Saccharopolyspora Pathumthaniensis Sp. Nov., a Novel Actinomycetes Isolated from Termite Guts (Speculitermes Sp.)

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 demonstrat- ed 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 meso-diaminopimelic acid, arabinose and galactose in their wall peptidoglycans (wall chemotype IV sensu The genus Saccharopolyspora was proposed by Lechevalier and Lechevalier, 1970). The organisms Lacey and Goodfellow (1975) for actinomycetes iso- have large amounts of phosphatidylglycerol, phos- lated from spontaneously heated sugarcane bagasse. phatidylcholine, phosphatidyl ethanolamine and phos- The genus currently encompasses aerobic, non-acid- phatidylmethylethanolamine (phospholipids pattern III fast organisms which form an extensively branched sensu Lechevalier et al. (1977)), are rich in iso- and substrate mycelium that fragments into rod-shaped, anteiso-branched chain fatty acids, and have a large non motile elements and an aerial mycelium that dif- amount of tetra-hydrogenated menaquinone with nine ferentiates into bead-like chains of spores. Saccha- isoprene units but lack mycolic acids (Embley et al., ropolyspora is also characterized by the presence of 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). * Address reprint requests to: Dr. Shinji Tokuyama, Depart- ment of Applied Biological Chemistry, Faculty of Agriculture, The genus Saccharopolyspora includes the follow- Shizuoka University, Shizuoka 422‒8529, Japan. ing validly described species: Saccharopolyspora Tel/Fax: +81‒54‒238‒4879 erythraea (Labeda, 1987), Saccharopolyspora gregorii E-mail: [email protected] (Goodfellow et al., 1989), Saccharopolyspora hordei 94 SINMA et al. Vol. 57

(Goodfellow et al., 1989), Saccharopolyspora rectivir- aerial hyphae and spore chain morphology, was ob- gula (Korn-Wendisch et al., 1989), Saccharopolyspora served on ISP media No. 2, 3, 4, 5, 6 and 7 (Shirling spinosa (Mertz and Yao, 1990), Saccharopolyspora and Gottlieb, 1966) using the coverslip technique. Ad- taberi (Korn-Wendisch et al., 1989), Saccharopolyspora ditional morphological properties were observed by hirsuta (Lacey and Goodfellow, 1975), Saccharopoly- examining gold-coated, dehydrated specimens of the spora spinosporotrichia (Zhou et al., 1998), Saccha- organism using a scanning electron microscope ropolyspora fl ava and Saccharopolyspora thermophila (Model JSM6060; JEOL, Ltd., Tokyo, Japan). (Lu et al., 2001), Saccharopolyspora antimicrobica Cultural, physiological, and biochemical characteris- (Yuan et al., 2008), Saccharopolyspora cebuensis (Pi- tics. The strain was examined for a range of pheno- mento-Elardo et al., 2008), Saccharopolyspora shan- typic properties following incubation for 14 d at 30°C dongensis (Zhang et al., 2008), Saccharopolyspora on various agar media according to procedures of endophytica (Qin et al., 2008), Saccharopolyspora Lacey and Goodfellow (1975) and Zhou et al. (1998). halophila (Tang et al., 2009), Saccharopolyspora ji- Decomposition of various compounds was examined angxiensis (Zhang et al., 2009), Saccharopolyspora using the basal medium recommended by Gordon et qujiaojingensis (Tang et al., 2009), Saccharopolyspora al. (1974). The temperature for growth and NaCl toler- rosea (Yassin, 2009), Saccharopolyspora tripterygii (Li ance were determined on ISP medium No. 2. Carbon et al., 2009) and Saccharopolyspora patthalungensis utilization was tested by using ISP medium No. 9 sup- (Duangmal et al. 2010). These taxa form a distinct plemented with a fi nal concentration of 1% of tested phyletic branch within the evolutionary radiation en- carbon sources. Nitrogen utilization was examined on compassed by the family Pseudonocardiaceae (Emb- the basal medium containing 10 g of glucose, 0.5 g of ley et al., 1988; Warwick et al., 1994). Members of the MgSO4･7H2O, 0.5 g of NaCl, 0.01 g of FeSO4･7H2O, genus Saccharopolyspora are a potentially rich source 1 g of K2HPO4 and 12 g of agar in (1 L) distilled water of natural products, but only erythromycin, produced (William et al., 1989). Gelatin liquefaction, peptoniza- by Saccharopolyspora erythraea, is currently commer- tion of milk, nitrate reduction, cellulose composition cially important (Embley, 1992). and starch hydrolysis were determined by cultivation Strain S582T was isolated during a search for ligno- on various media described by Hamada (2000). Chitin cellulosic compounds degrading actinomycetes from hydrolysis was detected on colloidal chitin agar (Hsu termite guts and found to produce a xylan-degrading and Lockwood, 1975). Lipid hydrolysis was tested on enzyme. The novel strain was isolated from the gut of Siera’s medium (Siera, 1957) supplemented with a grass-feeding termite, Speculitermes sp., collected Tween 80 (1% w/v). The organism was also examined from an orangery in Pathum Thani Province, Thailand. for its ability to grow at 4‒45°C. Strain S582T was found to have morphological proper- Chemotaxonomy. Biomass for chemotaxonomic ties consistent with its assignment to the genus Sac- and molecular systematic studies was prepared by charopolyspora. In the present investigation, this or- growing the strain in Tryptic Soy Broth (TSB) at 30°C ganism was examined for an array of genotypic and for 5 d. At maximum growth, the broth culture was phenotypic properties. This isolate represents a novel checked for purity, harvested by centrifugation, washed species of Saccharopolyspora, for which we propose three times with distilled water, and freeze-dried. Es- the name Saccharopolyspora pathumthaniensis. tablished procedures were used to determine the di- agnostic isomers of diaminopimelic acid (DAP), the Materials and Methods predominant whole-cell sugars and the major polar lipids (Hasegawa et al., 1983; Lechevalier and Leche- Isolation. Strain S582T was isolated from the gut of valier, 1980). Fatty acid methyl ester analysis was per- a Speculitermes sp. grass-feeding termite on a humic formed by GLC according to the instructions for the acid vitamin agar (Hayakawa and Nonomura, 1987). Microbial Identifi cation System (MIDI) (Sasser, 1990). The termite sample was collected from an orangery in Menaquinones were extracted and purifi ed according Pathum Thani Province, Thailand. The strain was main- to Tamura et al. (1994) and analyzed by LC-MS. tained on ISP medium No. 2 (Shirling and Gottlieb, Preparation of DNA, DNA base composition and 1966). DNA‒DNA hybridization. Genomic DNA was isolated Morphology. The characteristic of hyphae, notably from the strain by following the procedure described 2011 Novel Saccharopolyspora from termite guts 95 by Hopwood et al. (1985). The DNA base ratio of this son et al., 1997) and GenBank (Benson et al., 1997) strain was determined using the HPLC method of databases. The alignment was manually verifi ed and Tamaoka and Komagata (1984). DNA‒DNA related- adjusted prior to the construction of a phylogenetic ness was measured fl uorometrically using the mi- tree using MEGA version 4.1 (Kumar et al., 2008) and croplate hybridization method devised by Ezaki et al. neighbor joining (Saitou and Nei, 1987). Tree topolo- (1989). gies were evaluated by bootstrap analysis based on 16S rDNA sequencing analysis. 16S rDNA amplifi - 1,000 resamplings (Felsenstein, 1985). cation was performed by PCR using KOD plus (TOYO- Nucleotide sequence accession number. The BO, Japan) and universal bacteria 16S rDNA primers. GenBank/EMBL/DDBJ accession number for the 16S The forward primer, 9F (5′-GAGTTTGATCCTGGCTC rDNA sequence of strain S582T is HM067865. AG-3′), and reverse primer, 1541R (5′-AAGGAGGTG ATCCAGCC-3′) were used for the amplifi cation. Ampli- Results and Discussion fi cation was carried out using a DNA thermal cycler with the following program: 94°C for 2 min followed by Strain S582T exhibited a range of phenotypic and 25 cycles of denaturation (94°C for 15 s), primer an- chemotaxonomic properties consistent with its classi- nealing (50°C for 30 s) and primer extension (68°C for fi cation in the genus Saccharopolyspora (Lacey and 1.3 s) at the end of the cycle, with the reaction mixture Goodfellow, 1975). The phenotypic properties of strain kept at 4°C. The PCR products were purifi ed using S582T were typical of those of the genus Saccha- PCR purifi cation kits (MachereyNagel, Germany). The ropolyspora. The organism was an aerobic, Gram- purifi ed product was directly sequenced using a Big positive, non-acid alcohol-fast actinomycete that forms Dye® Terminator V3.1 cycle sequencing kit (Applied extensively branched substrate mycelia. Aerial hyphae Biosystems) and the universal primers 9F (5′-GAGTTT differentiate into short chains containing four to six GATCCTGGCTCAG-3′), 785F (5′-GGATTAGATACCCT smooth-surfaced and non-motile spores (Fig. 1). The GGTAGTC-3′), 802R (5′-TACCAGGGTATCTAATCC-3′) spores are oval and covered by a sheath. The color of and 1541R (5′-AAGGAGGTGATCCAGCC-3′). Se- the substrate mycelium is pale yellow. Soluble pig- quence gel electrophoresis was carried out and nucle- ment was not produced (Table 1). The temperature for otide sequences were automatically obtained using an growth is between 28 and 37°C. Applied Biosystems DNA sequencer (model 373A) The organism contains meso-DAP as the wall diami- and software provided by the manufacturer. no acid and contains arabinose and galactose as the Analysis of sequence data. The 16S rDNA se- major whole-cell sugars (wall chemotype IV pattern). A quence of the strain was multiply aligned using the phospholipid pattern contains phosphatidylcholine BioEdit program (version 7.0.5.3, Hall, 1999) against and phosphatidylethanolamine corresponding to corresponding nucleotide sequences of members of phospholipid type III (Lechevalier et al., 1977). Major

Saccharopolyspora retrieved from the DDBJ (Thomp- 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. 96 SINMA et al. Vol. 57

Table 1. Cultural characteristics of strain S582T.

Color of aerial Color of substrate Soluble Culture medium Growth mycelium mycelium pigment

Yeast extract-malt extract agar (ISP2) abundant white yellow ― Oatmeal agar (ISP3) abundant white light yellow ― Inorganic salt-starch agar (ISP4) poor white light yellow ― Glycerol asparagine agar (ISP5) moderate ― very pale yellow ― Peptone-yeast extract-iron agar (ISP6) moderate white pale yellow ― Tyrosine agar (ISP7) moderate white pale/light yellow ―

―: Not produced.

Table 2. Comparison of cellular composition of strain S582T with related species of the genus Saccharopolyspora.

S. endophytica S. tripterygii S. ﬂ ava Composition S582T YIM 61095T YIM 65359T AS 4.1520T

Major fatty acid iso-C15:0 (25.7) anteiso-C17:0 (24.9) iso-C16:0 (27.5) anteiso-C17:0 (27.8) iso-C16:0 (18.3) iso-C16:0 (18.2) anteiso-C17:0(17.0) iso-C16:0 (14.1) anteiso-C17:0 (17.9) iso-C17:0 (12.2) iso-C17:0 (12.5) iso-C17:0 (13.1) anteiso-C15:0 (10.2) iso-C15:0 (9.4) iso-C15:0 (11.5) iso-C15:0 (10.9) Phospholipid PC, PE PC, PE, PG, PI PG, PE, PC PC, PE Menaquinone MK-9(H4) MK-9(H4) MK-9(H4) MK-9(H4) G+C content (mol%) 70.2 66.2a 70.5b 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. ﬂ ava AS 4.1520T are from this study. Percent- ages 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 and 97.9%, respectively. The close relationship be- (10.2%). Strain S582T contains a tetrahydrogenated tween strain S582T and the type strain of S. endophyti- menaquinone with nine isoprene units as the predomi- ca YIM 61095T (Qin et al., 2008) is supported both by nant isoprenoid quinine. Comparison of the cellular treeing algorithms and by a high bootstrap value (Fig. compositions of strain S582T and other type strains of 2). validly described Saccharopolyspora species is shown Thus, despite the high 16S rDNA gene sequence in Table 2. This chemical profi le distinguishes strain similarity between strain S582 and S. endophytica YIM S582T from members of wall-chemotype-IV taxa, apart 61096T (99.5%), morphological and physiological from those classifi ed in the genus Saccharopolyspora characteristic of strain S582 indicated that it is not a (Zhou et al., 1998). In addition, strain S582T was found strain of S. endophytica YIM 61096T. Strain S582T pro- to have G+C-rich DNA (70.2 mol%). duced short chain aerial hyphae containing 4‒6 oval Almost complete 16S rDNA sequences were gener- spores covered by a sheath while S. endophytica YIM ated for strain S582T (1,495 nucleotides). Comparison 61096T produced a long chain of smooth surface of this nucleotide sequence with those of members of spores and lacked a spore-covering sheath. Further- genus Saccharopolyspora clearly showed that this more, S. endophytica YIM 61096T produced pink dif- strain belongs to the genus Saccharopolyspora (Lacey fusible pigment when grown on glycerol-asparagine and Goodfellow, 1975) (Fig. 2). The nucleotide se- agar (ISP No. 5) (Qin et al., 2008) while strain S582 did quence of strain S582T shows substantial differences not. from the corresponding sequences of its nearest In particular, degradation of xylan, uric acid and uti- neighbors including S. endophytica YIM 61095T, S. lization of D-lactose, sorbitol, acetate, L-histidine, L-val- tripterygii YIM 65359T and S. fl ava AS 4.1520T, and ine and L-arginine were effective for discrimination be- shares nucleotide similarity values of 99.5%, 99.0% tween S582T and related species. Furthermore, the 2011 Novel Saccharopolyspora from termite guts 97

Fig. 2. Neighbor-joining tree (Saitou and Nei, 1987) based on almost complete 16S rDNA sequences, showing relationships between strain S582T and representa- tives 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. Description of Saccharopolyspora pathumthaniensis endophytica YIM 61095T, S. tripterygii YIM 65359T and sp. nov. S. fl ava AS4.1520T were determined to be 53.3%, Saccharopolyspora pathumthaniensis sp. nov. (pa. 44.8% and 39.3%, respectively. The DNA‒DNA relat- thum.tha.ni’en.sis. N. L. fem. adj. pathumthaniensis edness values between strain S582T and related spe- pertaining to the Pathum Thani Province in Thailand cies was lower than the 70% recommended threshold where the type strain was collected). value for the delineation of genomic species (Stacke- Aerobic, Gram-positive, non-acid-alcohol-fast, non- brandt and Goebel, 1994). These data supported the motile actinomycete which forms an extensively fi nding that strain S582T represents a separated ge- branched, pale yellow substrate mycelium that frag- nomic species. Strain S582T can also be distinguished ments into rod-shaped elements after 3‒4 d at 30°C. from type strains of the validly described species of Aerial hyphae are produced upon prolonged cultiva- Saccharopolyspora by using a combination of pheno- tion on ISP medium No. 2. Grows well on ISP medium typic properties (Table 3). The chemotaxonomic, mo- No. 2 (yeast extract-malt extract agar) and ISP medium lecular systematic and phenotypic data showed that No. 3 (oatmeal agar). The aerial mycelium carries strain S582T should be given species status within the abundant chains of 4‒6 smooth-surfaced spores. Dif- genus Saccharopolyspora. It is proposed, therefore, fusible pigments are not produced in this strain. Ade- that strain S582T be classifi ed as Saccharopolyspora nine, casein, starch, tyrosine and uric acid are degrad- pathumthaniensis sp. nov. ed. Chitin and cellulose are not degraded. Nitrate is reduced. Uses L-arabinose, D-galactose, D-lactose, D- maltose, D-raffi nose, L-rhamnose, sucrose, fructose, glucose, mannose, melezitose, mannitol, D-xylose, erythritol and glycerol as sole carbon sources for en- 98 SINMA et al. Vol. 57

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

Microorganisms Characters S. endophytica S. tripterygii S. ﬂ ava S582T YIM 61095T YIM 65359T AS 4.1520T

Degradationa of Adenine + + + + Casein + --- Chitin - + -- Starch + + - + Tyrosine + + + - Cellulose - + - + Xylan + --+ Uric acid + --- Gelatin + + + + Tween 80 + + -- Nitrate reduction + + - + NaCl range for 0‒70‒15 0‒12 0‒7 growth (%, w/v) 0.1% phenol ---- Maximum growth 28‒37 20‒45 10‒37 28‒37 temperature b Utilization of L-Arabinose + + + - carbohydrate D-Galactose + + + + D-Lactose + --+ D-Maltose + + + + D-Rafﬁ nose + + - + L-Rhamnose + + - + Sucrose + + - + Fructose + + + + Glucose + + + + Inositol ---+ Mannose + + - + Melezitose + + -- Mannitol + + + + D-Xylose + + + + Erythritol + + + + Glycerol + + + + Sorbitol - + - + Acetate - +++ Pyruvate ---+ Dextran ---- b Utilization of L-Histidine + - ++ nitrogen L-Valine + --+ L-Arginine + - ++ L-Threonine + + + + L-Phenylalanine + + + + L-Proline + + + + L-Serine + + + + L-Methionine + + - + L-Cysteine + + + + KNO3 ++++

a Degradation: +, degraded; -, not degraded. b Growth: +, positive; -, negative. Data for strain S582T, S. endophytica YIM 61095T, S. tripterygii YIM 65359T and S. fl ava AS 4.1520T are from this study. 2011 Novel Saccharopolyspora from termite guts 99 ergy and growth but does not use inositol, sorbitol, Gordon, R. E., Barnett, D. A., Handerhan, J. E., and Pang, C. H. acetate, pyruvate or dextran. It grows at NaCl concen- N. (1974) Nocardia coeliaca, Nocardia autotrophica, and trations ranging from 0% to 7% (w/v). Growth is inhib- the nocardia strain. Int. J. Syst. Bacteriol., 24, 54‒63. Hall, T. A. (1999) BioEdit: A user-friendly biological sequence ited by lysozyme, vancomycin (50 µg/ml), streptomy- alignment editor and analysis. http://www.mbio.ncsu.edu/ cin sulphate (50 µg/ml), neomycin (50 µg/ml) and BioEdit/bioedit.html µ gentamicin (50 g/ml). The temperature for growth is Hamada, M. (2000) Morphological, physiological and biological between 25 and 37°C. The G+C content of the DNA is character of Actinomycetes. In Identifi cation Manual of Ac- 70.2 mol%. Type strain (S582T =NBRC 104112T =BCC tinomycetes. ed. by The Society for Actinomycetes Japan, 28624T) was isolated from the gut of Speculitermes sp. pp. 37‒47 (In Japanese). collected from Nongsua, Pathum Thani Province, Thai- Hasegawa, T., Takizawa, M., and Tanida, S. (1983) A rapid anal- land. ysis for chemical grouping of aerobic actinomycetes. J. Gen. Appl. Microbiol., 29, 319‒322. Hayakawa, M. and Nonomura, H. (1987) Humic acid‒vitamin Acknowledgments agar, a new medium for the selective isolation of soil actinomycetes. J. Ferment. Technol., 65: 501‒509. This research was supported in part by Grants-in-Aid for Sci- Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. entifi c Research from the Ministry of Education, Culture, Sports, J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Science and Technology of Japan (MEXT). This research was Schrempf, H. (1985) Preparation of chromosomal, plasmid done in cooperation with JSPS Asian Core Program (Yama- and phage DNA. In Genetic Manipulation of Streptomyces. guchi University). A Laboratory Manual, John Innes Foundation, Norwich, pp. 70‒74. References Hsu, S. C. and Lockwood, J. L. (1975) Powdered chitin as a selective medium for enumeration of actinomycetes in wa- Benson, D. A., Boguski, M. S., Lipman, D. J., and Ostell, J. ter and soil. Appl. Microbiol., 29, 422‒426. (1997) GenBank. Nucleic Acids Res., 25, 1‒6. Korn-Wendisch, F., Kempf, A., Grund, E., Kroppenstedt, R. M., Duangmal, K., Mingma, R., Thamchaipenet, A., Matsumoto, A., and Kutzner, H. J. (1989) Transfer of Faenia rectivirgula Ku- and Takahashi, Y. (2010) Saccharopolyspora phatthalung- rup and Agre 1983 to the genus Saccharopolyspora Lacey ensis sp. nov., isolated from rhizosphere soil of Hevea and Goodfellow 1975, elevation of Saccharopolyspora hir- brasiliensis. Int. J. Syst. Evol. Microbiol., 60, 1904‒1908. sute subsp. taberi Labeda 1987 to species level, and Embley, T. M. (1992) The family Pseudonocardiaceae. In the emended description of the genus Saccharopolyspora. Int. Prokaryotes, Vol. 1, ed. by Balows, A., Trüper, H. G., Dwor- J. Syst. Bacteriol., 39, 430‒441. kin, M., Harder, W., and Schleifer, K. H., Springer, Berlin. Kumar, S., Dudley, J., Nei, M., and Tamura, K. (2008) MEGA: A pp. 996‒1027. biologist-centric software for evolutionary analysis of DNA Embley, T. M., Roston, J., O’Donnell, A. G., and Goodfellow, M. and protein sequences. Brief. Bioinform., 9, 299‒306. (1987) Fatty acid composition in the classifi cation of Sac- Labeda, D. P. (1987) Transfer of the type strain of Streptomyces charopolyspora hirsuta. FEMS Microbiol. Lett., 41, 131‒ erythraeus (Waksman, 1923) Waksman and Henrici 1948 135. to the genus Saccharopolyspora Lacey and Goodfellow Embley, T. M., Smida, J., and Stackebrandt, E. (1988) Reverse 1975 as Saccharopolyspora erythraea sp. nov., and desig- transcriptase sequencing of 16S ribosomal RNA from Fae- nation of the neotype strain for Streptomyces erythraeus. nia rectivirgula, Pseudonocardia thermophila and Saccha- Int. J. Syst. Bacteriol., 37, 19‒22. ropolyspora hirsuta, three wall type IV actinomycetes which Lacey, J. and Goodfellow, M. (1975) A novel actinomycetes lack mycolic acids. J. Gen. Microbiol., 134, 961‒966. from sugar cane bagasse: Saccharopolyspora hirsuta gen. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric et sp. nov. J. Gen. Microbiol., 88, 75‒85. deoxyribonucleic acid‒deoxyribonucleic acid hybridization Lechevalier, M. P., De Bievre, C., and Lechevalier, H. A. (1977) in microdilution wells as an alternative to membrane fi lter Chemotaxonomy of aerobic actinomycetes: Phospholipids hybridization in which radioisotopes are used to determine composition. Biochem. Syst. Ecol., 5, 249‒260. genetic relatedness among bacterial strains. Int. J. Syst. Lechevalier, M. P. and Lechevalier, H. A. (1970) Chemical com- Bacteriol., 39, 224‒229. position as a criterion in the classifi cation of aerobic actino- Felsentein, J. (1985) Confi dence limits on phylogenies: An ap- mycetes. Int. J. Syst. Bacteriol., 20, 435‒443. proach using the bootstrap. Evolution, 39, 783‒791. Lechevalier, M. P. and Lechevalier, H. A. (1980) The chemotax- Goodfellow, M., Lacey, J., Athalye, M., Embley, T. M., and Bow- onomy of actinomycetes. In Actinomycete Taxonomy (So- en, T. (1989) Saccharopolyspora gregorii and Saccha- ciety for Industrial Microbiology Special Publication Num- ropolyspora hordei: Two new actinomycetes species from ber 6), ed. by Dietz, A. and Thayer, D. W., SIM, Arlington, fodder. J. Gen. Microbiol., 135, 2125‒2139. 100 SINMA et al. Vol. 57

pp. 277‒284. nov. and Couchioplanes caeruleus subsp. Azureus subsp. Li, J., Zhao, G. Z., Qin, S., Huang, H. Y., Zhu, W. Y., Xu, L. H., and nov. Int. J. Syst. Bacteriol., 44, 193‒203. Li, W. J. (2009) Saccharopolyspora tripterygii sp. nov., an Tang, S. K., Wang, Y., Cai, M., Zhi, X. Y., Lou, K., Xu, L. H., Jiang, endophytic actinomycete isolated from the stem of Trip- C. L., and Li, W. J. ( 2009) Saccharopolyspora halophila sp. terygium hypoglaucum. Int. J. Syst. Evol. Microbiol., 59, nov., a novel halophilic actinomycete isolated from a saline 3040‒3044. lake in China. Int. J. Syst. Evol. Microbiol., 59, 555‒558. Lu, Z., Liu, Z., Wang, L., Zhang, Y., Qi, W., and Goodfellow, M. Tang, S. K., Wang, Y., Wu, J. Y., Cao, L. L., Lou, K., Xu, L. H., (2001) Saccharopolyspora fl ava sp. nov. and Saccharopoly- Jiang, C. L., and Li, W. J. (2009) Saccharopolyspora qiji- spora thermophila sp. nov., novel actinomycetes from soil. aojingensis sp. nov., a halophilic actinomycete isolated Int. J. Syst. Evol. Microbiol., 51, 319‒325. from a salt lake. Int. J. Syst. Evol. Microbiol., 59, 2166‒ Mertz, F. P. and Yao, R. C. (1990) Saccharopolyspora spinosa 2170. sp. nov. isolated from soil collected in a sugar mill rum still. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Int. J. Syst. Bacteriol., 40, 34‒39. and Higgins, D. G. (1997) The CLUSTAL X windows inter- Pimentel-Elardo, S. M., Tiro, L. P., Grozdanov, L., and Hentschel, face: Flexible strategies for multiple sequence alignment U. (2008) Saccharopolyspora cebuensis sp. nov., a novel aided by quality analysis tools. Nucleic Acids Res., 25, actinomycete isolated from a Philippine sponge (Porifera). 4876‒4882. Int. J. Syst. Evol. Microbiol., 58, 628‒632. Warwick, S. T., Bowen, T., McVeigh, H., and Embley, T. M. (1994) Qin, S., Li, J., Zhao, G. Z., Chen, H. H., Xu, L. H., and Li, W. J. A phylogenetic analysis of the family Pseudonocardiaceae (2008) Saccharopolyspora endophytica sp. nov., an endo- and the genera Actinokineospora and Saccharothrix with phytic actinomycete isolated from the root of Maytenus 16S rRNA sequences and proposal to combine the genera austroyunnanensis. Syst. Appl. Microbiol., 31, 352‒357. Amycolata and Pseudonocardia in the emended genus Saitou, N. and Nei, M. (1987) The neighbor-joining method: A Pseudonocardia. Int. J. Syst. Bacteriol., 44, 293‒ new method for constructing phylogenetic trees. Mol. Biol. 299. Evol., 4, 406‒425. Williams, S. T., Sharpe, M. E., Holt, J. G., Murray, G. E., Brener, Sasser, M. (1990) Identifi cation of Bacteria by Gas Chromatog- D. J., Krieg, N. R., Mouldar, J. W., Pfennig, N. P., Sneath, H. raphy of Cellular Fatty Acids (MIDI Technical Note 101), A., and Staley, J. T. (1989) Bergey’s Manual of Systematic MIDI, Newark, DE. Bacteriology, Vol. 4, Williams & Wilkins, Baltimore. Shirling, E. B. and Gottlieb, D. (1966) Methods for characteriza- Yassin, A. F. (2009) Saccharopolyspora rosea sp. nov., isolated tion of Streptomyces species. Int. J. Syst. Bacteriol., 16, from a patient with bronchial carcinoma. Int. J. Syst. Evol. 313‒340. Microbiol., 59, 1148‒1152. Sierra, G. (1957) A simple method for the detection of lipolytic Yuan, L. J., Zhang, Y. Q., Guan, Y., Wei, Y. Z., Li, Q. P., Yu, L. Y., activity of microorganisms and some observation on the Li, W. J., and Zhang, Y. Q. (2008) Saccharopolyspora anti- infl uence of the contact between cells and fatty substrate. microbica sp. nov., an actinomycete from soil. Int. J. Syst. Antonie van Leeuwenhoek, 23, 15‒22. Evol. Microbiol., 58, 1180‒1185. Stackebrandt, E. and Goebel, B. M. (1994) Taxonomic note: A Zhang, J., Wu, D., and Liu, Z. (2009) Saccharopolyspora jiangx- place for DNA‒DNA reassociation and 16S rRNA sequence iensis sp. nov., isolated from grass-fi eld soil. Int. J. Syst. analysis in the present species defi nition in bacteriology. Evol. Microbiol., 59, 1076‒1081. Int. J. Syst. Bacteriol., 44, 846‒849. Zhang, J., Wu, D., Zhang, J., Liu, Z., and Song, F. (2008) Sac- Tamaoka, J. and Komagata, K. (1984) Determination of DNA charopolyspora shandongensis sp. nov., isolated from base composition by reversed-phase high-performance wheat-fi eld soil. Int. J. Syst. Evol. Microbiol., 58, 1094‒ liquid chromatography. FEMS Microbiol. Lett., 25, 125‒ 1099. 128. Zhou, Z. H., Liu, Z. H., Qian, Y. D., Kim, S. B., and Goodfellow, Tamura, T., Nakagaito, Y., Nishii, T., Hasegawa, T., Stackebrandt, M. (1998) Saccharopolyspora spinosporotrichia sp. nov., a E., and Yokota, A. (1994) A new genus of the order Actino- novel actinomycete from soil. Int. J. Syst. Bacteriol., 48, mycetales, Couchioplanes gen nov., with descriptions of 53‒58. Couchioplanes caeruleus (Horan and Brodsky 1986) comb.