Sphaerosporangium Gen. Nov., a New Member of the Family Streptosporangiaceae, with Descriptions of Three New Species As Sphaerosporangium Melleum Sp

Sphaerosporangium gen. nov., a new member of the family Streptosporangiaceae, with descriptions of three new species as Sphaerosporangium melleum sp. nov., Sphaerosporangium rubeum sp. nov. and Sphaerosporangium cinnabarinum sp. nov., and transfer of Streptosporangium viridialbum Nonomura and Ohara 1960 to Sphaerosporangium viridialbum comb. nov.

Ismet Ara1;2 and Takuji Kudo1 1Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 2Present address: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan (Received Sep. 12, 2006 / Accepted Dec. 20, 2006 / Published May 18, 2007)

The taxonomic status of five actinomycete strains, 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D-72(35)T, isolated from sandy soil was studied using the polyphasic approach. All isolates produced branching substrate mycelia and developed spherical spore vesicles on aerial hyphae containing non-motile spores. They contained meso-diaminopimelic acid and the N-acetyl type of peptidoglycan. The predominant menaquinones were MK-9(H4) and MK-9(H6). Madurose, mannose, ribose, galactose and glucose were Advancedetected in the whole-cell hydrolysate. The diagnostic phospholipids were phosphatidylethanolamine and ninhydrin-positive phosphoglycolipids, and iso-C16:0 and 10 methyl C17:0 were detected as the major cellular fatty acids. These morphological and chemotaxonomic data were related to those of the genus Streptosporangium in the family Streptosporangiaceae. Phylogenetic analysis based on 16S rDNA sequence data suggested that the strains belong to the family Streptosporangiaceae, but not to any known genus, and form a monophyletic clade with Streptosporangium viridialbum and ‘‘Streptosporangium cinnabarinum’’. The signature nucleotides of the members of thisView clade are different from those of any known genera of the family Streptosporangiaceae. On the basis of phylogenetic analysis and the characteristic patterns of signature nucleotides as well as the morphological and chemotaxonomic data, the genus Sphaerosporangium gen. nov. is proposed for our five isolates and the type strains of Streptosporangium viridialbum and ‘‘Streptosporangium cinnabarinum’’. DNA–DNA hybridization and phenotypic characterization indicate that ProofsT the new genus comprises four species, Sphaerosporangium melleum sp. nov. with the type strain 3-28(8) (=JCM 13064T =DSM 44954T), Sphaerosporangium rubeum sp. nov. with the type strain 3D-72(35)T (=JCM 13067T =DSM 44936T), Sphaerosporangium cinnabarinum sp. nov. (=JCM 3291 =DSM 44094) and Sphaerosporangium viridialbum comb. nov. (=JCM 3027T =DSM 43801T).

INTRODUCTION III, whole-cell sugar pattern B or C, fatty acid pattern 3c, major menaquinone MK-9(III, VIII-H4,H6,H2,H0) and The family Streptosporangiaceae was described for the phospholipid type PIV. The genus Streptosporangium redeﬁned maduromycete group1). At present, the Strepto- develops stable, branched mycelia and produces globose sporangiaceae, as amended by Stackebrandt et al.2), spore vesicles (usually 10 mm in diameter) on aerial includes the following genera: Streptosporangium3), Pla- mycelia. Sporangiospores are formed by septation of nomonospora4), Microtetraspora5), Planobispora6), Plano- coiled, unbranched hyphae within the sporangium. A tetraspora7), Herbidospora8), Microbispora9), Nonomur- comparative taxonomic study and phylogenetic analysis aea9) and Acrocarpospora10). The chemotaxonomic of Streptosporangium species indicated taxonomic hetero- properties of the genera in this family are more or less geneity of the genus Streptosporangium10,11). It was similar except for the menaquinone composition of the reported that the taxonomic position of Streptosporangium genus Herbidospora. The genera are distinguished by viridialbum and ‘‘Streptosporangium cinnabarinum’’ re- morphological features including the existence of spore mains ambiguous from the viewpoint of 16S rDNA vesicles (sporangia) and the number of spores per spore sequences10,12). vesicle or chain. Members of the family have cell wall type Five actinomycete strains that formed spherical spore

Author for correspondence: Ismet Ara. Tel & Fax: +81-3-5791-6133, E-mail: [email protected] The DDBJ accession numbers for the 16S rDNA sequences of strains 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D-72(35)T are AB208714, AB208715, AB208716, AB208717 and AB208718, respectively.

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vesicles on aerial mycelia were isolated from soil. mined by TLC as described by Staneck & Roberts21). Phylogenetic analysis based on 16S rDNA sequences Reducing sugars from whole-cell hydrolysates were ana- indicated that the isolates were distinct from previously lyzed by the HPLC method of Mikami & Ishida22). The N- described genera, but closely related to S. viridialbum and acyl group of muramic acid in peptidoglycan was deter- ‘‘ S. cinnabarinum’’. In this paper, we describe the morpho- mined by the method of Uchida & Aida23). Phospholipids in logical, physiological, chemotaxonomic and phylogenetic cells were extracted and identified by the method of characterization of strains 3-28(8)T, 4-20(13), 3D-70(20), Minnikin et al.24). Methyl esters of cellular fatty acids were 5-81(36) and 3D-72(35)T, S. viridialbum and ‘‘S. cinnabar- prepared and analyzed according to the instructions of the inum’’, and propose the new genus Sphaerosporangium Microbial Identification System (Sherlock Microbial Iden- gen. nov., with Sphaerosporangium melleum sp. nov., tification System; MIDI, Hewlett Packard, Palo Alto, CA, Sphaerosporangium rubeum sp. nov., Sphaerosporangium USA)25). Isoprenoid quinones were extracted by the method cinnabarinum sp. nov. and Sphaerosporangium viridialbum of Collins et al.26,27) and were analysed using an HPLC comb. nov. system equipped with a Cosmosil 5C18 column (4:6 150 mm; Nacalai Tesque, Kyoto, Japan)28) and mass spectrom- MATERIALS AND METHODS etry (Shimadzu GCMS QP 5050). Preparation and detection of methyl esters of mycolic acids were performed as Strains 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D- described by Tomiyasu29). 72(35)T were isolated from sandy soil collected at a forest- Genomic DNA extraction, PCR-mediated amplification side waterfall in Chokoria, Cox’s Bazar, Bangladesh. The of the 16S rRNA gene and sequencing of the PCR products strains were isolated using the dilution plate method with were performed as described by Nakajima et al.30). The humic acid-vitamin (HV) agar13) supplemented with cyclo- sequences were multiply aligned with selected sequences Advance1 1 heximide (50 mg l ), nystatin (50 mg l ) and nalidixic (Fig. 2) obtained from the GenBank/EMBL/DDBJ data- acid (20 mg l1)14). After 21 days of aerobic incubation at bases using the CLUSTAL X program31). The alignment 30 C, the strains were transferred and purified on yeast was manually verified and adjusted before construction extract-malt extract agar [(medium 2 of the International of a phylogenetic tree. The phylogenetic tree constructed Streptomyces Project (ISP medium 2)], and maintained as by the neighbour-joining method32) in the PAUP program working cultures on yeast-starch agar (JCM medium no. 61) (version 4.0 b10)33) was based on a comparison of 1223 containing soluble starch, 15.0 g; yeast extract,View 4.0 g; nucleotides present in all of the strains following elimi- K2HPO4, 0.5 g; MgSO4.7H2O, 0.5 g; and agar, 15.0 g in nation of gaps and ambiguous nucleotides from the se- 1 liter of distilled water (pH 7.2). quences between positions 34 and 1491 (Escherichia coli Strains 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D- position number), and Nocardia dassonvillei was used 72(35)T were grown on tap water agar, HV agar and as an outgroup. TheProofs confidence values for branches of sucrose-nitrate agar (Waksman no. 1) at 30 C for 21 days the phylogenetic tree were determined using bootstrap and then observed by light and scanning electron micro- analyses based on 1000 re-samplings34). Signature nucleo- scope (model S-2400; Hitachi, Tokyo, Japan). Motility was tides in the 16S rRNA gene of new taxon and members of observed with a light microscope using cells grown on agar the family Streptosporangiaceae were determined after medium at 30 C for 21 days and then incubated at 28 C manual verification of the CLUSTAL X alignment of the for 30–60 min in yeast-starch broth (NYS) (JCM medium sequences, and the nucleotide positions were numbered no. 61) and ISP-2 broth. The sample for scanning electron according to the corresponding position in the 16S rRNA microscopy was prepared as described by Itoh et al.15) and sequence of E. coli35). Ara & Kudo14). For culture characterization, the isolates DNA was isolated from biomass by the method of were grown for 21 days at 30 C on various agar media as Tamaoka36) and Saito & Miura37) with minor modification described by Waksman16), Shirling & Gottlieb17) and Asano as follows: achromopeptidase crude (Wako Pure Chem- & Kawamoto18). The Colour Harmony Manual19) was used icals), N-acetylmuramidase SG (Seikagaku Kogyo) and to determine the names and designations of colony colours. lysozyme were used to lyse the cells38). Cells that failed to The temperature range and NaCl tolerance for growth were be lysed by these enzymes were freeze-dried and mechan- determined on yeast-starch agar. Utilization of carbohy- ically ground as described by Raeder & Broda39). The G+C drates as sole carbon sources was tested by using content of the DNA was determined using the HPLC neutralized yeast nitrogen base without amino acids as a method of Tamaoka & Komagata40). An equimolar mixture basal medium according to the method of Stevenson20). of nucleotides for analysis of DNA base composition Production of melanoid pigments was examined using (Yamasa Shoyu, Choshi, Japan) was digested by bacterial tyrosine agar (ISP medium 7). alkaline phosphatase and used as the quantitative standard. The freeze-dried cells used for chemotaxonomic analysis DNA–DNA relatedness was measured fluorometrically were obtained from cultures grown in yeast-starch broth on using the microplate hybridization method devised by a rotary shaker at 30 C. The isomers of diaminopimelic Ezaki et al.41). Hybridization was performed at 55 C for acid (A2pm) in the cell wall peptidoglycan were deter- 2h.

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Table 1. Diﬀerential culture, physiological, chemotaxonomic characteristics and G+C (mol%) of strains 3-28(8)T, 3D-72(35)T, S. cinnabarinum JCM 3291T and S. viridialbum JCM 3027T S. cinnabarinum S. viridialbum Characteristic 3-28(8)T 3D-72(35)T JCM 3291T JCM 3027T Substrate mycelium colour on agar medium ISP 2 Honey gold (2ic) Coral red (6nc) Bamboo (2gc) Light tan (3gc) Oatmeal Mustard gold (2ne) Light coral red (6la) Light amber (3ic) Bamboo (2gc) Bennett Gold (2lc) Light coral red (6la) Light amber (3ic) Light wheat (2ea) Yeast-starch Mustard gold (2ne) Light coral red (6la) Light melon yellow (3ea) Bamboo (2gc)

Major MK-9(H4), MK-9(H6) MK-9(H6), MK-9(H4) MK-9(H4), MK-9(H6) MK-9(H4), MK-9(H2) menaquinones

Major fatty acids iso-C16:0 (47.6%), iso-C16:0 (14.6%), iso-C16:0 (49.4%), iso-C15:0 (20.2%), (%) 10 methyl C17:0 (15.8%) 10 methyl C17:0 (12.9%) 10 methyl C17:0 (17.5%) C17:0 (13.1%), C15:0 (12.3%), iso-C16:0 (11.5%), C17:0 (10.8%) C15:0 (11.2%), DNA G+C content 71.0 71.0 70.0 72.0 (mol%) AdvanceUtilization of: Adonitol + L-Rhamnose ++ + myo-Inositol +++++ Mannitol ++ + ++ /-Methyl-D- + ++ ++ ++ glucoside Lactose ++ ++View + ++ /-D-Melibiose ++ + ++ + Sucrose ++ ++ + ++ D-Raﬃnose + Proofs

RESULTS AND DISCUSSION but have a different phylogenetic position that is yet to be determined. The almost complete 16S rDNA sequences (1503, 1508, The results of DNA–DNA hybridization among strains 1498, 1499 and 1502 nt) of strains 3-28(8)T, 4-20(13), 3D- 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D-72(35)T, 70(20), 5-81(36) and 3D-72(35)T were determined. The ‘‘ S. cinnabarinum’’ and S. viridialbum are shown in phylogenetic position based on the 16S rDNA sequence of Table 3. The high DNA relatedness (83–98%) indicates all of the isolates was within the confines of the family that strains 3-28(8)T, 4-20(13), 3D-70(20) and 5-81(36) are Streptosporangiaceae, adjacent to S. viridialbum and ‘‘S. the same species under the new taxon Sphaerosporangium, cinnabarinum’’, which occupy a phylogenetic position and their morphological features are also similar to each distinct from other species of Streptosporangium10,12,42) other. The low level of DNA relatedness between the (Fig. 2). The sequence similarities among these strains isolate 3D-72(35)T and ‘‘S. cinnabarinum’’ and S. viridial- ranged from 96.8% to 98.7%. The strains 3-28(8)T,4- bum indicate that 3D-72(35)T and ‘‘S. cinnabarinum’’ are 20(13), 3D-70(20), 5-81(36) and 3D-72(35)T were mor- new species and S. viridialbum is new combination under phologically most closely related to the genus Streptospor- the genus Sphaerosporangium. It was reported by Wayne angium, and these organisms showed 94.7–95.8% 16S et al.43) that DNA–DNA relatedness has a 70% cut-off point rDNA similarity with this genus. Strains 3-28(8)T,4- for the delineation of genomic species. Based on culture, 20(13), 3D-70(20), 5-81(36) and 3D-72(35)T showed 93.9– physiological and chemotaxonomical characteristics, we 97.5% 16S rDNA similarity with other genera of the family propose the classification of three new species for the Streptosporangiaceae. On the basis of the phylogenetic strains 3-28(8)T, 3D-72(35)T and ‘‘S. cinnabarinum’’ and data as well as the chemotaxonomic data (Tables 1 and 2), one new combination for S. viridialbum under the new it is evident that these strains should be classified in a new genus Sphaerosporangium gen. nov. (Table 1). Table 2 genus. S. viridialbum and ‘‘S. cinnabarinum’’ contain all of summarizes the characteristics of the new genus and other the typical characteristics of the genus Streptosporangium, members of the family Streptosporangiaceae.

3 ACTINOMYCETOLOGICA ) ) S. 2 4 and ) 0 Þ 9 ’’ and pair of spores ) MK-9(H 4 Nonomuraea ) , 0 Þ 9 ) (III, VIII-H 2 S. cinnabarinum ,‘‘ T ++ MK-9(H ) MK-9 MK-9(III-H ) (III-H 2 4 ) (III, VIII-H Microbispora 0 , Þ 8 ) MK-9(III-H ) (III, VIII-H 2 4 ) MK-9(H 0 Herbidospora , Þ 7 ) MK-9(III-H ) (III, VIII-H 2 4 ) MK-9(H Advance 0 , 4-20(13), 3D-70(20), 5-81(36), 3D-72(35) T Planotetraspora , Þ 6 ) MK-9(III-H ) (III, VIII-H 2 View 4 Planobispora ++++++ on aerialmycelium spores on aerial short mycelium mycelium pairs on aerial containing a mycelium containing single spore longitudinal , Þ 5 ) (III, VIII-H ) MK-9(III-H 4 6 )

Proofs2 Microtetraspora , Þ 4 ) MK-10 MK-9(H 2 ) (III, IX-H 4 ) MK-10(H 6 Planomonospora , Þ ) MK-9(H ) (III,VIII-H 3 2 4 ) MK-9(III-H 0 Streptosporangium Acrocarpospora Herbidospora Planotetraspora Microtetraspora Nonomuraea Microbispora Planomonospora Planobispora Streptosporangiaceae ) MK-9(H ) (III, VIII-H 2 4 Streptosporangium )) MK-9(III-H ) (III-H 6 0 8 . Þ (III, VIII-H aerial mycelium mycelium quartet of spores containing four gium on aerial longitudinal sporangia, sporangia, MK-9(H MK-9(III-H MK-9(H MK-9(H 10 )} and the family Cell wall typeWhole-cell III sugar Bpattern Fatty-acid type 3c IIIMenaquinone (s) B MK-9 3c MK-9 III B, C MK-9 3c III B MK-10 III 3c B MK-9 3d III MK-9 B, C MK-9 3c III B, C MK-9 III 3c B, C MK-9 III B 3c MK-9 3c III B 3c Motile spores CharacteristicSporangium New taxon formation Globose sporangia on Globose sporangia on aerial mycelium Club or globose sporangia on aerial on sporophore Spore chains containing Sporangia typically Spore chains, Spore pseudosporan chain or Spores characteristic in clavate Cylindrical to Cylindrical to clavate Aerial mycelium + + + Phospholipidtype PIV PIV PIV, II PIV PIV PIV PIV PIV PIV PIV Table 2. Morphological features and chemotaxonomic characteristics of the new taxon {(isolates 3-28(8) Data were taken from viridialbum Acrocarpospora

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Table 3. DNA base composition and DNA–DNA relatedness (mean of six replications) among the isolates 3-28(8)T, 4-20(13), 3D- 70(20), 5-81(36), 3D-72(35)T,‘‘S. cinnabarinum JCM 3291’’ and S. viridialbum JCM 3027T

G+C Percentage DNA complimentarity with labeled DNA from: Taxon content (mol%) 3-28(8)T 4-20(13) 3D-70(20) 5-81(36) 3D-72(35)T 3291 3027T 3-28(8)T 71 100 96 96 98 39 50 42 4-20(13) 71 84 100 89 86 34 47 36 3D-70(20) 71 84 94 100 85 35 47 42 5-81(36) 71 83 92 88 100 32 47 41 3D-72(35)T 71 50 57 52 54 100 45 46 ‘‘ S. cinnabarinum’’ 70 66 67 64 65 34 100 46 JCM 3291 S. viridialbum 72 50 57 53 56 32 41 100 JCM 3027T

Signature nucleotides in the 16S rRNA gene of the new the formation of sporangia on aerial mycelia1,2) (Table 2). taxon and members of the family Streptosporangiaceae are The physiological properties of strains 3-28(8)T,4- shown in Table 4. The 16S rDNA sequence of strains 3- 20(13), 3D-70(20), 5-81(36) and 3D-72(35)T can be 28(8)T and 3D-72(35)T, S. viridialbum and ‘‘S. cinnabar- summarized as follows. They exhibited good growth on inum’’ contain all of the 16S rDNA signature nucleotides yeast extract-starch agar, oatmeal agar (ISP medium 3), Advance2) defined for the suborder Streptosporangineae . It is evident ISP medium 2, Bennett agar and glucose-yeast extract agar, from our study that, of the 12 signatures defined for the moderate growth on 1/5 yeast-starch agar, oatmeal-nitrate family Streptosporangiaceae2), all are present in the 16S agar, glucose-asparagine agar and Hickey-Tresner agar, rDNA sequence of the strains 3-28(8)T, 4-20(13), 3D- and poor growth on glycerol-asparagine agar, inorganic 70(20), 5-81(36) and 3D-72(35)T,‘‘S. cinnabarinum’’ and salts-starch agar, tyrosine agar, nutrient agar, HV agar, tap S. viridialbum. Furthermore, our results show several water agar and sucrose-nitrate agar. They formed aerial nucleotide pairs that differ from those of membersView of the hyphae and sporulated on oatmeal-nitrate and HV agar. No genus Streptosporangium, represented by the 16S rDNA strain produced soluble pigment on any agar medium nucleotide pairs at positions 263 (G), 264 (U), 595 (G), tested. They utilized D-glucose, L-arabinose, D-ribose, 600–638 (U-G), 602–636 (C-G), 603–635 (C-G), 627 (G), D-xylose, D-galactose, D-fructose, D-mannose, D-manni- 626 (U), 625 (G), 668–738 (C-G), 669–737 (A-U), 671– tol, salicin, lactose,Proofs D(+)melibiose, sucrose, maltose and 735 (G-C), 1012–1017 (G-C) and 1263–1272 (G-U) trehalose. They grew well at 20–37 C and pH 5–9, and (Table 4). none could grow on 3% NaCl. The isolates 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and Chemotaxonomic properties have been found to make 3D-72(35)T were Gram-positive, non-acid-fast, aerobic an important contribution to the polyphasic approach to organisms with branched hyphae. A non-fragmenting bacterial systematics, especially in the circumscription of substrate mycelium was formed. Morphological observa- phylogenetically coherent actinomycete taxa44,45). The tions by light microscope of a 21-day-old culture grown family Streptosporangiaceae is mainly defined by chemo- on oatmeal-nitrate, sucrose-nitrate, Hickey-Tresner or 1/5 taxonomy, and our isolates 3-28(8)T, 4-20(13), 3D-70(20), yeast-starch agar revealed the presence of single or 5-81(36) and 3D-72(35)T shared chemotaxonomic charac- clustered spherical spore vesicles on aerial hyphae. Ob- teristics with members of this family as follows. Their cell servation by scanning electron microscope indicated that walls contained meso-diaminopimelic acid and glucose, the spherical structures were variable in size (1.5–8.0 mm) madurose and mannose as the major whole-cell sugars in (Fig. 1). Spores in the spore vesicles were non-motile and addition to small amounts of ribose and galactose, indicat- formed by the separation of unbranched hyphae within the ing a whole-cell sugar pattern B according to Lechevalier spore vesicle. The spores were oval or spherical in shape & Lechevalier46). The major menaquinones MK-9(III, VIII- with a smooth, wrinkled and prominently ridged surface. H4) and MK-9(H6) and small amounts of MK-9(H2), MK- The surfaces of the spherical bodies were smooth at first, 9(H0) and MK-9(H8) were present. The predominant becoming wrinkled as the spores developed. Similar menaquinone pattern of our isolates differed from that of morphological features are observed in S. cinnabarinum other genera of the family Streptosporangiaceae, which and S. viridialbum. Although morphology is the sole have tetrahydrogenated, dehydrogenated and unsaturated criterion used to distinguish genera in the family Strepto- menaquinones with nine isoprene units [MK-9(III, VIII- sporangiaceae, the morphological characteristics and spore H4), MK-9(H2) and MK-9(H0), respectively, except for the development of our isolates were similar to those of genus Herbidospora, which has MK-10(III, IX-H4,H6,H2)] members of the genus Streptosporangium with respect to (Table 2). Tetrahydrogenation occurs at the sites of

5 ACTINOMYCETOLOGICA G-U G-C GG UC C-G C-G G-U Streptosporangiaceae A GG G C C-G/U-G A U C-UU-A C-U U-A C-U U-A C-U U-G G-U G-U G-U G-U G-U G-U G-U G-U Advance G-C G-C from other members of the family from the new taxon are shown in bold. G C-GC-G C-G C-G C-G C-G C-G C-G C-G C-G U-G U-G U-G U-G U-G G-U . Þ Nonomuraea Microtetraspora Microbispora Herbidospora Acrocarpospora View 35 - Sphaerosporangium G-C numbering Planobi ProofsU-G/C-G U-G U-G U-G U-G U-G Streptosporangiaceae - E. coli CGG C A A G AG G-CC-G G-C C-G G-C C-G C-G G-C G-C G-C C-G A-UU-A A-U U-A U-AA-U U-A A-UG-U U-A A-U G-C G-C GGGG G UUUU U U U U GGGG G G G G G G C-G C-G C-GG-C C-G G-C C-G G-C C-G G-C C-G G-C C-G G-C C-G G-C C-G G-C U-G C-U U-A A-U A-U A-U A-U A-U A-U A-U A-U G-U U-G G-U U-G spora spora spora G-C/G-U Planotetra- Planomono Ã C A C A CCCCC A U-A A-U A-U A-UA-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U A-U G-C G-U G-C Streptosporangium New taxon Signature nucleotides, which clearly diﬀerentiate members of the family Nucleotide positions of bases or base pairs are given according to the Position Ã 264 U 595 G 442–492594–645 G-C C-G603–635 G-C 625 C-G C-G G 263 G 627 G 600–638 U-G 602–636 C-G 626 U 657–749658–748659–746 G-C668–738 U-A669–737 A-U C-G G-C 835–851 A-U U-A A-U G-U G-U 671–735 G-C 990–1215 C-G C-G 1012–1017 G-C 1263–1272 G-U Table 4. 16S rDNA signature nucleotide positions that diﬀerentiate the new genus

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a b

c d Advance

Fig. 1. Scanning electron micrographs of globose sporangia on aerial mycelia of Sphaerosporangium melleum 3-28(8)T (a), Sphaerosporangium rubeum 3D-72(35)T (b), Sphaerosporangium cinnabarinum (=JCM 3291T) (c) and Sphaerosporangium viridialbum (=JCM 3027T) (d), grown on sucrose-nitrate agar except ViewS. rubeum 3D-72(35)T grown on oatmeal-nitrate agar for 21 days at 30 C.

isoprene unit III (the third unit from the 2-methyl-1,4- (Table 1). ‘‘S. cinnabarinum’’ possessed MK-9(H4), MK- napthoquinone moiety) and VIII [MK-9(III, VIII-H )]47). 9(H ) and MK-9(HProofs) and S. viridialbum possessed MK- 4 6 2 The position of tetrahydrogenation in Gram-positive bac- 9(H4), MK-9(H2) and MK-9(H0); the major fatty acid 47,48) teria is generally at the sites of units II and III . profile was branched iso-C16:0 (49.4%) and 10 methyl Although there is some difference between the predominant C17:0 (17.5%) for ‘‘S. cinnabarinum’’ and branched iso- menaquinone patterns of previously described members of C15:0 (20.2%), saturated C17:0 (13.1%), C15:0 (11.2%) and the family Streptosporangiaceae and our isolates, the branched iso-C16:0 (11.5%) for S. viridialbum (Table 1). In saturation of tetrahydrogenated menaquinones occurs in contrast, all Streptosporangium species are characterized the same position. The strains contained iso-C16:0 and 10 by the presence of MK-9(H2), MK-9(H4) and MK-9(H0)as 47,51) methyl C17:0 as the major cellular fatty acids, with small the major menaquinones , n-C17:1 as the major fatty amounts of saturated fatty acid C15:0,C16:0,C17:0, unsat- acid component, and small amounts of anteiso-C15:0, 49) urated fatty acid C17:1, iso-C15:0,C17:0,C14:0,C18:0, anteiso- anteiso-C17:0, iso-C15:0 and iso-C17:0 . Phosphatidyletha- C15:0,C17:0 and 10 methyl fatty acids C16:0. The fatty acid nolamine, diphosphatidylglycerol, ninhydrin-positive phos- pattern corresponds to fatty acid type 3c of Kroppenstedt49). phoglycolipids and phosphatidylinositol mannosides are Mycolic acids were absent. Phosphatidylethanolamine, detected but phosphatidylglycerol, phosphatidylinositol diphosphatidylglycerol, ninhydrin-positive phosphoglyco- and phosphatidylcholine are not detected, corresponding lipids and phosphatidylinositol mannosides were detected to phospholipid type IV of Lechevalier et al.50). Madurose but phosphatidylglycerol, phosphatidylinositol and phos- is the whole-cell sugar42). Although the isolates and ‘‘S. phatidylcholine were not detected, corresponding to phos- cinnabarinum’’ and S. viridialbum therefore differ in some pholipid type PIV of Lechevalier et al.50). The acyl type of chemotaxonomic characteristics, they appear to be closely the cell wall polysaccharides was acetyl. The G+C content related to each other. The results of morphological of the DNA was 70–72%. observation and phylogenetic analysis indicate that they In our study, ‘‘S. cinnabarinum’’ and S. viridialbum were belong to the same group. found to be species with morphological features similar to The distinctness of 16S rDNA sequences, the phyloge- those of the genus Streptosporangium. They showed some netic position and the specific signature nucleotide patterns differences in major menaquinones and fatty acid profile of strains 3-28(8)T, 4-20(13), 3D-70(20), 5-81(36) and 3D-

7 ACTINOMYCETOLOGICA

Sphaerosporangium rubeum 3D- 72(35)T (AB208718) 73 Sphaerosporangium melleum 3-28(8)T (AB208714) 96 83 Sphaerosporangium melleum 5-81(36) (AB208717) 51 Sphaerosporangium melleum 4-20(13) (AB208715) Sphaerosporangium melleum 3D-70(20) (AB208716) Sphaerosporangium cinnabarinum DSM 44094T (X89939) Sphaerosporangium viridialbum DSM 43801T (X89953) 64 T 100 Planotetrspora mira IFO 15435 (D85496) T Planotetraspora silvatica TT00-51 (AB112) Herbidospora cretacea IFO 15474T (D85485) T 100 Microtetraspora glauca IFO 14761 (D85490) 73 T 65 87 Microtetraspora malaysiensis H47-7 (AB062383) 83 Microbispora corallina DF-32T (AB018046) Microbispora rosea IFO 14044T (D86936) 100 Acrocarpospora macrocephala IFO 16266T (AB025318) T 68 Acrocarpospora pleiomorpha R-31 (AB006174) 100 Nonomuraea helvata IFO 14681T (U48975) 71 Nonomuraea polychroma IFO 14345T (U48977) Nonomuraea pusilla IFO 14684T (D85491) 100 AdvancePlanobispora longispora IFO 13918T (D85494) Planobispora rosea JCM 3166T (AB028654) Streptosporangium carneum DSM 44125T (X89938) 84 Streptosporangium roseum DSM 43021T (X89947) T 58 Streptosporangium fragile DSM 43847 (X89942) T 84 Planomonospora alba JCM 9373 (AB062381) T 100 PlanomonosporaView parontospora IFO 13880 (D85495) Planomonospora sphaerica JCM 9374T (AB062382) Nocardia dassonvillei IFO 14626T (D85492) 0.005 substitutions/site Proofs Fig. 2. Phylogenetic tree showing the position of the new genus Sphaerosporangium in the family Streptosporangiaceae based on almost complete 16S rDNA sequence comparison. The branching pattern was generated by the neighbour-joining method. The numbers on the tree indicate bootstrap values greater than 50%. The numbers in parentheses are accession numbers for the DDBJ/EMBL/ GenBank nucleotide sequence databases.

72(35)T,‘‘S. cinnabarinum’’ and S. viridialbum diﬀer- 9, and could not grow on 3% NaCl. It shows good growth entiate them from known actinomycetes belonging to the on yeast extract-starch agar, Bennett agar and oatmeal agar. family Streptosporangiaceae, and for this reason these In general, the vegetative mycelia are pale to brown-yellow isolates could merit new genus status. and the aerial mycelia are white. Melanin pigment production is negative. The cell wall contains meso- Description of Sphaerosporangium gen. nov. diaminopimelic acid; the wall chemotype is III. Madurose, Sphaerosporangium (S.pha.ero.spo.ran.gium. Gr. adj. glucose and mannose are detected as the major whole-cell Sphaero globose, spherical; Gr. fem. n. spora a seed, sugars in addition to small amounts of ribose and galactose, spore; Gr. n. argeion vessel; sporangium pl. sporangia, indicating that the whole-cell sugar pattern is B. The major spore-bearing. N.L. n. Sphaerosporangium an organism menaquinones are MK-9(H4) and MK-9(H6), and small with globose or spherically shaped, spore-containing amounts of MK-9(H2), MK-9(H0) and MK-9(H8) are vessels, i.e. sporangia). present. In general, the major cellular fatty acids are iso- The cells are Gram-positive and form branching hyphae. C16:0 and 10 methyl C17:0, with small amounts of saturated Non-fragmentary substrate mycelia are present. Spherically fatty acid C15:0,C16:0,C17:0, unsaturated fatty acid C17:1, shaped structures are borne on aerial mycelia. These iso-C15:0,C17:0,C14:0,C18:0, anteiso-C15:0,C17:0 and 10 structures contain coiled spore chains. The spores are oval methyl fatty acids C16:0. Mycolic acids are absent. or spherical (0.4–0.9 0.6–1.2 mm) with a smooth, wrin- Phosphatidylethanolamine, diphosphatidylglycerol, ninhy- kled and prominently ridged surface and are non-motile. drin-positive phosphoglycolipids and phosphatidylinositol The organism is aerobic, grows well at 20–37 C and pH 5– mannosides are detected, but phosphatidylglycerol, phos-

8 ACTINOMYCETOLOGICA

phatidylinositol and phosphatidylcholine are not detected menaquinones are MK-9(H6) and MK-9(H4), and small (phospholipid type PIV). The genus is placed in the family amounts of MK-9(H2), MK-9(H8) and MK-9(H0) are Streptosporangiaceae on the basis of the phylogenetic present. The major cellular fatty acids are iso-C16:0 position of the strains based on their distinct 16S rDNA (14.6%), 10 methyl C17:0 (12.9%), saturated C15:0 sequences. All family-speciﬁc nucleotide signatures are (12.3%), C16:0 (10.3%) and C17:0 (10.8%), with small present except for a G-C pair at position 1012–1017 instead amounts of iso-C15:0 (6.7%), anteiso-C15:0 (5.0%), anteiso- of an A-U pair. The acyl type of the cell wall polysaccha- C17:0 (4.6%), 10 methyl C16:0 (3.0%) and C17:0 (3.1%). The rides is acetyl. The G+C content of the DNA is 70–72%. G+C content of the DNA is 71%. The habitat is soil. The The type species is Sphaerosporangium melleum. type strain is 3D-72(35)T (=JCM 13067T =DSM 44936T).

Description of Sphaerosporangium melleum sp. nov. Description of Sphaerosporangium cinnabarinum sp. Sphaerosporangium melleum (me.lle.um. N.L. n. adj. nov. melleum honey coloured). Sphaerosporangium cinnabarinum {(cin.na.bari.num. N.L. Morphological, chemotaxonomic and general character- adj. cinnabarinus of cinnabar, referring to the vermilion istics are as given above for the genus descriptions. The (bright red) colour of vegetative mycelium and diﬀusible organism shows good growth on yeast extract-starch agar, pigment)}. yeast extract-malt extract agar, glucose-yeast extract agar, Morphological, chemotaxonomic and general character- Bennett agar and oatmeal agar, moderate growth on istics are as given above for the genus. The organism shows glucose asparagine agar, Hickey-Tresner agar, 1/5 yeast good growth on oatmeal agar and yeast extract-starch agar, extract-starch agar, oatmeal-nitrate agar and humic acid- moderate growth on glucose asparagine agar, inorganic Advancevitamin agar, and poor growth on glycerol-asparagine agar, salts-starch agar, Bennett agar, glucose-yeast extract agar, inorganic salts-starch agar, tyrosine agar, nutrient agar, tap 1/5 yeast extract-starch agar, oatmeal-nitrate agar and water agar and sucrose-nitrate agar. Abundant sporulation humic acid-vitamin agar, and poor growth on yeast extract- occurs on tap water agar, sucrose-nitrate agar, inorganic malt extract agar, Hickey-Tresner agar, nutrient agar, salts-starch agar agar and 1/5 yeast extract-starch agar. The glycerol-asparagine agar, tyrosine agar, tap water agar vegetative mycelia are light wheat to honey gold in colour and sucrose-nitrate agar. Sporulation occurs on sucrose- and the aerial mycelia are white. Glycerol, erythritol and nitrate agar, oatmeal-nitrate agar and humic acid-vitamin myo-inositol are poorly utilized. The major menaquinonesViewagar. The vegetative mycelia are light wheat to melon are MK-9(H4) and MK-9(H6), and small amounts of MK- yellow in colour and the aerial mycelia are white. Glycerol, 9(H2), MK-9(H8) and MK-9(H0) are present. The major erythritol, adonitol and D-mannitol are poorly utilized. The cellular fatty acids are iso-C16:0 (47.6%) and 10 methyl major menaquinones are MK-9(H4) and MK-9(H6), and C (15.8%), with small amounts of iso-C (8.0%), small amounts of MK-9(HProofs), MK-9(H ) and MK-9(H ) are 17:0 15:0 2 8 0 saturated fatty acid C15:0 (3.6%), C16:0 (3.0%), C17:0 (3.0%), present. The major cellular fatty acids are iso-C16:0 (49.4%) anteiso-C17:0 (3.3%) and 10 methyl fatty acids C16:0 (3.5%). and 10 methyl C17:0 (17.5%), with small amounts of The G+C content of the DNA is 71%. The habitat is unsaturated C17:1 (!8c) (5.0%), saturated C15:0 (4.1%), T T soil. The type strain is 3-28(8) (=JCM 13064 =DSM C17:0 (3.7%) iso-C15:0 (3.0%), anteiso-C15:0 (5.0%) and T 44954 ). anteiso-C17:0 (3.5%). The G+C content of the DNA is 70%. The habitat is soil. The type strain is JCM 3291T (=DSM Description of Sphaerosporangium rubeum sp. nov. 44094T). Sphaerosporangium rubeum (ru.be.um. N.L. n. adj. rubeus red coloured). Description of Sphaerosporangium viridialbum Morphological, chemotaxonomic and general character- (Nonomura and Ohara 1960) comb. nov. istics are as given above for the genus descriptions. The Sphaerosporangium viridialbum (vi.ri.di.al’bum. N.L. adj. organism shows good growth on yeast extract-starch agar, viridis green; L. adj. album white; N.L. neut. adj. yeast extract-malt extract agar, glucose-yeast extract agar, viridialbum greenish white). Bennett agar, Hickey-Tresner agar and oatmeal agar, Descriptions of this taxon have been published previ- moderate growth on inorganic salts-starch agar, nutrient ously by Nonomura & Ohara52) and Nonomura53). The agar, 1/5 yeast extract-starch agar, oatmeal-nitrate agar and descriptions below are based on our study. Morphological, humic acid-vitamin agar, and poor growth on glucose chemotaxonomic and general characteristics are as given asparagine agar, glycerol-asparagine agar, tyrosine agar, above for the genus. The organism shows good growth on tap water agar and sucrose-nitrate agar. Scanty sporulation oatmeal agar, Bennett agar, glucose-yeast extract agar, occurs on humic acid-vitamin agar, sucrose-nitrate agar, Hickey-Tresner agar and yeast extract-starch agar, moder- oatmeal-nitrate agar and water agar. The vegetative ate growth on nutrient agar, yeast extract-malt extract agar, mycelia are light coral pink to coral red in colour and the 1/5 yeast extract-starch agar, oatmeal-nitrate agar and aerial mycelia are white. Glycerol, erythritol, adonitol, humic acid-vitamin agar, and poor growth on glucose myo-inositol and D-raﬃnose are poorly utilized. The major asparagine agar, inorganic salts-starch agar, glycerol-

9 ACTINOMYCETOLOGICA asparagine agar, tyrosine agar, tap water agar and sucrose- biol. 16: 369–372, 1993 nitrate agar. The vegetative mycelia are light wheat to 12) Ward-Rainey, N.; F. A. Rainey & E. Stackebrandt: The bamboo yellow in colour and the aerial mycelia are white. phylogenetic structure of the genus Streptosporangium. Syst. Sporulation occurs on oatmeal agar, Hickey-Tresner agar, Appl. Microbiol. 19: 50–55, 1996 sucrose-nitrate agar, 1/5 yeast extract-starch agar, oatmeal- 13) Hayakawa, M. & H. Nonomura: Humic acid-vitamin agar, a new medium for selective isolation of soil actinomycetes. J. nitrate agar and humic acid-vitamin agar. Glycerol, Ferment. Technol. 65: 501–509, 1987 erythritol and D-raffinose are poorly utilized. The major 14) Ara, I. & T. Kudo: Three novel species of the genus menaquinones are MK-9(H4) and MK-9(H2), and small Catellatospora, Catellatospora chokoriensis sp. nov., Cat- amounts of MK-9(H0) and MK-9(H6) are present. The ellatospora coxensis sp. nov. and Catellatospora banglade- major cellular fatty acids are iso-C15:0 (20.2%), saturated shensis sp. nov. and transfer of Catellatospora citrea subsp. C17:0 (13.1%), C15:0 (11.2%), iso-C16:0 (11.5%) and unsat- methionotrophica Asano and Kawamoto 1988 to Catellato- urated C17:1 (!8c) (10.3%), with small amounts of 10 spora methionotrophica sp. nov. comb. nov. Int. J. Syst. Evol. methyl C17:0 (8.4%) and saturated C16:0 (6.9%). The G+C Microbiol. 56: 393–400, 2006 content of the DNA is 72%. The habitat is acidic volcanic 15) Ito, T.; T. Kudo, F. Parenti & A. Seino: Amended description ash. The type strain is JCM 3027T (=DSM 43801T). of the genus Kineosporia, based on chemotaxonomic and morphological studies. Int. J. Syst. Bacteriol. 39: 168–173, 1989 ACKNOWLEDGEMENT 16) Waksman, S. A.: Classification, Identification, and Descrip- tion of Genera and Species. In The Actinomycetes, vol. 2. The authors are indebted to the Japan Society for the Baltimore: Williams & Wilkins, 1961 Promotion of Science forfinancial support. 17) Shirling, E. B. & D. Gottlieb: Methods for characterization of Advance Streptomyces species. Int. J. Syst. Bacteriol. 16: 313–340, REFERENCES 1966 18) Asano, K. & I. Kawamoto: Catellatospora, a new genus of 1) Goodfellow, M.; L. J. Stanton, K. E. Simpson & D. E. the Actinomycetales. Int. J. Syst. Bacteriol. 36: 512–517, Minnikin: Numerical and chemical classification of Actino- 1986 planes and some related actinomycetes. J. Gen. Microbiol. 19) Jacobson, E.; W. C. Grauville & C. E. Fogs: Colour 136: 19–36, 1990 Harmony Manual,4th ed. Container Corporation of America, 2) Stackebrandt, E.; F. A. Rainey & N. L. Ward-Rainey:ViewChicago, 1958 Proposal for a new hierarchic classification system, Actino- 20) Stevenson, I. L.: Utilization of aromatic hydrocarbons by bacteria classis nov. Int. J. Syst. Bacteriol. 47: 479–491, Arthrobacter spp. Can. J .Microbiol. 13: 205–211, 1967 1997 21) Staneck, J. L. & G. D. Roberts: Simplified approach to 3) Couch, J. N.: A new genus and family of the Actino- identification of aerobic actinomycetes by thin-layer chro- mycetales Actinoplanes J. Appl.Proofs Microbiol. , with a revision of the genus . matography. 28: 226–231, 1974 Elisha Mitchell Sci. Soc. 71: 148–155, 1955 22) Mikami, H. & Y. Ishida: Post-column fluorometric detection 4) Thiemann, J. E.; H. Pagani & G. Beretta: A new genus of the of reducing sugar in high-performance liquid chromatogra- Actinoplanaceae: Planomonospora gen. nov. G. Microbiol. phy using arginine. Bunseki Kagaku 32: E207–E210, 1983 15: 27–38, 1967 23) Uchida, K. & K. Aida: An improved method for the glycolate 5) Thiemann, J. E.; H. Pagani & G. Beretta: A new genus of the test for simple identification of the acyl type of bacterial cell Actinomycetales: Microtetraspora gen. nov. J. Gen. Micro- walls. J. Gen. Appl. Microbiol. 30: 131–134, 1984 biol. 50: 295–303, 1968 24) Minnikin, D. E.; A. G. O’Donnell, M. Goodfellow, G. 6) Thiemann, J. E. & G. Beretta: A new genus of the Alderson, M. Athalye, A. Schaal & J. H. Parlett: An Actinoplanaceae: Planobispora gen. nov. Arch. Mikrobiol. integrated procedure for the extraction of bacterial isoprenoid 62: 157–166, 1968 quinones and polar lipids. J. Microbiol. Methods. 2: 233–241, 7) Runmao, H.; W. Guizhen & L. Junying: A new genus of 1984 actinomycetes, Planotetraspora gen. nov. Int. J. Syst. 25) Sasser, M.: Identification of bacteria by gas chromatography Bacteriol. 43: 468–470, 1993 of cellular fatty acids (MIDI Technical Note 101). New 8) Kudo, T.; T. Itoh, S. Miyadoh, T. Shomura & A. Seino: York, DE: MIDI, 1990 Herbidospora gen. nov., a new genus of the family 26) Collins, M. D.; T. Pirouz, M. Goodfellow & D. E. Minnikin: Streptosporangiaceae Goodfellow et al., 1990. Int. J. Syst. Distribution of menaquinones in actinomycetes and coryne- Bacteriol. 43: 319–328, 1993 bacteria. J. Gen. Microbiol. 100: 221–230, 1977 9) Zhang, Z.; Y. Wang & J. Ruan: Reclassification of 27) Collins, M. D.; M. Faulkner & M. Keddie: Menaquinone Thermomonospora and Microtetraspora. Int. J. Syst. Bacter- composition of some sporeforming actinomycetes. Syst. iol. 48: 411–422, 1998 Appl. Microbiol. 5: 20–29, 1984 10) Tamura, T.; S. Suzuki & K. Hatano: Acrocarpospora gen. 28) Tamaoka, J.; Y. Katayama-Fujimura & H. Kuraishi: Analysis nov., a new genus of the order Actinomycetales. Int. J. Syst. of bacterial menaquinone mixtures by high performance Bacteriol. 50: 1163–1171, 2000 liquid chromatography. J. Appl. Bacteriol. 54: 31–36, 1983 11) Kemmerling, C.; H. Gurtler, R. T. Kroppenstedt, R. Toalster: 29) Tomiyasu, I.: Mycolic acid composition and thermally & E. Stackebrandt: Evidence for the phylogenetic hetero- changes in Nocardia asteroides. J. Bacteriol. 151: 828– geneity of the genus Streptosporangium. Syst. Appl. Micro- 837, 1982

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30) Nakajima, Y.; V. Kitpreechavanich, K. Suzuki & T. Kudo: comb. nov., Kutzneria kofuensis comb. nov., and Kutzneria Microbispora corallina sp. nov., a new species of the genus albida comb. nov., and emendation of the genus Strepto- Microbispora isolated from Thai soil. Int. J. Syst. Bacteriol. sporangium. Int. J. Syst. Bacteriol. 44: 265–269, 1994 49: 1761–1767, 1999 43) Wayne, L. G.; D. J. Brenner, R. R. Colwell & 9 other 31) Thompson, J. D.; T. J. Gibson, F. Plewniak, F. Jeanmougin & authors: International Committee on Systematic Bacteriol- D. G. Higgins: The Clustal X windows interface: flexible ogy. Report of the ad hoc committee on reconcilation of strategies for multiple sequence alignment aided by quality approaches to bacterial systematics. Int. J. Syst. Bacteriol. analysis tools. Nucleic. Acids Res. 24: 4876–4882, 1997 37: 463–464, 1987 32) Saitou, N. & M. Nei: The neighbor-joining method: a new 44) Kroppenstedt, R. & M. Goodfellow: The family Thermomo- method for reconstructing phylogenetic trees. Mol. Biol. nosporaceae.InThe prokaryotes, pp. 1085–1114, Edited by Evol. 4: 406–425, 1987 A. Balows, H. G. Truper, M, Dworkin, W. Harder, and K. H. 33) Swofford, D. L.: PAUP. Phylogenetic Analysis Using Schleifer, 2nd ed. Springer, New York, 1991 Parsimony (and Other Methods). Version 4. Sinauer 45) Goodfellow, M.: The family Streptosporangiaceae.InThe Associates, Sunderland, Massachusetts, 2001 prokaryotes, pp. 1115–1138. Edited by A. Balows, H. G. 34) Felsenstein, J.: Confidence limits on phylogenies: an ap- Truper, M. Dworkin, W. Harder, and K. H. Schleifer, 2nd ed. proach using the bootstrap. Evolution 39: 783–791, 1985 Springer, New York, 1991 35) Brosius, J.; J. L. Palmer, J. P. Kennedy & H. F. Noller: 46) Lechevalier, M. P. & H. A. Lechevalier: Chemical compo- Complete nucleotide sequence of a 16S ribosomal RNA gene sition as a criterion in the classification of aerobic actino- from Escherichia coli. Proc. Natl. Acad. Sci. USA 75: 4801– mycetes. Int. J. Syst. Bacteriol. 20: 435–443, 1970 4805, 1978 47) Collins, M. D.; R. M. Kroppenstedt, J. Tamaoka, K. 36) Tamaoka, J.: Determination of DNA Base Composition. In Komagata & T. Kinoshita: Structures of tetrahydrogenated Chemical Methods in Prokaryotic Systematics, pp. 463–470. menaquinones from Actinomadura angiospora, Faenia rec- AdvanceEdited by M. Goodfellow & A. G. O’Donnell. Chichester: tivirgula, and Saccharothrix australiensis. Curr. Microbiol. John Wiley & Sons, 1994 17: 275–279, 1988 37) Saito, H. & K. Miura: Preparation of transforming deoxy- 48) Yamada, Y.; G. Inouye, Y. Tahara & K. Kondo: On the ribonucleic acid by phenol treatment. Biochim. Biophys. chemical structure of menaquinones with the tetrahydrogen- Acta. 72: 619–629, 1963 ated isoprenoid side chain. Biochem. Biophys. Acta. 486: 38) Kudo, T.; K. Matsushima, T. Itoh, J. Sasaki & K. Suzuki: 195–203, 1977 Description of four new species of the genus Kineosporia: 49) Kroppenstedt, R. M.: Fatty acid and menaquinone analysis of Kineosporia succinea sp. nov., kineosporia rhizophilaViewsp. actinomycetes and related organisms. In Chemical Methods nov., kineosporia mikuniensis sp. nov. and kineosporia in Bacterial Systematics, pp. 173–199. Edited by M. Good- rhamnosa sp. nov., isolated from plant samples, and amended fellow & D. E. Minnikin. London: Academic Press, 1985 description of the genus Kineosporia. Int. J. Syst. Bacteriol. 50) Lechevalier, M. P.; A. E. Stern & H. A. Lechevalier: 48: 1245–1255, 1998 Phospholipids inProofs the taxonomy of actinomycetes. Zentbl. 39) Raeder, U. & P. Broda: Rapid preparation of DNA from Bakteriol. Hyg. Abt. 1 Suppl. 11: 111–116, 1981 filamentous fungi. Lett. Appl. Microbiol. 1: 17–20, 1985 51) Kudo, T. & A. Seino: Transfer of Streptosporangium 40) Tamaoka, J. & K. Komagata: Determination of DNA base indianense Gupta 1965 to the genus Streptomyces as composition by reversed-phase high-performance liquid Streptomyces indiaensis (Gupta 1965) comb. nov. Int. J. chromatography. FEMS Microbiol. Lett. 25: 125–128, 1984 Syst. Bacteriol. 37: 241–244, 1987 41) Ezaki, T.; Y. Hashimoto & E. Yabuuchi: Fluorometric 52) Nonomura, H. & Y. Ohara: Distribution of the actinomycetes deoxyribonucleic acid- deoxyribonucleic acid hybridization in soil. V. The isolation and classification of the genus in microdilution wells as an alternative to membrane filter Streptosporangium. J. Ferment. Technol. 38: 405–409, 1960 hybridization in which radioisotopes are used to determine 53) Nonomura, H.: Genus Streptosporangium Couch 1955, genetic relatedness among bacterial strains. Int. J. Syst. 148AL. In Bergey’s Manual of Systematic Bacteriology, Bacteriol. 39: 224–229, 1989 vol. 4, pp. 2545–2551. Edited by S. T. Williams, M. E. 42) Stackebrandt, E.; R. M. Kroppenstedt, K. D. Jahnke.: C. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins, 1989 Kemmerling & H. Gurtler: Transfer of Streptosporangium 54) Kroppenstedt, R. M.; E. Stackebrandt & M. Goodfellow: viridogriseum (Okuda et al. 1996), Streptosporangium Taxonomic revision of the actinomycete genera Actinoma- viridogriseum subsp. kofuense (Nonomura and Ohara dura and Microtetraspora. Syst. Appl. Microbiol. 13: 148– 1969), and Streptosporangium albidum (Furumai et al. 160, 1990 1968) to Kutzneria gen. nov. as Kutzneria viridogrisea