose Xylan Uric acid Gelatin Tween 80

L-Arabinose D-Galactose D-Lactose D-Maltose D-Raffinose L-Rhamnose

Utilizationb of nitrogen

Sucrose Fructose Glucose Inositol Mannose Melezitose Mannitol D-Xylose Erythritol Glycerol Sorbitol Acetate Pyruvate Dextran L-Histidine L-Valine L-Arginine L-Threonine L-Phenylalanine L-Proline L-Serine L-Methionine L-Cysteine KNO3

S582T

S. endophytica YIM 61095T

S. tripterygii YIM 65359T

S. flava AS 4.1520T

+ + ­ + + ­ + + + + +

+ ­ + + + + ­ ­ + + +

+ ­ ­ ­ + ­ ­ ­ + ­ ­

+ ­ ­ + ­ + + ­ + ­ +

0 7

0 15

0 12

0 7

­

­

­

­

28 37

20 45

10 37

28 37

+ + + + + + + + + ­ + + + + + + ­ ­ ­ ­ + + + + + + + + + +

+ + ­ + + + + + + ­ + + + + + + + + ­ ­ ­ ­ ­ + + + + + + +

+ + ­ + ­ ­ ­ + + ­ ­ ­ + + + + ­ + ­ ­ + ­ + + + + + ­ + +

­ + + + + + + + + + + ­ + + + + + + + ­ + + + + + + + + + +

 a Degradation: +, degraded; ­, not degraded. b Growth: +, positive; ­, negative.  Data for strain S582T, S. endophytica YIM 61095T, S. tripterygii YIM 65359T and S. flava AS 4.1520T are from this study.

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ergy and growth but does not use inositol, sorbitol, acetate, pyruvate or dextran. It grows at NaCl concentrations ranging from 0% to 7% (w/v). Growth is inhibited by lysozyme, vancomycin (50 µg/ml), streptomycin sulphate (50 µg/ml), neomycin (50 µg/ml) and gentamicin (50 µg/ml). The temperature for growth is between 25 and 37 C. The G+C content of the DNA is 70.2 mol%. Type strain (S582T =NBRC 104112T =BCC 28624T) was isolated from the gut of Speculitermes sp. collected from Nongsua, Pathum Thani Province, Thailand. Acknowledgments  This research was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). This research was done in cooperation with JSPS Asian Core Program (Yamaguchi University). References Benson, D. A., Boguski, M. S., Lipman, D. J., and Ostell, J. (1997) GenBank. Nucleic Acids Res., 25, 1 6. Duangmal, K., Mingma, R., Thamchaipenet, A., Matsumoto, A., and Takahashi, Y. (2010) Saccharopolyspora phatthalungensis sp. nov., isolated from rhizosphere soil of Hevea brasiliensis. Int. J. Syst. Evol. Microbiol., 60, 1904 1908. Embley, T. M. (1992) The family Pseudonocardiaceae. In the Prokaryotes, Vol. 1, ed. by Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K. H., Springer, Berlin. pp. 996 1027. Embley, T. M., Roston, J., O Donnell, A. G., and Goodfellow, M. (1987) Fatty acid composition in the classification of Saccharopolyspora hirsuta. FEMS Microbiol. Lett., 41, 131 135. Embley, T. M., Smida, J., and Stackebrandt, E. (1988) Reverse transcriptase sequencing of 16S ribosomal RNA from Faenia rectivirgula, Pseudonocardia thermophila and Saccharopolyspora hirsuta, three wall type IV actinomycetes which lack mycolic acids. J. Gen. Microbiol., 134, 961 966. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol., 39, 224 229. Felsentein, J. (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39, 783 791. Goodfellow, M., Lacey, J., Athalye, M., Embley, T. M., and Bowen, T. (1989) Saccharopolyspora gregorii and Saccharopolyspora hordei: Two new actinomycetes species from fodder. J. Gen. Microbiol., 135, 2125 2139.

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