J. Gen. Appl. Microbiol., 53, 39–51 (2007)

Full Paper

Luedemannella gen. nov., a new member of the family Micromonosporaceae and description of Luedemannella helvata sp. nov. and Luedemannella flava sp. nov.

Ismet Ara* and Takuji Kudo

Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351–0198, Japan

(Received September 14, 2006; Accepted December 12, 2006)

Three actinomycete strains were isolated from soil samples collected in Bangladesh. The cul- tures formed spherical sporangia on short sporangiophores directly above the surface of the substrate mycelium. The sporangia developed singly or in clusters and each sporangium con- tained several nonmotile spherical to oval spores with a smooth surface. The strains 3-9(24)T, 3-21(27) and 7-40(26)T contained meso-diaminopimelic acid in the cell walls, predominant

menaquinone MK-9(H6) and MK-9(H4) and glucose, xylose, galactose, mannose, rhamnose, ribose and arabinose in the whole-cell hydrolysates. Diagnostic phospholipid is phos-

phatidylethanolamine and branched anteiso-C17 : 0 (30.0–38.0%), anteiso-C15 : 0 (12.5–14.0%), iso-

C16 : 0 (10.0–15.0%) and iso-C15 : 0 (10.0–12.0%) were detected as the major cellular fatty acids. The acyl type of the peptidoglycan was glycolyl and mycolic acids were not detected. The GC con- tent of the DNA was 71 mol%. The chemotaxonomic data indicate that these strains belong to the family Micromonosporaceae. Phylogenetic analysis based on 16S rRNA gene sequence data suggested that the strains 3-9(24)T, 3-21(27) and 7-40(26)T fall within the family Micromonospo- raceae. On the basis of phylogenetic analysis and characteristic patterns of signature nu- cleotides as well as morphological and chemotaxonomic data, Luedemannella gen. nov. is pro- posed for our 3 isolates. DNA-DNA hybridization experiment and phenotypic characterization in- dicated that the new genus was constituted of 2 species, as Luedemannella helvata sp. nov. for the strain 3-9(24)T (JCM 13249TMTCC 8091T) and Luedemannella flava for the strain 7-40(26)T (JCM 13250TMTCC 8095T) in the family Micromonosporaceae.

Key Words——actinomycetes; Micromonosporaceae; polyphasic taxonomy

Introduction and Goodfellow et al. (1990) emended the family and added the genera Actinoplanes, Dactylosporangium The family Micromonosporaceae was first described and Pilimelia on the basis of numerical and chemical by Krasil’nikov (1938) based on morphological basis analyses. Koch et al. (1996) emended the description of the family to reflect its phenotypic and chemotaxo- *Present address and address reprint requests to: Dr. Ismet nomic heterogeneity. Later Stackebrandt et al. (1997) Ara, Kitasato Institute for Life Sciences, Kitasato University, emended the family on the basis of 16S rDNA/rRNA 5–9–1 Shirokane, Minato-ku, Tokyo 108–8641, Japan. sequence phylogenetic clustering and the presence of Tel/Fax: 81–3–5791–6133 taxon-specific 16S rDNA/rRNA signature nucleotides. E-mail: [email protected] The DDBJ accession numbers for the 16S rDNA sequences At present, the family Micromonosporaceae consists of of strains 3-9(24)T, 3-21(27) and 7-40(26)T are AB236957, 15 genera, each genus having distinctive morphologi- AB236958 and AB236959, respectively. cal features and/or chemotaxonomic characteristics. 40 ARA and KUDO Vol. 53

Micromonospora and Salinispora species form single lated from sandy soil collected in Chokoria and Cox’s spores on short or long sporophores; Actinoplanes, Bazar, Bangladesh. Strains were isolated using the Dactylosporangium, Pilimelia and Virgisporangium standard dilution plate method and grown on humic species form sporangia; Catellatospora, Asanoa, acid-vitamin agar (HV) (Hayakawa and Nonomura, Catenuloplanes, Couchioplanes, Longispora, Actino- 1987) supplemented with cycloheximide (50 mg L1), catenispora, Polymorphospora and Spirilliplanes nystatin (50 mg L1) and nalidixic acid (20 mg L1). species form spore chains; Verrucosispora species do After 21 days of aerobic incubation at 30°C, the strains not form aerial mycelium or sporangia; Actinoplanes, were transferred and purified on yeast extract-malt ex- Dactylosporangium, Pilimelia, Catenuloplanes, Couch- tract agar [(medium 2 of the International Strepto- ioplanes and Spirilliplanes spores show motility; myces Project (ISP medium 2)] and maintained as Catenuloplanes and Couchioplanes species have ly- working cultures on yeast-starch agar containing solu- sine instead of meso-diaminopimelic acid as the di- ble starch, 15.0 g; yeast extract, 4.0 g; K2HPO4, 0.5 g; amino acid of the peptidoglycan. The genus Actino- MgSO4 ·7H2O, 0.5 g; and agar, 15.0 g in 1 L of distilled planes (Couch, 1950) emend. Stackebrandt and Krop- water (pH 7.2) (JCM medium 61). penstedt 1987 (Stackebrandt and Kroppenstedt, 1987) Strains 3-9(24)T, 3-21(27) and 7-40(26)T were grown is a member of the family Micromonosporaceae on sucrose-nitrate agar (Waksman medium 1) and 1/5 (Krasil’nikov, 1938) emend. Stackebrandt et al. 1997 yeast extract-starch agar media at 30°C for 21 days (Stackebrandt et al., 1997). Organisms placed in the and then observed by light and scanning electron mi- genus Actinoplanes are characterized by the presence croscope (model S-2400 Hitachi, Tokyo, Japan). The of spherical, cylindrical, digitate, lobate, bottle or flask- sample for scanning electron microscopy was pre- shaped or very irregular sporangia; sporangiospores pared as described by Itoh et al. (1989) and Ara and are motile with tufts of polar flagella; aerial hyphae are Kudo (2006). The phenotypic properties were exam- scant. The peptidoglycan contains meso-diamino- ined using several standard methods. For cultural pimelic acid, which may be replaced by hydroxydi- characterization, the isolates were grown for 21 days aminopimelic acid. Xylose is the diagnostic sugar of at 30°C on various agar media as described by Waks- whole-cell hydrolysates and small amounts of galac- man (1950, 1961); Shirling and Gottlieb (1966) and tose and/or arabinose are also frequently found. Asano and Kawamoto (1986) (Table 1). The Color Har- Phosphatidylethanolamine is the diagnostic phospho- mony Manual (Jacobson et al., 1958) was used to de- lipid. Iso-/anteiso-branched and monounsaturated fatty termine the names and designations of colony colors. acids and/or cis-9,10-octadecanoic acid (oleic acid) The temperature range and NaCl tolerance for growth are predominant. The predominant isoprenoid quinone were determined on yeast-starch agar (JCM medium is MK-9(H4). The G C content of DNA ranges from 72 61). Utilization of carbohydrates as sole carbon to 73 mol%. The type species is Actinoplanes philip- sources was tested by using neutralized yeast nitrogen pinensis Couch. base without amino acids as a basal medium accord- Morphological and chemotaxonomic properties of ing to the method of Stevenson (1967). Production of these three strains, 3-9(24)T, 3-21(27) and 7-40(26)T, melanoid pigments was examined using tyrosine agar that formed spherical sporangial structures on sub- (ISP medium 7). strate mycelia resembling the genus Actinoplanes, The freeze-dried cells used for chemotaxonomic have been isolated from sandy soil samples from analyses were obtained from cultures grown in yeast- Chokoria and Kasturi Ghat, Cox’s Bazar, Bangladesh. starch broth (JCM medium no. 61) on a rotary shaker On the basis of their 16S rDNA sequences, phyloge- at 30°C. The isomers of diaminopimelic acid (DAP) in netically they formed a lineage within the family Mi- the cell wall peptidoglycan were determined by using cromonosporaceae but not within any existing genera TLC as described by Staneck and Roberts (1974). Re- of the family. Therefore, we propose that the isolates ducing sugars from whole-cell hydrolysates were ana- should be included in a new genus Luedemannella. lyzed by the HPLC method of Mikami and Ishida (1983). The N-acyl group of muramic acid in peptido- Materials and Methods glycan was determined by the method of Uchida and Aida (1984). Phospholipids in cells were extracted and Strains 3-9(24)T, 3-21(27) and 7-40(26)T were iso- identified by the method of Minnikin et al. (1984). 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 41

Methyl esters of cellular fatty acids were prepared and and were analyzed by a HPLC equipped with a Cos- analyzed according to the instructions of the Microbial mosil 5C18 column (4.6 by 150 mm; Nacalai Tesque, Identification System (MIDI) (Sherlock Microbial Identi- Kyoto, Japan). Preparation and detection of methyl es- fication System; MIDI, Hewlett Packard, Palo Alto, CA, ters of mycolic acids were carried out as described by USA) (Sasser, 1990). Isoprenoid quinones were ex- Tomiyasu (1982). tracted by the method of Collins et al. (1977, 1984) Genomic DNA extraction, PCR-mediated amplifica-

Fig. 1. Light micrographs and scanning electron micrographs of globose sporangia on substrate mycelia of Luede- mannella helvata 3-9(24)T (a, b), L. helvata 3-21(27) (c) and Luedemannella flava 7-40(26)T (d, e, f) grown on sucrose- nitrate agar for 21 days at 30°C. 42 ARA and KUDO Vol. 53 tion of the 16S rRNA gene, and sequencing of the minor modification as follows: achromopeptidase PCR products were carried out as described by Naka- crude (Wako Pure Chemicals), N-acetylmuramidase jima et al. (1999). The sequences were multiply SG (Seikagaku Kogyo) and lysozyme were used for aligned with selected sequences (Fig. 1) obtained from lysing cells (Kudo et al., 1998). In case that cells failed the GenBank/EMBL/DDBJ databases by using the to be lysed by these enzymes, the cells were freeze- CLUSTAL X program package (Thompson et al., dried and mechanically ground as described by 1997). The alignment was manually verified and ad- Raeder and Broda (1985). The GC content of the justed prior to construction of a phylogenetic tree. A DNA was determined using the HPLC method of phylogenetic tree constructed by the neighbor-joining Tamaoka and Komagata (1984). An equimolar mixture method (Saitou and Nei, 1987) in the CLUSTAL X pro- of nucleotides for analysis of DNA base composition gram package (Thompson et al., 1997) was based on (Yamasa Shoyu, Choshi, Japan) was digested by bac- the comparison of 1,339 nucleotides present in all the terial alkaline phosphatase and used as the quantita- strains as a result of elimination of gaps and ambigu- tive standard. DNA-DNA relatedness was measured ous nucleotides from the sequences (Escherichia coli fluorometrically using the microplate hybridization position number) and Streptomyces ambofaciens was method devised by Ezaki et al. (1989). Hybridization used as an outgroup. The confidence values of was carried out at 55°C for 2 h. Signature nucleotides branches of the phylogenetic tree were determined in the 16S rRNA gene of new taxa and members of the using bootstrap analyses based on 1,000 resamplings family Micromonosporaceae were determined after (Felsenstein, 1985). manually verification of the CLUSTAL X alignment of DNA was isolated from biomasses by the method of sequences and the nucleotide positions were num- Tamaoka (1994) and Saito and Miura (1963) with bered according to the corresponding position in the

Fig. 2. Phylogenetic tree showing the position of the new genus Luedemannella under the family Micromonosporaceae based on nearly complete 16S rDNA analysis. Numbers at nodes indicate the level (%) of bootstrap support based on neighbor-joining analysis of 1,000 resampled datasets. Only values 40% are shown. Bar, 0.01 nucleotide substitution per 100 nucleotides. Fig. 2. Details. 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 43

16S rRNA sequence of E. coli (Brosius et al., 1978). in the sporangium were nonmotile. The spores were The nucleotide sequence data reported in this paper spherical to oval shaped with a smooth surface and will appear in the DDBJ, EMBL and GenBank nu- loosely arranged in sporogenous hyphae (Fig. 1). Al- cleotide sequence databases with the accession num- though morphology is the sole criterion used to distin- bers listed in Fig. 2. guish genera in the family Micromonosporaceae, strain 3-9(24)T, 3-21(27) and 7-40(26)T formed irregular to Results and Discussion globose sporangia on substrate mycelium, which re- sembled the sporangia of the genus Actinoplanes The isolates 3-9(24)T, 3-21(27) and 7-40(26)T were under light microscope and scanning electron micro- gram-positive, non-acid-fast and aerobic organisms scope. The isolates 3-9(24)T, 3-21(27) and 7-40(26)T with branched hyphae. A non-fragmenting substrate were distinguished from the genus Actinoplanes based mycelium was formed. Morphological observations by on 16S rDNA sequence analysis and by the absence light microscope of a 21-d-old culture grown on su- of motile spores (Table 5). Further, 3-9(24)T, 3-21(27) crose-nitrate agar and 1/5 yeast extract-starch agar re- and 7-40(26)T differ from the genus Actinoplanes in vealed the presence of single or clustered spherical to menaquinone pattern and fatty acid contents (Table 3). irregular sporangia on substrate mycelium (Fig. 1). Ob- Unlike members of the genus Actinoplanes, however, servation by scanning electron microscope indicated which usually develop motile arthrospores within the that the spherical to irregular structures were variable substrate mycelia arranged in chains inside sporangial in size ( 3.0–5.0 mm). Spores in sporogenous hyphae wall, they contain MK-9(H4) and MK-10(H4) in their cell Table 1. Cultural characteristics of isolates 3-9(24)T, 3-21(27) and 7-40(26)T.

3-9(24)T 3-21(27) 7-40(26)T

Agar medium Growth/Reverse color/ Growth/Reverse color/ Growth/Reverse color/ Sporulation Sporulation Sporulation

Glucose-asparagine /Shell(3ca)//Light wheat(2ea)//Colorless/ Glycerol-asparagine /Colorless//Colorless//Colorless/ Salts-starch /Colorless//Shell(3ca)//Colorless/ Tyrosine /Colorless//Colorless/ /Cream(11/2)/ Nutrient /Shell(3ca)/ /Maize(2ga)//Light wheat(2ea)/ Yeast extract-malt extract /Honey gold(2ic)//Honey gold(2ic)//Maize(2ga)/ Oatmeal /Shell(3ca)//Light wheat(2ea)//Dusty coral(6gc)/ Bennett /Melon yellow(3ga)//Light wheat(2ea)//Light wheat(2ea)/ Glucose-yeast extract /Brown maize(3la)//Maize(2ga)//Light wheat(2ea)/ Hickey-Tresner /Shell(3ca)//Light wheat(2ea)//Light wheat(2ea)/ Water /Colorless//Colorless//Colorless/ Sucrose-nitrate /Shell(3ca)/ /Colorless/ /Colorless/ Yeast extract-starch /Colorless/ /Light wheat(2ea)//Light wheat(2ea)/ Oatmeal-nitrate /Shell(3ca)//Shell(3ca)//Colorless/ 1/5 yeast extract-starch /Colorless/ /Colorless/ /Dusty coral(6gc)/ Sucrose-beef extract /Shell(3ca)//Honey gold(2ic)/ ND ISP6 //// ISP1 /Shell(3ca)//Light wheat(2ea)/ 1/20 V8 juice* /Shell(3ca)/ /Shell(3ca)/ Humic acid-vitamin /Colorless//Colorless/ 1/10 yeast-malt extract ////

Cultures were incubated at 30°C for 3 weeks. Aerial mycelium and pigmentation were not formed on all of the agar media tested. Color designations and codes in parentheses were taken from the Color Harmony Manual (Jacobson et al., 1958). Growth and sporulation on aerial mycelium are scored as: (), good; (), moderate; (), doubtful/poor; (), no growth and no spore formed;

*, V8 canned vegetable juice (Campbell Soup Co.); ND, not detected. 44 ARA and KUDO Vol. 53 wall (Table 5). Table 2. Physiological characteristics of the isolates 3-9(24)T The physiological properties of strains 3-9(24)T, 3- and 7-40(26)T. 21(27) and 7-40(26)T can be summarized as follows: T T Strains 3-9(24)T, 3-21(27) exhibited good growth on Characteristic 3-9(24) 7-40(26) yeast-extract-malt extract agar, Bennett agar, glucose- Utilization of: yeast extract agar and moderate growth on inorganic D-Glucose salt-starch agar, oatmeal agar, Hickey-Tresner agar, Glycerol sucrose-nitrate agar, yeast extract-starch agar, oat- Erythritol meal-nitrate agar, 1/5 yeast extract-starch agar, su- Adonitol crose-beef extract agar, ISP medium 1 agar, and 1/20 L-Arabinose T D-Ribose V8 juice agar (Table 1). Strain 7-40(26) exhibited good growth on oatmeal agar (ISP medium 3), Bennett agar, D-Xylose glucose-yeast extract agar, Hickey-Tresner agar, yeast D-Galactose D-Fructose extract-starch agar (JCM medium 61) and 1/5 yeast D-Mannose extract-starch agar (JCM medium 202) (Table 1). The L-Rhamnose T T strains 3-9(24) , 3-21(27) and 7-40(26) did not pro- myo-Inositol duce soluble pigment on any agar medium tested and D-Mannitol T aerial hyphae were not formed. The strain 3-9(24) uti- a-Methyl-D-glucoside lized D-glucose, L-arabinose, maltose and trehalose; Salicin grew well at 20–37°C and pH 5–9 and could not grow Lactose on 3% NaCl. On the other hand the strain 7-40(26)T a-D( )Melibiose Sucrose utilized D-glucose, L-arabinose, D-xylose, D-galactose, D()Raffinose D-mannose, L-rhamnose, salicin, lactose, a-D()meli- Maltose biose, sucrose, maltose and trehalose; grew well at Trehalose 20–30°C and pH 6–9 and could not grow on 2% NaCl Growth at (°C): 20–37 20–30 (Table 2). Growth at (pH): 5–9 6–9 Chemotaxonomic properties have been found to be Growth on 2% NaCl an important contribution to the polyphasic approach Melanin pigment to bacterial systematics, especially in the circumscrip- Characteristics are scored as (), positive; (), moderate; tion of phylogenetically coherent actinomycete taxa (), doubtful/poor; (), negative. (Goodfellow, 1991; Kroppenstedt and Goodfellow, 1991). The family Micromonosporaceae is a taxon mainly defined by chemotaxonomy and our isolates, 3- C17 : 0 (3.0–10.0%), unsaturated C17 : 1 w8c (3.0–5.0%) T T 9(24) , 3-21(27) and 7-40(26) , shared chemotaxo- and branched iso-C17 : 0 (2.5–3.4%), indicating fatty nomic characteristics with members of this family as acid type 2d of Kroppenstedt (1985) (Table 4). Mycolic follows: cell walls contained meso-diaminopimelic acid acids were absent. Phosphatidylethanolamine, diphos- and glucose, xylose galactose, mannose, rhamnose, phatidylglycerol, phosphatidylglycerol and phosphatidyl- ribose and arabinose as the whole cell sugars indicat- inositol and phosphatidylinositol mannosides were de- ing the whole-cell sugar pattern D according to tected, corresponding to phospholipid type PII of Lechevalier and Lechevalier (1970). The major Lechevalier et al. (1981). The acyl type of the cell wall menaquinones MK-9(H6), and MK-9(H4) and small was glycolyl. The G C contents of DNA were 71 T T amounts of MK-9(H2) and MK-9(H8) were present. Our mol%. Strains 3-9(24) , 3-21(27) and 7-40(26) are isolates have different predominant menaquinone pat- wall chemotype II organisms. The genera Micromono- terns from those of the other genera of the family Mi- spora, Actinoplanes, Dactylosporangium and Pilimelia cromonosporaceae (Table 3). are wall chemotype II genera, but genera The strains 3-9(24)T, 3-21(27) and 7-40(26)T com- Actinoplanes, Dactylosporangium and Pilimelia have posed of branched anteiso-C17 : 0 (30.0–38.0%), an- motile spores and a different menaquinone pattern teiso-C15 : 0 (12.5–14.0%), iso-C16 : 0 (10.0–15.0%) and (Table 5). iso-C15 : 0 (10.0–12.0%) and small amount of saturated The almost complete 16S rRNA gene sequences 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 45

Table 3. Chemotaxonomic characteristics of isolates 3-9(24)T, 3-21(27) and 7-40(26)T.

Characteristics 3-9(24)T 3-21(27) 7-40(26)T

Cell wall diamino acid meso-DAP meso-DAP meso-DAP Characteristic sugar (s) Glucose, xylose, galactose, Glucose, xylose, rhamnose, Glucose, xylose, mannose, mannose, rhamnose, ribose, mannose, galactose, ribose, galactose, ribose, arabinose, arabinose arabinose rhamnose Phospholipid (s) DPG, PE, PG, PI, PIMs DPG, PE, PG, PI, PIMs DPG, PE, PG, PI, PIMs

Menaquinone (s) MK-9(H6), MK-9(H4), MK-9(H2), MK-9(H6), MK-9(H4), MK-9(H2), MK-9(H6), MK-9(H8)

MK-9(H8) MK-9(H8) N-acyl group of muramic acid Glycolyl Glycolyl Glycolyl Mycolic acid Negative Negative Negative

Abbreviations: DAP, diaminopimelic acid; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;

PI, phosphatidylinositol; PIMs, phosphatidylinositol mannosides; Abbreviations for menaquinones are exemplified by MK-9(H6): a hexahydrogenated menaquinone with nine isoprene units; (), scored as negative.

Table 4. Cellular fatty acid compositions of isolates 3-9(24)T, edness (85%) (mean of six replications, 16S rRNA 3-21(27) and 7-40(26)T. gene sequence similarities is 100%) (data not shown) indicates that the phylogenetically closest strains 3- a T T Fatty acid 3-9(24) 3-21(27) 7-40(26) 9(24)T and 3-21(27) are the same species under the new taxon Luedemannella and their morphological Saturated b features are also similar to each other. The low level C15 : 0 1.30 1.08 1.83 (50%) (mean of six replications species, 16S rRNA C16 : 0 tr tr 2.44 gene sequence similarities is 98.9%) of DNA-DNA re- C17 : 0 3.07 2.79 10.10 T T C18 : 0 1.16 1.49 2.38 latedness between the isolates 3-9(24) and 7-40(26) Unsaturated indicate that these two isolates are new under the 2OH-C16 : 1 4.26 3.19 tr genus Luedemannella. It was reported by Wayne et al. C 8c 3.65 2.52 5.15 17 : 1 w (1987) that value of DNA-DNA relatedness is 70% cut- C 9c 1.42 1.54 3.74 18 : 1 w off point for the delineation of genomic species, and Branched therefore we propose the classification of two new i-C 10.19 11.60 11.92 15 : 0 species for the strains 3-9(24)T and 7-40(26)T under a-C15 : 0 13.75 12.47 14.00 the new genus Luedemannella gen. nov. Table 5 sum- i-C16 : 0 12.78 14.77 9.62

i-C17 : 0 2.48 3.38 2.72 marizes the differential characteristics of the new

a-C17 : 0 37.86 36.74 29.93 genus and other members of the family Micromono-

a-C17 : 1 1.88 1.77 sporaceae. Signature nucleotide patterns in the 16S rRNA gene a Values are percentages of total cellular fatty acids. Trace of the new taxon and members of the family Mi- amounts (less than 1.0%) are not shown. cromonosporaceae are presented in Table 6. It is evi- b Number of carbon atoms : number of double bonds. Abbre- viations: i, iso; a, anteiso. dent in our study that of the 11 signatures defined for the family Micromonosporaceae (Stackebrandt et al., 1997), all are present in the 16S rRNA gene se- (1,500 nt, 1,496 nt and 1,506 nt ) of strains 3-9(24)T, 3- quences of the strains 3-9(24)T, 3-21(27) and 7- 21(27) and 7-40(26)T were determined in this study. 40(26)T except for the “U-A” pair at position 139–224, The phylogenetic position of the isolates is within the “A” at position 381, the “A/G-C” pair at position confines of the family Micromonosporaceae (Fig. 2) 656–750 and the C-U pair at position 999–1041. Fur- but form a lineage distinct from described organisms. ther that several nucleotide pairs differentiating the Table 6 summarizes the differential characteristics of new taxon Luedemannella from the members of the the new genus and other members of the family Mi- family Micromonosporaceae are shown in Table 6. cromonosporaceae. Further, the high DNA-DNA relat- The distinctness of the 16S rRNA sequences, phylo- 46 ARA and KUDO Vol. 53 ) - 4,6,8 meso 10(H Xyl Ara, Gal, - ) 4 . meso 9(H ) - 4 meso 10(H ) - 4,2 Micromonosporaceae 9(H meso , absent; ND, not determined; Xyl, xylose; t al. (1994, 1997, 2001, 2006); Rheims et ) - 4,6,8 and Maldonado et al. (2005). 9(H meso , present; and the family ) 4 -Lys L ) 8 ), 9(H 8 -Lys Luedemannella 10(H L ) ) or 9(H - 4,6 4,6 meso Rham, Rib 9(H 10(H Catellato- Catenul- Couchio- Dactylo- Pilim- Spirilli- Verruco- Virgispo- ) - 6,8 meso Asanoa ) - 4 ), 10(H 4 meso 10(H ), 9(H ) - 6,4 6,4 meso 10(H 9(H ) - 4,6 meso 9(H Actinocat- Polymor- Actino- - - ) 4 meso ), 9(H ) - 4,6 4,6 meso 10(H ) - 4,6 meso 10(H ) - 6,4,2,8 able 5. Morphological features and chemotaxonomic characteristics of the new taxon T DAP DAP DAPRib, Ara DAP DAP DAP DAP DAP DAP DAP DAP DAP DAP DAP Rham, nnella spora nospora sporameso enispora phospora planes spora oplanes planes porangium elia planes sispora rangium Luedema- Longi- Micromo- Salini C content 71 70 71–72 70–73 72 71 72–73 71–72 70–71 71–73 69–73 71–73 ND 69 70 71 type** (MK-) type* menaquinone(s) 9(H (mol%) Data were taken from, Vobis (1989), Stackebrandt & Kroppenstedt (1987); Goodfellow et al. (1990); Yokota et al. (1993);Tamura e et al. (1993);Tamura (1989), Stackebrandt & Kroppenstedt (1987); Goodfellow et al. (1990); Yokota Data were taken from, Vobis *, according to the classification of Lechevalier et al. (1977); **, Kroppenstedt (1985); Fatty-acid 2d 2d 3b 3a 3b 2a 2d 2d 3b 2c 2c 3b 2d 2d 2b 2d Whole-cell Xyl, Gal, Ara, Gal, Ara, Xyl Ara, Gal, Ara, Gal, Xyl Ara, Xyl Ara, Gal, Xyl,Man, Xyl Ara, Gal, Ara, Xyl Ara, Xyl Ara, Xyl Man, sugars Man, Xyl Xyl Xyl XylAra, Gal, Xyl Xyl Phospholipid PIIG PII PII PII PII PII PII PII PII PIII PII PII PII PII PII PII Sporangium Spore motility Diamino acid Characteristic Major 9(H al. (1998); Kane, 1966; Kudo et (1999); Lee (2000); & Hah (2002); Matsumoto (2003); Thawai (2006) Gal, galactose; Man, mannose; Rham, rhamnose; Rib, ribose; Ara, arabinose. 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 47 . Spirilli- Verrucosi- Virgospo- Micromonosporaceae Pilimelia from family Luedemannella AAAAAAA CAUACCA/C /C numbering (Brosius et al., 1978). E. coli AG UA GGGGGAGGG GGGGGAGAA UAUAUUAAUA Catenul- Couchio- Catellato- Dactylospor- Longi- Micromono- AA AG GA GA C/ Asanoa AAAAUUUAAAAA CCCCCACCCCCC GGGGGGGGG/AGGG GGGGGGGGAGGG able 6. new genus 16S rDNA signature nucleotide positions that differentiate T A A AUAUAAA/UAAUUUA CUUUCUUCCUUCU CUUUUCCUCUCCC UC UCCCCUCCCCCCC GA GA GA *A nella planes oplanes planes spora angium spora spora planes spora rangium Luedeman- Actino- a Nucleotide positions of bases or base pairs are given according to the 19–1154 U–G U–A U–A U–G U–A U–G U–A U–G U–A U–A/G U–A C–G C–G a 121–1152 C–G G–C G–C C–G G–C C–G G–C C–G G–C C–G G–C G–C C/G–G/C Position 129 139–224140–223 *U–A144–178222 G–C232 U–G A–U G–U U–A G–U G–C U–A A–U G–C U–G G–U G–C U–G U–G G–U U–A/G A–U G–C U–A U–G C–G U–G A–U G–U U–A U–G G/ C–C/G U–G U–G A–U U–A G–U A–U G–U/C U–A U/G–A U–A 1445–1457 U–G U–G C–G C–G U–G C–G C–G C–G C–G C–G U–A C–G C–G 262 381 415 456 594.645602–636616–624 U.G615–625 C–G656–750 G–C C.G836.850 C–G A–U *A/G–C859 G–U968 G.C G–C G–C C.G C–G G–U U–G G.U G–C C.G A–U G–U U–G G–C G.U C.G C–G G–U C–G G–C G.U C–G U.G G–U G–C G–C G.U C–G U.G A–U C–G G–C C–G G.U C.G G–U A–U G–C C–G G.U C.G G–C G–C C–G C–G G.U C.G A–U C–G A–U C–G G.C G–U C.G C–G G–C C–G G.U G–U C.G U–G G–C C–G A–U G.U C.G C–G G–C G.U G.U 546 998–1043999–1041 G–C1001–1039 *C–U1003 U–G1006 C–G C–G1009–1020 G–C1010 C–G C–G U 1011–1018 C–G1012–1017 G–C U–A C–G11 C–G G–C A C–G1 G–C C–G U–G C–G A–U C–G C–G G C–G G–C/G U–G A–U C–G G/C–C/G G–C C–G C–G C G–C A–U G–U G–C C–G C–G G–C A–U G–U C C/G–G/C G–C C–G G–C A–U C–G C–G G/U–C C–G G G–C A/C–U A–U U–G C–G C–G C–G G–C G A–U U–A C–G C–G C–G G–C C–G A–U G–C U/C–A/G G C–G G–C C–G U–G A–U A C–G C–G A–U C–G G A–U A–U G A G G 1252 A A U 48 ARA and KUDO Vol. 53 genetic position and the specific signature nucleotide iso-C16 : 0 (10.0–15.0%) and iso-C15 : 0 (10.1–12.0%) T patterns of the 16S rRNA gene of strains 3-9(24) , 3- and small amounts of saturated C17 : 0 (3.0–10.0%), un- T 21(27) and 7-40(26) differentiated them from known saturated C17 : 1 w8c (3.0–5.0%) and branched iso- actinomycetes belonging to the family Micromonospo- C17 : 0 (2.5–3.4%) are present, indicating fatty acid type raceae and for that reason these isolates merit new 2d. Mycolic acids are absent. Phosphatidylethanol- genus status. Moreover, on the basis of morphological, amine, diphosphatidylglycerol, phosphatidylglycerol, physiological, chemotaxonomic, and phylogenetic cri- phosphatidylinositol and phosphatidylinositol manno- teria and signature nucleotide patterns of the 16S sides are present. The acyl type of the cell wall is gly- rRNA gene, the strains 3-9(24)T, 3-21(27) and 7- colyl. The GC contents of DNA are 71 mol%. The 40(26)T are readily distinguishable from the closest genus is placed in the family Micromonosporaceae on genera Catellatospora and Actinoplanes and other the basis of phylogenetic position of the strains based motile actinomycetes mentioned above and warrant a on the distinct 16S rRNA gene sequences. All family- new taxon. Therefore, we propose the strains 3-9(24)T, specific nucleotide signatures of 16S rRNA gene are 3-21(27) and 7-40(26)T be classified in a new genus, present except for the “U-A” pair at position 139–224, the genus Luedemannella, whose the type species is “A” at position 381, the “A/G-C” pair at position Luedemannella helvata sp. nov. 656–750 and the C-U pair at position 999–1041. The type species is Luedemannella helvata. Description of Luedemannella gen. nov. Luedemannella (Lue.de.mann.ella. N.L. adj. Luede- Description of Luedemannella helvata sp. nov. mannella, referring to G. M. Luedemann, a Russian Luedemannella helvata (hel.va’ta. N.L. adj. helvata actinomycetologist who contributed to the taxonomy of honey yellow referring to the color of the substrate the family Micromonosporaceae). mycelium). Cells are gram-positive, non-acid-fast, aerobic or- Morphological, chemotaxonomic and general char- ganisms with branching hyphae. Non-fragmenting light acteristics are as given above for the genus descrip- wheat- to honey gold-colored substrate mycelia are tions. The organism shows moderate growth on ISP present. Spores are found borne in spherical shaped medium 4, ISP medium 3, Hickey-Tresner agar, su- sporangia (3.0 to 5.0 mm) on substrate mycelium. crose-nitrate agar, yeast extract-starch agar (JCM Several spores are present per sporangium, and the medium 61), oatmeal-nitrate agar, 1/5 yeast extract- spores are spherical to oval (0.2 to 0.4 mm) with a starch agar (JCM medium 202), sucrose-beef extract smooth surface and nonmotile when they are sus- agar, ISP medium 1, and 1/20 V8 juice agar; poor pended in sterile distilled water. The organism utilizes growth on glucose-asparagine agar, glycerol-as- D-glucose, L-arabinose, D-xylose, D-galactose, D-man- paragine agar, tyrosine agar (ISP medium 7), nutrient nose, L-rhamnose, salicin, lactose, a-D()melibiose, agar, tap water agar, and humic acid-vitamin agar; and sucrose, maltose and trehalose. The organism grows negative growth on ISP medium 6 and 1/10 yeast ex- well at 20–37°C and pH 5–9 and neither could grow on tract-malt extract agar. Melanin pigment production on 3% NaCl. The organism shows good growth on yeast ISP medium 7 is negative. Abundant sporulation oc- extract-malt extract agar (ISP medium 2), oatmeal curs on nutrient agar, sucrose-nitrate agar, yeast ex- agar (ISP medium 3), Bennett agar, glucose-yeast ex- tract-starch agar, 1/5 yeast extract-starch agar, and tract agar, Hickey-Tresner agar, yeast extract-starch 1/20 V8 juice agar, and moderate sporulation occurs agar (JCM medium 61) and 1/5 yeast extract-starch on glucose-asparagine agar, glycerol-asparagine agar, agar (JCM medium 202). The cell wall contains meso- tyrosine agar, yeast extract-malt extract agar, oatmeal diaminopimelic acid, and Wall chemotype is II. Galac- agar, Bennett agar, glucose-yeast extract agar, Hickey- tose, mannose, glucose, xylose and rhamnose are de- Tresner agar, tap water agar, and oatmeal-nitrate agar. tected as the major whole cell sugars in addition to In general the vegetative mycelia are shell to melon small amounts of ribose and arabinose, indicating that yellow in color and aerial mycelia are not present. D- the whole-cell sugar pattern is D. Major menaquinones Xylose, lactose, sucrose and D-raffinose are moder- are MK-9(H6) and MK-9(H4) and small amounts of MK- ately utilized, erythritol, D-galactose and salicin are

9(H2) and MK-9(H8) are present. In general, branched poorly utilized, and glycerol, adonitol, D-ribose, D-fruc- anteiso-C17 : 0 (30.0–38.0%), anteiso-C15 : 0 (12.5–14.0%), tose, D-mannose, L-rhamnose, myo-inositol, D-manni- 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 49 tol, a -methyl-D-glucoside, and a -D-melibiose are not (1978) Complete nucleotide sequence of a 16S ribosomal utilzed. The GC content of DNA is 71 mol%. The RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. habitat is soil. The type strain is 3-9(24)T (JCM USA, 75, 4801–4805. 13249TMTCC 8091T). Collins, M. D., Faulkner, M., and Keddie, M. (1984) Menaquinone composition of some sporeforming actino- mycetes. Syst. Appl. Microbiol., 5, 20–29. Description of Luedemannella flava sp. nov. Collins, M. D., Pirouz, T., Goodfellow, M., and Minnikin, D. E. Luedemannella flava (fla’va. N.L. adj. flava yellow (1977) Distribution of menaquinones in actinomycetes and referring to the color of the substrate mycelium). corynebacteria. J. Gen. Microbiol., 100, 221–230. Morphological, chemotaxonomic and general char- Couch, J. N. (1950) Actinoplanes a new genus of the Actino- acteristics are as given above for the genus descrip- mycetales. J. Elisha Mitchell Sci. Soc., 66, 87–92. tions. The organism shows moderate growth on yeast Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric extract-malt extract agar, and oatmeal-nitrate agar, deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter and poor growth on glucose-asparagine agar, glycerol- hybridization in which radioisotopes are used to determine asparagine agar, ISP medium 4, ISP medium 7, nutri- genetic relatedness among bacterial strains. Int. J. Syst. ent agar, tap water agar, and sucrose-nitrate agar. Bacteriol., 39, 224–229. Melanin pigment production on ISP medium 7 is nega- Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- tive. Abundant sporulation occurs on glucose-as- proach using the bootstrap. Evolution, 39, 783–791. paragine agar, ISP medium 5, ISP medium 4, tap Goodfellow, M. (1991) The family Micromonosporaceae. In The water agar, and 1/5 yeast extract-starch agar; moder- Prokaryotes, 2nd ed., ed. by Balows, A., Truper, H. G., ate sporulation on ISP medium 7, glucose-yeast ex- Dworkin, M., Harder, W., and Schleifer, K. H., Springer, tract agar, Hickey-Tresner agar, sucrose-nitrate agar, New York, pp. 1115–1138. Goodfellow, M., Stanton, L. J., Simpson, K. E., and Minnikin, D. and oatmeal-nitrate agar; and no sporulation on nutri- E. (1990) Numerical and chemical classification of Actino- ent agar, yeast extract-malt extract agar, ISP medium planes and some related actinomycetes. J. Gen. Microbiol., 3, Bennett agar, or yeast extract-starch agar. In gen- 136, 19–36. eral the vegetative mycelia are cream yellow to wheat Hayakawa, M. and Nonomura, H. (1987) Humic acid-vitamin yellow in color and aerial mycelia are not present. agar, a new medium for selective isolation of soil actino- Glycerol, D-ribose, D-fructose, myo-inositol, D-mannitol, mycetes. J. Ferment. Technol., 65, 501–509. and a-methyl-D-glucoside are poorly utilized and ery- Ito, T., Kudo, T., Parenti, F., and Seino, A. (1989) Amended de- scription of the genus Kineosporia, based on chemotaxo- thritol, adonitol, and D-raffinose are not utilized. The nomic and morphological studies. Int. J. Syst. Bacteriol., GC content of DNA is 71 mol%. The habitat is soil. 39, 168–173. The type strain is 7-40(26)T (JCM 13250TMTCC Jacobson, E., Grauville, W. C., and Fogs, C. E. (1958) Color T 8095 ). Harmony Manual, 4th ed., Container Corporation of Amer- ica, Chicago. Acknowledgments Kane, W. D. (1966) A new genus of Actinoplanaceae, Pilimelia, with a description of two species, Pilimelia terevasa and The authors are indebted to Japan Society for Promotion of Pilimelia anulata. J. Elisha Mitchell Sci. Soc., 82, 220–230. Science (JSPS) for financial support. Koch, C., Kroppenstedt, R. M., Rainey, F. A., and Stackebrandt, E. (1996) 16S ribosomal DNA analysis of the genera Mi- References cromonospora, Actinoplanes, Catellatospora, Catenulo- planes, Couchioplanes, Dactylosporangium, and Pilimelia Ara, I. and Kudo, T. (2006) Three novel species of the genus and emendation of the family Micromonosporaceae. Int. J. Catellatospora, Catellatospora chokoriensis sp. nov., Catel- Syst. Bacteriol., 46, 765–768. latospora coxensis sp. nov. and Catellatospora Krasil’nikov, N. A. (1938) Ray Fungi and Related Organisms bangladeshensis sp. nov. and transfer of Catellatospora cit- Actinomycetales, Izdatel’stvo Akademii Nauk SSSR, rea subsp. methionotrophica Asano and Kawamoto 1988 to Moscow. Catellatospora methionotrophica sp. nov. comb. nov. Int. J. Kroppenstedt, R. M. (1985) Fatty acid and menaquinone analy- Syst. Evol. Microbiol., 56, 393–400. sis of actinomycetes and related organisms. In Chemical Asano, K. and Kawamoto, I. (1986) Catellatospora, a new Methods in Bacterial Systematics, ed. by Goodfellow, M. genus of the Actinomycetales. Int. J. Syst. Bacteriol., 36, and Minnikin, D. E., Academic Press, London, pp. 512–517. 173–199. Brosius, J., Palmer, J. L., Kennedy, J. P., and Noller, H. F. Kroppenstedt, R. and Goodfellow, M. (1991) The family Ther- 50 ARA and KUDO Vol. 53

momonosporaceae. In The Prokaryotes, 2nd ed., ed. by (1999) Microbispora corallina sp. nov., a new species of the Balows, A., Truper, H. G., Dworkin, M., Harder, W., and genus Microbispora isolated from Thai soil. Int. J. Syst. Schleifer, K. H., Springer, New York, pp. 1085–1114. Bacteriol., 49, 1761–1767. Kudo, T., Matsushima, K., Itoh, T., Sasaki, J., and Suzuki, K. Raeder, U. and Broda, P. (1985) Rapid preparation of DNA from (1998) Description of four new species of the genus Ki- filamentous fungi. Lett. Appl. Microbiol., 1, 17–20. neosporia: Kineosporia succinea sp. nov., Kineosporia rhi- Rheims, H., Schumann, P., Rohde, M., and Stackebrandt, E. zophila sp. nov., Kineosporia mikuniensis sp. nov. and Ki- (1998) Verrucosispora gifhornensis gen. nov., sp. nov., a neosporia rhamnosa sp. nov., isolated from plant samples, new member of the actinobacterial family Micromonospo- and amended description of the genus Kineosporia. Int. J. raceae. Int. J. Syst. Bacteriol., 48, 1119–1127. Syst. Bacteriol., 48, 1245–1255. Saito, H. and Miura, K. (1963) Preparation of transforming de- Kudo, T., Nakajima, Y., and Suzuki, K. (1999) Catenuloplanes oxyribonucleic acid by phenol treatment. Biochim. Biophys. crispus (Petrolini et al. 1993) comb. nov.: Incorporation of Acta, 72, 619–629. the genus Planopolyspora Petrolini 1993 into the genus Saitou, N. and Nei, M. (1987) The neighbor-joining method: A Catenuloplanes Yokota et al. 1993 with an amended de- new method for reconstructing phylogenetic trees. Mol. scription of the genus Catenuloplanes. Int. J. Syst. Bacte- Biol. Evol., 4, 406–425. riol., 49, 1853–1860. Sasser, M. (1990) Identification of Bacteria by Gas Chromatog- Lechevalier, M. P., DeBievre, C., and Lechevalier, H. A. (1977) raphy of Cellular Fatty Acids (MIDI Technical Note 101), Chemotaxonomy of aerobic actinomycetes: Phospholipid MIDI, New York, DE. composition. Biochem. Syst. Ecol., 5, 249–260. Shirling, E. B. and Gottlieb, D. (1966) Methods for characteriza- Lechevalier, M. P. and Lechevalier, H. A. (1970) Chemical com- tion of Streptomyces species. Int. J. Syst. Bacteriol., 16, position as a criterion in the classification of aerobic actino- 313–340. mycetes. Int. J. Syst. Bacteriol., 20, 435–443. Stackebrandt, E. and Kroppenstedt, R. M. (1987) Union of the Lechevalier, M. P., Stern, A. E., and Lechevalier, H. A. (1981) genera Actinoplanes Couch, Ampullariella Couch, and Phospholipids in the taxonomy of actinomycetes. Zentralbl. Amorphosporangium Couch in a redefined genus Actino- Bakteriol. Hyg. Abt. (Suppl. 1), 11, 111–116. planes. Syst. Appl. Microbiol., 9, 110–114. Lee, S. D. and Hah, Y. C. (2002) Proposal to transfer Catel- Stackebrandt, E., Rainey, F. A., and Ward-Rainey, N. L. (1997) latospora ferruginea and ‘Catellatospora ishikariense’ to Proposal for a new hierarchic classification system, Acti- Asanoa gen. nov. as Asanoa ferruginea comb. nov. and nobacteria classis nov. Int. J. Syst. Bacteriol., 47, 479–491. Asanoa ishikariensis sp. nov., with the emended descrip- Staneck, J. L. and Roberts, G. D. (1974) Simplified approach to tion of the genus Catellatospora. Int. J. Syst. Evol. Micro- identification of aerobic actinomycetes by thin-layer chro- biol., 52, 967–972. matography. Appl. Microbiol., 28, 226–231. Lee, S. D., Kang, S.-O., and Hah, Y. C. (2000) Catellatospora Stevenson, I. L. (1967) Utilization of aromatic hydrocarbons by koreensis sp. nov., a novel actinomycete isolated from a Arthrobacter spp. Can. J. Microbiol., 13, 205–211. gold-mine cave. Int. J. Syst. Evol. Microbiol., 50, 1103– Tamaoka, J. (1994) Determination of DNA base composition. In 1111. Chemical Methods in Prokaryotic Systematics, ed. by Maldonado, L. A., Fenical, W., Jensen, P. R., Kauffman, C. A., Goodfellow, M. and O’Donnell, A. G., John Wiley and Sons, Mincer, T. J., Ward, A. C., Bull, A. T., and Goodfellow, M. Chichester, pp. 463–470. (2005) Salinispora arenicola gen. nov., sp. nov. and Salinis- Tamaoka, J. and Komagata, K. (1984) Determination of DNA pora tropica sp. nov., obligate marine actinomycetes be- base composition by reversed-phase high-performance liq- longing to the family Micromonosporaceae. Int. J. Syst. uid chromatography. FEMS Microbiol. Lett., 25, 125–128. Evol. Microbiol., 55, 1759–1766. Tamura, T., Hatano, K., and Suzuki, K. (2006) A new genus of Matsumoto, A., Takahashi, Y., Shinose, M., Seino, A., Iwai, Y., the family Micromonosporaceae, Polymorphospora gen. and Omura, S. (2003) Longispora albida gen. nov., a novel nov., with description of Polymorphospora rubra sp. nov. genus of the family Micromonosporaceae. Int. J. Syst. Evol. Int. J. Syst. Evol. Microbiol., 56, 1959–1964. Microbiol., 53, 1553–1559. Tamura, T., Hayakawa, M., and Hatano, K. (1997) A new genus Mikami, H. and Ishida, Y. (1983) Post-column fluorometric de- of the order Actinomycetales, Spirilliplanes gen. nov., with tection of reducing sugar in high-performance liquid chro- description of Spirilliplanes yamanashiensis sp. nov. Int. J. matography using arginine. Bunseki Kagaku, 32, E207– Syst. Bacteriol., 47, 97–102. E210. Tamura, T., Hayakawa, M., and Hatano, K. (2001) A new genus Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., of the order Actinomnycetales, Virgisporangium gen. nov., Athalye, M., Schaal, A., and Parlett, J. H. (1984) An inte- with descriptions of Virgisporangium ochraceum sp. nov. grated procedure for the extraction of bacterial isoprenoid Int. J. Syst. Evol. Microbiol., 51, 1809–1816. quinines and polar lipids. J. Microbiol. Methods, 2, 233– Tamura, T., Nakagaito, Y., Nishii, T., Hasegawa, T., Stacke- 241. brandt, E., and Yokota, A. (1994) A new genus of the order Nakajima, Y., Kitpreechavanich, V., Suzuki, K., and Kudo, T. Actinomycetales, Couchioplanes gen. nov., with descrip- 2007 Luedemannella gen. nov., Luedemannella helvata sp. nov., and Luedemannella flava sp. nov. 51

tions of Couchioplanes caeruleus (Horan and Brodsky atic Bacteriology, Vol. 4, ed. by Williams, S. T., Sharpe, M. 1986) comb. nov. and Couchioplanes caeruleus subsp. E., and Holt, J. G., Williams and Wilkins, Baltimore, pp. azureus subsp. nov. Int. J. Syst. Bacteriol., 44, 193–203. 2418–2450. Thawai, T., Tanasupawat, S., Itoh, T., and Kudo T. (2006) Waksman, S. A. (1950) The Actinomycetes: Their Nature, Oc- Actinocatenispora thailandica gen. nov., sp. nov., a new currence, Activities and Importance, Chronica Botanica, member of the family Micromonosporaceae. Int. J. Syst. Waltham, MA. Evol. Microbiol., 56, 1789–1794. Waksman, S. A. (1961) Classification, identification, and de- Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., scription of genera and species. In The Actinomycetes, Vol. and Higgins, D. G. (1997) The Clustal X windows interface: 2, Williams and Wilkins, Baltimore. Flexible strategies for multiple sequence alignment aided Wayne, L. G., Brenner, D. J., Colwell, R. R., and 9 other authors by quality analysis tools. Nucleic Acids Res., 24, 4876– (1987) International Committee on Systematic Bacteriology. 4882. Report of the ad hoc committee on reconcilation of ap- Tomiyasu, I. (1982) Mycolic acid composition and thermally proaches to bacterial systematic. Int. J. Syst. Bacteriol., 37, changes in Nocardia asteroides. J. Bacteriol., 151, 828– 463–464. 837. Yokota, A., Tamura, T., Hasegawa, T., and Huang, L. H. (1993) Uchida, K. and Aida, K. (1984) An improved method for the gly- Catenuloplanes japonicus gen. nov., sp. nov., nom. rev., a colate test for simple identification of the acyl type of bacte- new genus of the order Actinomycetales. Int. J. Syst. Bac- rial cell walls. J. Gen. Appl. Microbiol., 30, 131–134. teriol., 43, 805–812. Vobis, G. (1989) Actinoplanetes. In Bergey’s Manual of System-