Actinomycetologica Copyright 2007 The Society for Actinomycetes Japan

Krasilnikovia gen. nov., a new member of the family and description of Krasilnikovia cinnamonea sp. 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 Nov. 24, 2006 / Published May 18, 2007)

A novel actinomycete strain was isolated from sandy soil collected in Bangladesh. The culture formed pseudosporangia on short sporangiophores directly above the surface of the substrate mycelium. The pseudosporangia developed singly or in clusters and each pseudosporangium contained many non-motile oval to reniform spores with a smooth surface. The strain 3-54(41)T contained meso-diaminopimelic acid in the cell wall, predominant menaquinone MK-9(H6), and galactose, mannose, xylose and arabinose in the whole-cell hydrolysate. The diagnostic phospholipid was phosphatidylethanolamine, and branched iso-C16:0 (44.0%), iso-C14:0 (13.0%) and unsaturated C18:1 (!9c) (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 G+C Advancecontent of the DNA was 71 mol%. The chemotaxonomic data indicate that this strain belongs to the family Micromonosporaceae. Phylogenetic analysis based on 16S rDNA sequence data also suggested that the strain 3-54(41)T falls within this family. On the basis of phylogenetic analysis and the characteristic patterns of signature nucleotides as well as the morphological and chemotaxonomic data, our isolate is proposed to be Krasilnikovia gen. nov., and this strain should be classified as the species Krasilnikovia cinnamonea sp. nov. in the family Micromonosporaceae. The type strainView is 3-54(41)T (=JCM 13252T = MTCC 8094T). INTRODUCTION species form spore chains; Verrucosispora species do not form aerial mycelia or sporangia; Actinoplanes, Dactylo- The family Micromonosporaceae was first described by sporangium, Pilimelia, Catenuloplanes, Couchioplanes and Krasil’nikov1) on a morphological basis and was amended Spirilliplanes sporesProofs show motility; and Catenuloplanes by Goodfellow et al.2), who added the genera Actinoplanes, and Couchioplanes species have lysine instead of meso- Dactylosporangium and Pilimelia on the basis of numerical diaminopimelic acid as the diamino acid of the peptido- and chemical analyses. Koch et al.3) amended the descrip- glycan. tion of the family to reflect its phenotypic and chemo- During taxonomic studies of rare actinomycetes, strain taxonomic heterogeneity. Later, Stackebrandt et al.4) 3-54(41)T, which formed spherical pseudosporangial further amended the family on the basis of phylogenetic structures on substrate mycelia, was isolated from sandy clustering of 16S rDNA/rRNA sequences and the presence soil samples from Chokoria, Cox’s Bazar, Bangladesh. of taxon-specific 16S rDNA/rRNA signature nucleotides. On the basis of its 16S rDNA sequences, the isolate falls At present, the family Micromonosporaceae comprises 15 phylogenetically within the family Micromonosporaceae genera: Micromonospora5), Actinoplanes6), Pilimelia7), adjacent to the genus Couchioplanes and Actinoplanes Dactylosporangium8), Catellatospora9), Catenuloplanes10), globisporus. Therefore, we propose that the isolate Couchioplanes11), Spirilliplanes12), Verrucosispora13), should be included in a new genus, Krasilnikovia, the Virgisporangium14), Asanoa15), Longispora16), Salinis- name being derived from the Russian microbiologist N. A. pora17), Actinocatenispora18) and Polymorphospora19), each Krasil’nikov. genus having distinctive morphological and/or chemo- taxonomic characteristics. Micromonospora and Salinis- MATERIALS AND METHODS pora species form single spores on short or long spor- ophores; Actinoplanes, Dactylosporangium, Pilimelia and Strain 3-54(41)T was isolated from sandy soil collected Virgisporangium species form sporangia; Catellatospora, at a forest-side waterfall in Chokoria, Cox’s Bazar, Asanoa, Catenuloplanes, Couchioplanes, , Bangladesh. The strain was isolated using the standard Spirilliplanes, Actinocatenispora and Polymorphospora dilution plate method and grown on humic acid-vitamin

Corresponding author. Phone & Fax: +81-3-5791-6133. E-mail: [email protected] The DDBJ accession number for the 16S rDNA sequences of strain 3-54(41)T is AB236956.

1 ACTINOMYCETOLOGICA

agar (HV)20) supplemented with cycloheximide (50 mg l1), Table 1. Culture characteristics of isolate 3-54(41)T nystatin (50 mg l1) and nalidixic acid (20 mg l1). After 21 Cultures were incubated at 30C for 3 weeks. Aerial mycelia and days of aerobic incubation at 30C, the strain was trans- pigmentation were not formed on all of the agar media tested. ferred and purified on yeast extract-malt extract agar Colour designations and codes in parentheses were taken from the 26Þ [(medium 2 of the International Streptomyces Project (ISP Colour Harmony Manual . Growth and sporulation on substrate medium 2)] and maintained as working cultures on yeast- mycelium are scored as: (++), good; (+), moderate; ( ), doubtful/poor; (), no growth and no spores formed; ,V8 starch agar containing soluble starch, 15.0 g; yeast extract, canned vegetable juice (Campbell Soup Co.). 4.0 g; K2HPO4, 0.5 g; MgSO4.7H2O, 0.5 g; and agar, 15.0 g in 1 liter of distilled water (pH 7.2). Agar medium 3-54(41)T Strain 3-54(41)T was grown on tap water agar, sucrose- Growth/Reverse nitrate agar (Waksman medium 1) and HV agar media at color/Sporulation 30C for 21 days and then observed by light and scanning Glucose-asparagine ++/Light wheat (2ea)/++ electron microscope (model S-2400; Hitachi, Tokyo, Glycerol-asparagine +/Melon yellow (3ga)/+ Japan). The sample for scanning electron microscopy was Inorganic salts-starch ++/Melon yellow (3ga)/ prepared as described by Itoh et al.21) and Ara & Kudo22). Tyrosine +/Orange rust (4pe)/+ For the spore motility test, the isolate was inoculated on Nutrient /Light amber (3ic)/+ sucrose-nitrate agar at 30C for 21 days. Spore suspensions Yeast extract-malt extract ++/Amber (3lc)/ were obtained by gentle scraping of the agar surface and Oatmeal ++/Mustard gold (2ne)/ mixed with 10 ml of sterile tap water. The spore suspension Bennett ++/Mustard gold (2ne)/ was incubated at 30C for about 1 h. The suspension was Glucose-yeast extract ++/Melon yellow (3ga)/ Advanceshaken at irregular intervals and allowed to settle down. Hickey-Tresner ++/Amber (3pe)/+ About 0.1 ml supernatant was taken with a sterile Pasteur Tap water /Colorless/++ pipette, spread onto a clean slide and covered with a cover Sucrose-nitrate ++/Bamboo (2gc)/++ slip. The motility of spores was observed under a light Yeast extract-starch ++/Cinnamon (3le)/+ microscope at 100 and 200 magnification. Oatmeal-nitrate ++/Light wheat (2ea)/ The phenotypic properties were examined using several 1/5 yeast-starch +/Light wheat (2ea)/+ standard methods. For cultural characterization, the isolates Sucrose-beef extract +/Mustard gold (2ne)/ were grown for 21 days at 30C on various agarView media ISP medium 6 +/Cinnamon (3le)/ as described by Waksman23,24); Shirling & Gottlieb25), and ISP medium 1 +/Mustard gold (2ne)/+ Asano & Kawamoto9) (Table 1). The Colour Harmony 1/20 V8 juice +/Light wheat (2ea)/+ Manual26) was used to determine the names and desig- Humic acid-vitamin +/Cinnamon (3le)/++ nations of colony colours. The temperature range and NaCl 1/10 yeast extract-maltProofs extract +/Light wheat (2ea)/+ tolerance for growth were determined on yeast extract- starch agar (JCM medium 61). Utilization of carbohydrates as sole carbon sources was tested using neutralized yeast with a Cosmosil 5C18 column (4:6 150 mm; Nacalai nitrogen base without amino acids as a basal medium Tesque, Kyoto, Japan). Preparation and detection of methyl according to the method of Stevenson27). Production of esters of mycolic acids were performed as described by melanoid pigments was examined using tyrosine agar Tomiyasu35). (ISP medium 7). Genomic DNA extraction, PCR-mediated amplification The freeze-dried cells used for chemotaxonomic analysis of the 16S rRNA gene and sequencing of the PCR products were obtained from cultures grown in yeast-starch broth were performed as described by Nakajima et al.36) and Ara (JCM medium no. 61) on a rotary shaker at 30C. The & Kudo22). The sequences were multiply aligned with isomers of diaminopimelic acid (A2pm) in the cell wall selected sequences (Fig. 2) obtained from the GenBank/ peptidoglycan were determined by TLC as described by EMBL/DDBJ databases using the CLUSTAL X pro- Staneck & Roberts28). Reducing sugars from whole-cell gram37). The alignment was manually verified and adjusted hydrolysates were analyzed by the HPLC method of before construction of a phylogenetic tree. The phyloge- Mikami & Ishida29). The N-acyl group of muramic acid netic tree constructed by the neighbour-joining method38) in peptidoglycan was determined by the method of Uchida in the CLUSTAL X program37) was based on a comparison & Aida30). Phospholipids in cells were extracted and of 1356 nucleotides present in all strains following the identified by the method of Minnikin et al.31). Methyl elimination of gaps and ambiguous nucleotides using a esters of cellular fatty acids were prepared and analyzed sequence alignment editor (Se-Al v2.0A11, 1997–2002 according to the instructions of the Microbial Identification Andrew Rambout and Mike Charleston, University of System (Sherlock Microbial Identification System; MIDI, Oxford, UK). Streptomyces ambofaciens was used as an Hewlett Packard, Palo Alto, CA, USA)32). Isoprenoid outgroup. The confidence values of the branches of the quinones were extracted by the method of Collins et phylogenetic tree were determined using bootstrap analyses al.33,34) and were analysed using an HPLC system equipped based on 1000 re-samplings39).

2 ACTINOMYCETOLOGICA

C D

Advance

Fig. 1. Light micrograph (A) and scanning electron micrographs (B, C and D) of spherical pseudosporangia on substrate mycelia of Krasilnikovia cinnamonea 3-54(41)T grown on sucrose-nitrateView agar for 21 days at 30C. DNA was isolated from biomass by the method of on the substrate mycelium (Fig. 1A). Observation by Tamaoka40) and Saito & Miura41) with minor modification. scanning electron microscope revealed that the structures Cells that failed to be lysed by the enzymes were freeze- were pseudosporangia (without sporangial wall) and varied dried and mechanically ground as described by Raeder & in size (2.0–5.0 mProofsm) (Fig. 1B, C, D). Spores or spore Broda42,43). The G+C content of the DNA was determined chains in the pseudosporangium were non-motile and using the HPLC method of Tamaoka & Komagata44). formed by the unbranched hyphae. The spores were oval An equimolar mixture of nucleotides for analysis of DNA or reniform in shape with a smooth surface. base composition (Yamasa Shoyu, Choshi, Japan) was digested by bacterial alkaline phosphatase and used as Physiological properties the quantitative standard. Signature nucleotides in the The physiological properties of strain 3-54(41)T can 16S rRNA gene of the new taxon and members of the be summarized as follows. They exhibited good growth family Micromonosporaceae were determined after manual on glucose-asparagine agar, inorganic salts-starch agar verification of the CLUSTAL X alignment of sequences, (ISP medium 4), oatmeal-nitrate agar (JCM medium 52), and the nucleotide positions were numbered according sucrose-nitrate agar, yeast extract-starch agar (JCM to the corresponding position in the 16S rRNA sequence medium 61), oatmeal agar (ISP medium 3), ISP medium 2, of Escherichia coli45). The nucleotide sequence data Bennett agar, Hickey-Tresner agar and glucose-yeast reported in this paper will appear in the DDBJ, EMBL extract agar, and aerial hyphae were not formed. Soluble and GenBank nucleotide sequence databases with the pigment was not produced on any agar medium tested. accession numbers listed in Fig. 2. The strain utilized D-glucose, glycerol, D-xylose, D- galactose, D-fructose, D-mannose, salicin and maltose. It RESULTS grew well at 20–37C and pH 5–9, and could not grow on 3% NaCl. Culture characteristics The isolate 3-54(41)T comprised Gram-positive, non- Chemotaxonomic analysis acid-fast, aerobic organisms with branched hyphae. A non- Chemotaxonomic properties have been found to make fragmenting substrate mycelium was formed. Morpholog- an important contribution to the polyphasic approach to ical observations by light microscope indicated the pres- bacterial systematics, especially in the circumscription of ence of single or clustered spherical to irregular structures phylogenetically coherent actinomycete taxa49,50). The

3 ACTINOMYCETOLOGICA

0.01

Krasilnikovia cinnamonea 3-54(41) T(AB236956)

55 Couchioplanes caeruleus sub. sp. caeruleus IFO 13939 T (D85479) 97 42 Couchioplanes caeruleus sub. sp. azureus IFO 13993 T (D85478)

Actinoplanes globisporus IFO 13912 T (AB037003)

Spirilliplanes yamanashiensis IFO 15828 T (D63912)

Actinoplanes philippinensis IFO 13940 T (AB047499)

Dactylosporangium aurantiacum DSM 43157 T (X93191) 40 Advance Virgisporangium aurantiacum YU438-5 T (AB006169)

Catellatospora citrea IMSNU 22008 T (AF152106) 53

Longispora albida K97-0003 T (AB089241)

ViewT Polymorphospora rubra TT 97-42 (AB223089)

Salinispora arenicola CNH-643 T (AY040619) 62 Proofs

Micromonospora chalcea DSM 43026 T (X92594)

Asanoa ferruginea IMSNU 22009 T (AF152108)

Verrucosispora gifhornensis HR1-2 T (Y15523) 95

Catenuloplanes japonicus DSM 44102 T (X93201)

Pilimelia terevasa DSM 43040 T (X93190)

Actinocatenispora thailandica TT2-10 T (AB107233)

Streptomyces ambofaciens ATCC 23877 T (M27245)

Fig. 2. Phylogenetic tree showing the position of the new genus Krasilnikovia in the family Micromonosporaceae based on nearly complete 16S rDNA analysis. Numbers at nodes indicate the level (%) of bootstrap support based on neighbour-joining analysis of 1000 re-sampled datasets. Only values >40% and above are shown. Bar, 0.01 nucleotide substitution per 100 nucleotides.

4 ACTINOMYCETOLOGICA

Table 2. Physiological characteristics of the isolate 3-54(41)T Table 3. Cellular fatty acid composition of isolate 3-54(41)T Characteristics are scored as: (++), positive; (+), moderate; (), Fatty acida 3-54(41)T doubtful/poor; (), negative. Saturated T Characteristic 3-54(41) b C16:0 2.29 Utilization of: C17:0 1.10 D-Glucose ++ C18:0 6.34 Glycerol ++ Unsaturated

Erythritol C18:1 (!9c) 12.42 Adonitol Branched

L-Arabinose i-C14:0 12.60 D-Ribose i-C15:0 3.41 D-Xylose ++ a-C15:0 2.88 D-Galactose ++ i-C16:0 44.16 D-Fructose ++ i-C16:1 1.14 D-Mannose ++ a-C17:0 2.27 L-Rhamnose i-C18:0 2.73 myo-Inositol 10 Methyl

D-Mannitol C17:0 3.80 -Methyl-D-glucoside aValues are percentages of total cellular fatty acids. Trace Salicin ++ amounts (less than 1.0%) are not shown. AdvanceLactose bNumber of carbon atoms:number of double bonds. Abbrevia- -D(+)Melibiose tions: i, iso; a, anteiso. Sucrose + D(+)Raffinose Maltose ++ Trehalose 16S rDNA phylogenetic analysis Growth at: 20–37 The almost complete 16S rDNA sequence (1511 nt) of Viewstrain 3-54(41)T was determined in this study, and the Growth at (pH): 5–9 Growth on 3% NaCl): phylogenetic position based on the 16S rDNA sequence Melanin pigment: of the isolate is within the confines of the family Micro- monosporaceae (Fig. 2), near to the genus Couchioplanes and Actinoplanes globisporusProofs. Actinoplanes globisporus contains all of the typical characteristics of the genus family Micromonosporaceae is mainly defined by chemo- Actinoplanes but has a different phylogenetic position that , and our isolate 3-54(41)T shared chemotaxo- has yet to be determined. The sequence similarities among nomic characteristics with members of this family as the genus Couchioplanes and Actinoplanes globisporus follows. The cell walls contained meso-diaminopimelic range from 96.1% to 97.0%. Strain 3-54(41)T showed 94.2– acid and galactose, mannose, xylose, arabinose, ribose and 97.5% 16S rDNA similarity with the other genera of the glucose as the whole-cell sugars, indicating a whole-cell family Micromonosporaceae. On the basis of the phyloge- sugar pattern D according to Lechevalier & Lechevalier51). netic data as well as the morphological and chemotaxo- The major menaquinone MK-9(H6) and small amounts of nomic data, it is evident that the strain should be classified MK-9(H4) and MK-9(H8) were present. Iso-C16:0, iso-C14:0 as a new genus. Table 4 summarizes the differential and unsaturated-C18:1 (!9c) were the major cellular fatty characteristics of the new genus and other members of acids, with small amounts of the saturated fatty acid C18:0, the family Micromonosporaceae. Signature nucleotide 10 methyl fatty acids C17:0, iso-C15:0, anteiso-C15:0, anteiso- patterns in the 16S rRNA gene of the new taxon and C17:0, and saturated C16:0, indicating fatty acid type 2d the Micromonosporaceae are presented in Table 5. It is of Kroppenstedt52) (Table 3). Mycolic acids were absent. evident from our study that, of the 11 signatures defined Phosphatidylethanolamine, diphosphatidylglycerol, phos- for the Micromonosporaceae4), all are present in the 16S phatidylglycerol, and phosphatidylinositol and phosphati- rDNA sequence of the strain 3-54(41)T. Furthermore, dylinositol mannosides were detected, corresponding to several nucleotide pairs differing from the members of phospholipid type PII of Lechevalier et al.53). The acyl type the family Micromonosporaceae, and particularly Couchio- of the cell wall polysaccharides was glycolyl. The G+C planes, Actinoplanes and Actinoplanes globisporus, are content of the DNA was 71%. The predominant menaqui- shown in Table 5. none pattern of our isolate differed from that of the other genera of the family Micromonosporaceae (Table 4).

5 ACTINOMYCETOLOGICA , Þ Þ ) - 14 8 13 ; DISCUSSION 6 ; - 4

et al. T + meso DAP Ara, Gal, Xyl Virgispo rangium Taxonomic position of the strain 3-54(41) Þ

- T 13 Strain 3-54(41) formed globose pseudosporangia on - ) 10(H 4 substrate mycelium that resembled the pseudosporangia of , Rheims Þ meso DAP Verruco sispora 19

; the genus Couchioplanes and the sporangia of the genus Þ 14 - 12 ; )9(H Actinoplanes - 4 under light microscope. The chains of very 12 ;

11 long, coiled and aggregated sporogenous hyphae without meso DAP Spirilli planes zoospores appeared to be sporangium-like structures under et al. ) 10(H - 2 - ; Þ the light microscope. Under the scanning electron micro- 4 7 scope, however, these structures were found to be highly meso DAP Pilim elia Þ - 8 aggregated hyphae, not true sporangia. Furthermore, ; Tamura ) 9(H Þ T 8 ; 48

6 the isolate 3-54(41) was distinguished from the genera -DAP ; 4 Couchioplanes and Actinoplanes globisporus by the ++ meso Dactylosp orangium 9(H T

Þ absence of motile spores. Strain 3-54(41) is a wall - 10

) chemotype II organism. The genera Micromonospora, 8 ), 8 Actinoplanes, Dactylosporangium and Pilimelia are wall -Lys ++ ++ l Xyl Ara, Xyl Ara, Xyl Ara, Xyl Man, Xyl 10(H Catenul oplanes 9(H chemotype II genera, but Actinoplanes, Dactylosporan- - . Þ Þ ) 22 ; Tamura & Hatano ; 18 6 gium and Pilimelia have motile spores and a different )or Þ ; 9 6 - 4 Micromonosporaceae ;

10 T 4 menaquinone pattern (Table 4). Strain 3-54(41) is also et al. meso DAP Xyl, Man, Gal, Ara, Rham, Rib Catellato spora 9(H 10(H

, Absent; ND, not determined; Xyl, Xylose; Gal, Galactose; Man, Mannose; distinguished from these genera in that its spores are not et al. Þ 15 Advance)

8 enclosed in a sporangial wall. ; - 6 The distinctness of the 16S rDNA sequence, phyloge- meso DAP Ara, Gal, Xyl 10(H Asanoa

; Yokota netic position and specific signature nucleotide patterns Þ and the family - 2 T ; and Thawai of the 16S rRNA gene of strain 3-54(41) differentiate it Þ ; +, Present; ), Þ Þ 22 6 ) ; 5 52 et al. 6 -DAP 4 from known actinomycetes belonging to the family Micro- ; 4 monosporaceae, and thus this isolate could merit new meso Ara, Xyl 10(H Micromono spora 9(H

Þ Viewgenus status. Moreover, on the basis of morphological, 18 - Krasilnikovia physiological, chemotaxonomic and phylogenetic criteria ) 6 - ; 4

; Ara & Kudo and the signature nucleotide pattern of the 16S rRNA gene, ; Goodfellow Þ Þ

17 T 47 meso DAP Ara, Gal, Xyl Actinoca tenispora the strain 3-54(41) is readily distinguishable from the ) 9(H Þ

6 closest genera Couchioplanes and Actinoplanes and the ;

- Proofs 16 et al. - 4 species Actinoplanes glogisporus and other motile actino- meso DAP Ara, Gal, Xyl Longi spora mycetes mentioned above, and thus warrants a new taxon.

Þ T T - 17 We propose that the strain 3-54(41) (=JCM 13252 = - ) 10(H 4 MTCC 8094T) be classified in a new genus, Krasilnikovia, ; Maldonado meso DAP 9(H Ara, Gal, Xyl Salini spora ; Horan & Brodsky Þ Þ

Þ the type species of which is Krasilnikovia cinnamonea 16 46 19 -

) sp. nov. 4 ) ; 4 -DAP 6 et al. ; 6 , According to the classification of Kroppenstedt ; meso 9(H Polymor phospora 10(H Description of Krasilnikovia gen. nov. Þ Þ 57 ,

54 Krasilnikovia (kra.sil.ni.kov.ia. N.L. adj. Krasilnikovia, ; referring to N. A. Krasil’nikov, a Russian actinomycetol- 14 et al. - 46{48 ) -DAP 4 ), ; Matsumoto 4

Þ ogist who contributed to the taxonomy of the family 15 + 10(H 9(H Actino planes Ara, Xyl Xyl meso Micromonosporaceae). Þ

11 The cells are Gram-positive, non-acid-fast, aerobic - )

4 organisms with branching hyphae. Non-fragmenting light , Stackebrandt & Kroppenstedt -Lys Þ ++ Ara, Gal, Xyl l Couchi oplanes yellow to cinnamon coloured substrate mycelia are present. 54 ; Lee & Hah Þ Large and spherically shaped structures contain aggrega- 56

) 9(H tions of spore chains and substrate mycelium resembling 8 ; 4 -DAP ; et al. 6 sporangia, but true sporangia with a sporangial wall are not 2d9(H 2cPII71 2d PII 69–73 2a PII 72–73 3a PII 71 2d PII 70–73 3b 70 PII 72 3b PII 71–72 PII 2d 71–72 3b PII 70–71 71–73 PII 2c 71–73 PIII 3b ND 69 PII 2d 70 PII 2d 71 PII 2b PII 2d PII Gal, Man, Xyl, Ara, Rib New taxon Krasilnikovia meso observed. Twenty-one-day-old cultures grown on glucose- , Lee Þ asparagine agar, sucrose-nitrate agar and HV agar develop 55 substrate mycelia with long spore chains that are hooked

et al. and highly aggregated. Several spores are present per spore

chain, and the spores are oval to short rods (0:2{0:4 , According to the classification of Lechevalier Rham, Rhamnose; Rib, Ribose; Ara, Arabinose. Major menaquinone(s) (MK-) G+C content (mol%) Spore motility Whole-cell sugars Table 4. Morphological features and chemotaxonomic characteristics of the new taxon Characteristic Sporangium Diamino acid Fatty-acid type Data were taken from Vobis Phospholipid type Kudo 0:8{1:0 mm) with a smooth surface and are non-motile

6 ACTINOMYCETOLOGICA

Table 5. 16S rDNA signature nucleotide positions that differentiate the new genus Krasilnikovia of the family Micromonosporace Actinoplanes Position Krasilnikovia Couchioplanes Actinoplanes globisporus 129 U C UU 139–224 A-U G-U A-U U-G 140–223 U-G G-C G-U C-G 144–178 U-G U-G U-A U-G 153–168 C-G G/C-G C-G C-G 154–167 C-G G/C-G C-G C-G 155–166 C-G G/C-G C-G C-G 222 C UUU 232 G G A G 262 G G A G 445–489 C-G G-C G-C G-C 446–488 C-G G-C G-C G-C 456 A UCU 457 G AAG 602–636 C-G C-G A-U C-G 614–626 G-C G-G/C G-C A-U Advance615–625 C-G C-G G-C C-G 616–624 G-C G-U G-U A-U 629 G C/G GG 631 C U/C U U 632 U G/U UU 832.854 G.U G.U G.C G.U 837.849 G.C G.C G.U G.C 1001–1039 C-GView C-G G-C C-G 1006 A A U A 1011–1018 U-A C-G C-G C-G 1119–1154 U-A U-A U-AProofsC-G 1121–1152 G-C G-C G-C C-G 1133–1141 G-C G-C G-C A-U 1256 U U C U 1263–1272 G-U A-U A-U C-G Nucleotide positions of bases or base pairs are given according to the E. coli numbering45Þ. Signature nucleotides that clearly differentiate the genera Couchioplanes and Actinoplanes and the species Actinoplanes globisporus from the new taxon Krasilnikovia are shown in bold. , All family-specific nucleotides are present except for these pairs.

when suspended in sterile distilled water. The organism MK-9(H4) and MK-9(H8) are present. In general, iso-C16:0, utilizes D-glucose, glycerol, D-xylose, D-galactose, D- iso-C14:0 and unsaturated C18:1 (!9c) are the major cellular fructose, D-mannose, salicin, maltose and sucrose. It grows fatty acids, with small amounts of saturated C18:0,10 well at 20–37 C and pH 5–9, and could not grow on 3% methyl C17:0, iso-C15:0, anteiso-C15:0, anteiso-C17:0 and NaCl. The organism shows good growth on glucose- saturated-C16:0. Mycolic acids are absent. Phosphatidyl- asparagine agar, inorganic salts-starch agar (ISP medium ethanolamine, diphosphatidylglycerol, phosphatidylglyc- 4), oatmeal-nitrate agar, sucrose-nitrate agar, yeast extract- erol, phosphatidylinositol and phosphatidylinositol manno- starch agar, oatmeal agar (ISP medium 3), ISP medium 2, sides are present. The acyl type of the cell wall Bennett agar, Hickey-Tresner agar and glucose-yeast polysaccharides is glycolyl. The G+C content of the extract agar. The cell wall contains meso-diaminopimelic DNA is 71%. The genus is placed in the family Micro- acid; the wall chemotype is II. Galactose, mannose and monosporaceae on the basis of the phylogenetic position of glucose are detected as the major whole-cell sugars in the strain based on its distinct 16S rDNA sequence. All addition to small amounts of ribose, arabinose and xylose, family-specific nucleotide signatures are present except indicating that the whole-cell sugar pattern is D. The for a C-G pair at position 445–489 and 446–488 instead of major menaquinone is MK-9(H6), and small amounts of C-G, U-A at position 1011–1018 instead of C-G, and G-U

7 ACTINOMYCETOLOGICA at position 1263–1272 instead of A-U. The type species is 5) Ørskov, J.: Investigations into the Morphology of the Ray Krasilnikovia cinnamonea. Fungi. Levin and Munksgaard, Copenhagen, 1923 6) Couch, J. N.: Actinoplanes a new genus of the Actino- Description of Krasilnikovia cinnamonea sp. nov. mycetales. J. Elisha Mitchell Sci. Soc. 66: 87–92, 1950 Krasilnikovia cinnamonea (cin.na.mo’nea L. n. cinna- 7) Kane, W. D.: A new genus of Actinoplanaceae, Pilimelia, with a description of two species, Pilimelia terevasa and mum cinnamon; N.L. adj. cinnamonea cinnamon-coloured). Pilimelia anulata. J. Elisha Mitchell Sci. Soc. 82: 220–230, Morphological, chemotaxonomic and general character- 1966 istics are as given above for the genus description. 8) Thiemann, J. E.; H. Pagani & G. Beretta: A new genus of the The organism shows moderate growth on 1/5 yeast- Actinoplanaceae: Dactylosporangium, gen. nov. Archiv fur starch agar, glycerol-asparagine agar, tyrosine agar, Mikrobiologie, 1967, 58, 42–52. sucrose-beef extract agar, ISP medium 6, ISP medium 1, 9) Asano, K. & I. Kawamoto: Catellatospora, a new genus of 1/20 V8 juice agar, HV agar, and 1/10 yeast-malt extract the Actinomycetales. Int. J. Syst. Bacteriol. 36: 512–517, agar, and poor growth on nutrient agar and tap water agar. 1986 Melanin pigment production on ISP medium 7 is negative. 10) Yokota, A.; T. Tamura, T. Hasegawa & L. H. Huang: Abundant sporulation occurs on glucose-asparagine agar, Catenuloplanes japonicus gen. nov., sp. nov., nom. rev., a sucrose-nitrate agar and HV agar media, moderate spor- new genus of the order Actinomycetales. Int. J. Syst. Bacteriol. 43: 805–812, 1993 ulation on glycerol asparagine agar, tyrosine agar, nutrient 11) Tamura, T.; Y. Nakagaito, T. Nishii, T. Hasegawa, E. agar, Hickey-Tresner agar, yeast extract-starch agar, 1/5 Stackebrandt & A. Yokota: A new genus of the order yeast extract-starch agar, ISP medium 1 agar, 1/20 V8 Actinomycetales, Couchioplanes gen. nov., with descriptions juice agar and 1/10 yeast extract-malt extract agar, and no of Couchioplanes caeruleus (Horan and Brodsky 1986) Advancesporulation on inorganic salts-starch agar, yeast extract- comb. nov. and Couchioplanes caeruleus subsp. azureus malt extract agar, oatmeal agar, Bennett agar, glucose-yeast subsp. nov. Int. J. Syst. Bacteriol. 44: 193–203, 1994 extract agar, oatmeal-nitrate agar, sucrose-beef extract agar 12) Tamura, T.; M. Hayakawa & K. Hatano: A new genus of and ISP medium 6 agar media. In general, the vegetative the order Actinomycetales, Spirilliplanes gen. nov., with mycelia are light melon yellow to cinnamon yellow description of Spirilliplanes yamanashiensis sp. nov. Int. J. in colour and aerial mycelia are not present. Adonitol, Syst. Bacteriol. 47: 97–102, 1997 L-rhamnose, D-mannitol, -D(+)melibiose, D-raffinose 13) Rheims, H.; P. Schumann, M. Rohde & E. Stackebrandt: Verrucosispora gifhornensis gen. nov., sp. nov., a new and trehalose are poorly utilized, and erythritol, L-arabi-View member of the actinobacterial family Micromonosporaceae. nose, D-ribose, myo-inositol, -methyl-D-glucoside and Int. J. Syst. Bacteriol. 48: 1119–1127, 1998 lactose are not utilized. The G+C content of the DNA is 14) Tamura, T.; M. Hayakawa & K. Hatano: A new genus of T 71%. The habitat is soil. The type strain is 3-54(41) the order Actinomnycetales, Virgisporangium gen. nov., with (= JCM 13252T = MTCC 8094T). VirgisporangiumProofs ochraceum Int. J. descriptions of sp. nov. Syst. Evol. Microbiol. 51: 1809–1816, 2001 ACKNOWLEDGEMENT 15) Lee, S. D. & Y. C. Hah: Proposal to transfer Catellatospora ferruginea and ‘Catellatospora ishikariense’toAsanoa gen. The authors are indebted to the Japan Society for the nov. as Asanoa ferruginea comb.nov. and Asanoa ishikar- Promotion of Science for financial support. iensis sp. nov., with the emended description of the genus Catellatospora. Int. J. Syst. Evol. Microbiol. 52: 967–972, 2002 REFERENCES 16) Matsumoto, A.; Y. Takahashi, M. Shinose, A. Seino, Y. Iwai & S. Omura: Longispora albida gen. nov., a novel genus 1) Krasil’nikov, N. A.: Ray Fungi and Related Organisms of the family Micromonosporaceae. Int. J. Syst. Evol. Actinomycetales. Moscow: Izdatel’stvo Akademii Nauk Microbiol. 53: 1553–1559, 2003 SSSR, 1938 17) Maldonado, L. A.; W. Fenical, P. R. Jensen, C. A. Kauffman, 2) Goodfellow, M.; L. J. Stanton, K. E. Simpson & D. E. T. J. Mincer, A. C. Ward, A. T. Bull & M. Goodfellow: Minnikin: Numerical and chemical classification of Actino- Salinispora arenicola gen. nov., sp. nov. and Salinispora planes and some related actinomycetes. J. Gen. Microbiol. tropica sp. nov., obligate marine actinomycetes belonging 136: 19–36, 1990 to the family Micromonosporaceae. Int. J. Syst. Evol. 3) Koch, C.; R. M. Kroppenstedt, F. A. Rainey & E. Microbiol. 55: 1759–1766, 2005 Stackebrandt: 16S ribosomal DNA analysis of the genera 18) Thawai, T.; S. Tanasupawat, T. Itoh & T. Kudo: Actino- Micromonospora, Actinoplanes, Catellatospora, Catenulo- catenispora thailandica gen. nov., sp. nov., a new member planes, Couchioplanes, Dactylosporangium, and Pilimelia of the family Micromonosporaceae. Int. J. Syst. Evol. and emendation of the family Micromonosporaceae. Int. J. Microbiol. 56: 1789–1794, 2006 Syst. Bacteriol. 46: 765–768, 1996 19) Tamura, T.; K. Hatano & K. Suzuki: A new genus of the 4) Stackebrandt, E.; F. A. Rainey & N. L. Ward-Rainey: family Micromonosporaceae, Polymorphospora gen.nov., Proposal for a new hierarchic classification system, Actino- with description of Polymorphospora rubra sp. nov. Int. J. classis nov. Int. J. Syst. Bacteriol. 47: 479–491, Syst. Evol. Microbiol. 56: 1959–1964, 2006 1997 20) Hayakawa, M. & H. Nonomura: Humic acid-vitamin agar,

8 ACTINOMYCETOLOGICA

a new medium for selective isolation of soil actinomycetes. 38) Saitou, N. & M. Nei: The neighbor-joining method: a new J. Ferment. Technol. 65: 501–509, 1987 method for reconstructing phylogenetic trees. Mol. Biol. 21) Ito, T.; T. Kudo, F. Parenti & A. Seino: Amended description Evol. 4: 406–425, 1987 of the genus Kineosporia, based on chemotaxonomic and 39) Felsenstein, J.: Confidence limits on phylogenies: an ap- morphological studies. Int. J. Syst. Bacteriol. 39: 168–173, proach using the bootstrap. Evolution 39: 783–791, 1985 1989 40) Tamaoka, J.: Determination of DNA Base Composition. In 22) Ara, I. & T. Kudo: Three novel species of the genus Chemical Methods in Prokaryotic Systematics, pp. 463–470. Catellatospora, Catellatospora chokoriensis sp. nov., Cat- Edited by M. Goodfellow & A. G. O’Donnell. Chichester: ellatospora coxensis sp. nov. and Catellatospora banglade- John Wiley & Sons, 1994 shensis sp. nov. and transfer of Catellatospora citrea subsp. 41) Saito, H. & K. Miura: Preparation of transforming deoxy- methionotrophica Asano and Kawamoto 1988 to Catellato- ribonucleic acid by phenol treatment. Biochim. Biophys. spora methionotrophica sp. nov. comb. nov. Int. J. Syst. Evol. Acta. 72: 619–629, 1963 Microbiol. 56: 393–400, 2006 42) Kudo, T.; K. Matsushima, T. Itoh, J. Sasaki & K. Suzuki: 23) Waksman, S. A.: The Actinomycetes: Their Nature, Occur- Description of four new species of the genus Kineosporia: rence, Activities and Importance. Waltham, MA: Chronica Kineosporia succinea sp. nov., kineosporia rhizophila sp. Botanica, 1950 nov., kineosporia mikuniensis sp. nov. and kineosporia 24) Waksman, S. A.: Classification, Identification, and Descrip- rhamnosa sp. nov., isolated from plant samples, and amended tion of Genera and Species. In The Actinomycetes, vol. 2. description of the genus Kineosporia. Int. J. Syst. Bacteriol. Baltimore: Williams & Wilkins, 1961 48: 1245–1255, 1998 25) Shirling, E. B. & D. Gottlieb: Methods for characterization of 43) Raeder, U. & P. Broda: Rapid preparation of DNA from Streptomyces species. Int. J. Syst. Bacteriol. 16: 313–340, filamentous fungi. Lett. Appl. Microbiol. 1: 17–20, 1985 1966 44) Tamaoka, J. & K. Komagata: Determination of DNA 26) Jacobson, E.; W. C. Grauville & C. E. Fogs: Colour base composition by reversed-phase high-performance Advanceth Harmony Manual,4 ed. Container Corporation of America, liquid chromatography. FEMS Microbiol. Lett. 25: 125– Chicago, 1958 128, 1984 27) Stevenson, I. L.: Utilization of aromatic hydrocarbons by 45) Brosius, J.; J. L. Palmer, J. P. Kennedy & H. F. Noller: Arthrobacter spp. Can. J. Microbiol. 13: 205–211, 1967 Complete nucleotide sequence of a 16S ribosomal RNA 28) Staneck, J. L. & G. D. Roberts: Simplified approach to gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75: identification of aerobic actinomycetes by thin-layer chro- 4801–4805, 1978 matography. Appl. Microbiol. 28: 226–231, 1974 View46) Stackebrandt, E. & R. M. Kroppenstedt: Union of the genera 29) Mikami, H. & Y. Ishida: Post-column fluorometric detection Actinoplanes Couch, Ampullariella Couch, and Amorpho- of reducing sugar in high-performance liquid chromatogra- sporangium Couch in a redefined genus Actinoplanes. Syst. phy using arginine. Bunseki Kagaku 32: E207–E210, 1983 Appl. Microbiol. 9: 110–114, 1987 30) Uchida, K. & K. Aida: An improved method for the glycolate 47) Horan, A. C. & B. Brodsky: Actinoplanes caeruleus sp. nov., ProofsActinoplanes Int. J. test for simple identification of the acyl type of bacterial cell a blue-pigmented species of the genus . walls. J. Gen. Appl. Microbiol. 30: 131–134, 1984 Syst. Bacteriol. 36: 187–191, 1986 31) Minnikin, D. E.; A. G. O’Donnell, M. Goodfellow, G. 48) Tamura, T. & K. Hatano: Phylogenetic analysis of the genus Alderson, M. Athalye, A. Schaal & J. H. Parlett: An Actinoplanes and transfer of Actinoplanes minutisporangius integrated procedure for the extraction of bacterial isoprenoid Ruan et al. 1986 and Actinoplanes aurantiacus to Crypto- quinones and polar lipids. J. Microbiol. Methods. 2: 233–241, sporangium minutisporangius comb. nov. and Cryptospor- 1984 angium aurantiacum sp. nov. Int. J. Syst. Evol. Microbiol. 51: 32) Sasser, M.: Identification of bacteria by gas chromatography 2119–2125, 2001 of cellular fatty acids (MIDI Technical Note 101). New 49) Kroppenstedt, R. & M. Goodfellow: The family Thermomo- York, DE: MIDI, 1990 nosporaceae.InThe prokaryotes, pp. 1085–1114, Edited by 33) Collins, M. D.; T. Pirouz, M. Goodfellow & D. E. Minnikin: A. Balows, H. G. Truper, M, Dworkin, W. Harder, and K. H. Distribution of menaquinones in actinomycetes and coryne- Schleifer, 2nd ed. Springer, New York, 1991 bacteria. J. Gen. Microbiol. 100: 221–230, 1977 50) Goodfellow, M.: The family Streptosporangiaceae.InThe 34) Collins, M. D.; M. Faulkner & M. Keddie: Menaquinone prokaryotes, pp. 1115–1138. Edited by A. Balows, H. G. composition of some sporeforming actinomycetes. Syst. Truper, M. Dworkin, W. Harder, and K. H. Schleifer, 2nd ed. Appl. Microbiol. 5: 20–29, 1984 Springer, New York, 1991 35) Tomiyasu, I.: Mycolic acid composition and thermally 51) Lechevalier, M. P. & H. A. Lechevalier: Chemical compo- changes in Nocardia asteroides. J. Bacteriol. 151: 828– sition as a criterion in the classification of aerobic actino- 837, 1982 mycetes. Int. J. Syst. Bacteriol. 20: 435–443, 1970 36) Nakajima, Y.; V. Kitpreechavanich, K. Suzuki & T. Kudo: 52) Kroppenstedt, R. M.: Fatty acid and menaquinone analysis Microbispora corallina sp. nov., a new species of the genus of actinomycetes and related organisms. In Chemical Microbispora isolated from Thai soil. Int. J. Syst. Bacteriol. Methods in Bacterial Systematics, pp. 173–199. Edited by 49: 1761–1767, 1999 M. Goodfellow & D. E. Minnikin. London: Academic Press, 37) Thompson, J. D.; T. J. Gibson, F. Plewniak, F. Jeanmougin & 1985 D. G. Higgins: The Clustal X windows interface: flexible 53) Lechevalier, M. P.; A. E. Stern & H. A. Lechevalier: strategies for multiple sequence alignment aided by quality Phospholipids in the taxonomy of actinomycetes. Zentbl. analysis tools. Nucleic. Acids Res. 24: 4876–4882, 1997 Bakteriol. Hyg. Abt. 1 Suppl. 11: 111–116, 1981

9 ACTINOMYCETOLOGICA

54) Vobis, G.: Actinoplanetes. In Bergey’s Manual of Systematic Catenuloplanes. Int. J. Syst. Bacteriol. 49: 1853–1860, 1999 Bacteriology, vol. 4, pp. 2418–2450. Edited by S. T. 56) Lee, S. D.; S. –O. Kang & Y. C. Hah: Catellatospora Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams koreensis sp. nov., a novel actinomycete isolated from a & Wilkins, 1987 gold-mine cave. Int. J. Syst. Evol. Microbiol. 50: 1103–1111, 55) Kudo, T.; Y. Nakajima & K. Suzuki: Catenuloplanes crispus 2000 (Petrolini et al. 1993) comb. nov.: incorporation of the genus 57) Lechevalier, M. P.; C. DeBievre & H. A. Lechevalier: Planopolyspora Petrolini 1993 into the genus Catenuloplanes Chemotaxonomy of aerobic actinomycetes: phospholipid Yokota et al. 1993 with an amended description of the genus composition. Biochem. Syst. Ecol. 5: 249–260, 1977

Advance View Proofs

10