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

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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 en- dophytica 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 dis- tinguished from the phylogenetically closest relatives. Based on these genotypic and pheno- typic 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 compo- sitions 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)
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