International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1315–1323 Printed in Great Britain Saccharothrix violacea sp. nov., isolated from a gold mine cave, and Saccharothrix albidocapillata comb. nov. Soon Dong Lee,1 Eun Suk Kim,1 Jung-Hye Roe,1 Jae-heon Kim,2 Sa-Ouk Kang1 and Yung Chil Hah1 Author for correspondence: Yung Chil Hah. Tel: j82 2 880 6700. Fax: j82 2 888 4911. e-mail: hahyungc!snu.ac.kr 1 Department of The generic position of two isolates from soils inside a gold mine cave in Microbiology, College of Kongju, Korea, was determined by 16S rDNA sequencing and chemotaxonomic Natural Sciences and Research Center for characteristics. Phylogenetic analysis indicated that both of the isolates Molecular Microbiology, formed a clade with Lentzea albidocapillata and members of the genus Seoul National University, Saccharothrix of the family Pseudonocardiaceae. The chemical composition of Seoul 151-742, Republic of Korea the isolates and of Lentzea albidocapillata was consistent with that of the genus Saccharothrix, which is characterized by a type III cell wall (the meso- 2 Department of Microbiology, College of isomer of diaminopimelic acid, and galactose and rhamnose as characteristic Natural Sciences, whole-cell sugars), MK-9(H4) as the major menaquinone, and a phospholipid Dan Kook University, type PII pattern (phosphatidylethanolamine as a diagnostic phospholipid). The Cheon An 330-180, Republic of Korea combination of morphological features, chemotaxonomic characters and phylogenetic data supported the proposal that Lentzea albidocapillata, the only and type strain of the genus Lentzea, should be transferred to the genus Saccharothrix. On the basis of physiological properties, cellular fatty acid composition and DNA–DNA hybridization data, two new species within the genus Saccharothrix are proposed: Saccharothrix violacea sp. nov., type strain LM 036T (lIMSNU 50388T), and Saccharothrix albidocapillata comb. nov., type strain DSM 44073T (lIMSNU 21253T). Keywords: Saccharothrix violacea sp. nov., Saccharothrix albidocapillata comb. nov., soil bacteria, phylogeny INTRODUCTION traliensis, Saccharothrix coeruleofusca, Saccharothrix coeruleoviolacea, Saccharothrix cryophilis, Saccharo- The genus Saccharothrix was described by Labeda et thrix espanaensis, Saccharothrix flava, Saccharothrix al. (1984) for amycolate actinomycetes that are charac- longispora, Saccharothrix mutabilis, Saccharothrix terized by fragmentation of both the vegetative and syringae, Saccharothrix texasensis and Saccharothrix aerial mycelia into ovoid elements and the following waywayandensis (Labeda & Lechevalier, 1989; Grund cell chemistry: a type III cell wall (the meso-isomer of & Kroppenstedt, 1989). diaminopimelic acid, and rhamnose and galactose as whole-cell sugars); absence of mycolic acids; a major Yassin et al. (1995) proposed Lentzea albidocapillata menaquinone of MK-9(H%); and a phospholipid type gen. nov., sp. nov. for a mesophilic actinomycete PII (phosphatidylethanolamine) or PIV (phosphatidyl- which forms a well-developed, fragmenting aerial ethanolamine and glucosamine-containing phos- mycelium and a branched substrate mycelium. The pholipids) pattern (Labeda & Lechevalier, 1989). genus Lentzea has a type III cell wall (meso-isomer of The genus currently contains 12 described species: diaminopimelic acid and no characteristic sugar), Saccharothrix aerocolonigenes, Saccharothrix aus- phosphatidylethanolamine as a diagnostic phospho- lipid (the phospholipid type PII pattern) and a non- ................................................................................................................................................. hydrogenated menaquinone with nine isoprene units Abbreviation: ISP, International Streptomyces Project. (MK-9) as a major menaquinone. This genus currently The EMBL accession numbers for the 16S rDNA nucleotide sequences of contains the only and type species, Lentzea albido- isolates LM 036T and LM 044 are AJ242633 and AJ242634, respectively. capillata, which was isolated from a tissue specimen 01329 # 2000 IUMS 1315 S. D. Lee and others taken from a patient suffering from peritoneal car- extract\iron agar (ISP medium 6) and tyrosine agar (ISP cinomatosis. medium 7). For scanning electron microscopy, agar blocks on which growth had occurred were fixed in 1% osmium The family Pseudonocardiaceae Embley et al. 1988 was tetroxide, dehydrated with an ethanol series and then recently emended by Stackebrandt et al. (1997), based critical-point-dried under liquid CO#. Each specimen was on the 16S-rDNA-sequence-based phylogenetic clus- sputter-coated with gold and observed with a scanning tering and the presence of family-specific signature electron microscope (Stereoscan 260; Cambridge Instru- nucleotides. According to cell wall chemotype, the 13 ments). genera of the family Pseudonocardiaceae are divided Physiological characteristics. Utilization of carbohydrates into two groups as follows: the type IV cell wall genera and of organic acids as carbon sources was determined as comprise Actinokineospora, Actinopolyspora, Amy- described by Pridham & Gottlieb (1948) and Gordon et al. colatopsis, Kibdelosporangium, Pseudonocardia, Sac- (1974), respectively. Acid production from carbohydrates charomonospora and Saccharopolyspora (Warwick was determined by a colour change in Bacto OF basal medium (Difco) supplemented with the substrates at a final et al., 1994), whereas the type III cell wall genera concentration of 1% (w\v). Decomposition of adenine, comprise Actinosynnema (Hasegawa et al., 1978), guanine, hypoxanthine, tyrosine and xanthine were de- Kutzneria (Stackebrandt et al., 1994), Lentzea (Yassin termined as described by Gordon et al. (1974), and arbutin et al., 1995), Saccharothrix (Labeda et al., 1984), and aesculin decomposition and casein hydrolysis were Streptoalloteichus (Tomita et al., 1987) and Thermo- determined as described by Williams et al. (1983). De- crispum (Korn-Wendisch et al., 1995). The type III cell composition of DNA was determined using Bacto DNase wall genera and the genus Actinokineospora, a member test agar (Difco). Nitrate reduction, hydrogen sulfide of the type IV cell wall genera, form a coherent cluster production, gelatin liquefaction, starch hydrolysis and within the radiation of the family Pseudonocardiaceae hippurate hydrolysis were studied as described by Mac (Lee et al., 2000b; Yassin et al., 1995). Faddin (1980). Urease activity was determined by a colour change from red to pink in Bacto urea broth (Difco). During the taxonomic study of soil actinomycetes, we Catalase activity was checked with 3% hydrogen peroxide. obtained two isolates from soils collected inside a gold Oxidase production was tested by examining the oxidation mine cave in Kongju, Republic of Korea. Both of the of 1% tetramethyl-p-phenylenediamine. NaCl tolerance isolates formed well-developed aerial mycelia that studies were performed on nutrient agar containing NaCl at final concentrations of 0, 2, 4, 7 and 10% (w\v). Thallium fragmented into rod-shaped elements. The compara- acetate tolerance was determined on nutrient agar at a final tive analysis of 16S rDNA sequences supported the concentration of 0n01% (w\v). To determine sensitivity to conclusion that our isolates were related to members lysozyme, a 0n1% (w\v) solution of lysozyme was filter- of the genera Lentzea and Saccharothrix. In this paper sterilized and added to nutrient agar at a final concentration we describe the characterization and classification of of 0n01%. Growth was tested at temperatures ranging from the isolates, and we propose the new taxa Saccharo- 10 to 42 mC. thrix violacea sp. nov. and Saccharothrix albidocapil- Growth in the presence of various antibiotics was tested T lata comb. nov. The isolates LM 036 and LM 044 using the standard disk diffusion technique (National Com- have been deposited in the Culture Collection Center mittee for Clinical Laboratory Standards, 1994). Disks of the Institute of Microbiology, Seoul National Uni- containing the different antibiotics (Susceptibility test versity (IMSNU) under the numbers IMSNU 50388T discs; Difco) were applied to Mueller–Hinton agar (Difco) and IMSNU 50393, respectively. plates which had been inoculated with 100 µl mycelial suspension in 20% glycerol with a Dispense-O-Disc dis- penser (Difco). The inverted plates were incubated for 7 d at METHODS 30 mC. T To determine the production of antibiotic substances, the Micro-organisms and culture conditions. Strains LM 036 ability of the isolates to inhibit the growth of reference and LM 044 were isolated from soils collected inside a gold organisms was examined using an overlay technique mine cave in Kongju, Korea, on tap water agar and (Williams et al., 1983). Reference organisms selected were oligotrophic medium (M5) by using the dilution plating Bacillus subtilis IMSNU 10011, Micrococcus luteus IMSNU method. The M5 medium contained (per litre of tap water) 20371, Streptomyces murinus IMSNU 20248T, Staphyl- glucose, 0n1g; K#HPO%,0n5g; Na#HPO%,0n7 g; KNO$, ococcus aureus subsp. aureus IMSNU 11089, Escherichia coli 0n1 g; NaCl, 0n3 g; MgSO%;7H#O, 0n1 g; CaCl#;2H#O, IMSNU 10080, Enterobacter aerogenes IMSNU 10256, 0n02 g; FeSO%;7H#O, 200 mg; ZnSO%;7H#O, 180 µg; Saccharomyces cervisiae IMSNU 30102, Aspergillus niger MnSO%;4H#O, 20 µg; CuSO%;5H#O, 90 µg; CoSO%;7H#O, IMSNU 31067 and Candida albicans IMSNU 30018. Spot- 10 µg;H$BO$, 200 µg; and (NH%)'Mo(O#%;4H#O, 5 µg. For inoculated cells of the isolates grown on nutrient agar