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Krasilnikovia Gen. Nov., a New Member of the Family Micromonosporaceae and Description of Krasilnikovia Cinnamonea Sp

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Krasilnikovia Gen. Nov., a New Member of the Family Micromonosporaceae and Description of Krasilnikovia Cinnamonea Sp Actinomycetologica Copyright Ó 2007 The Society for Actinomycetes Japan Krasilnikovia gen. nov., a new member of the family Micromonosporaceae and description of Krasilnikovia cinnamonea sp. nov. Ismet Ara1;2à 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, Longispora, Bangladesh. The strain was isolated using the standard Spirilliplanes, Actinocatenispora and Polymorphospora dilution plate method and grown on humic acid-vitamin ÃCorresponding 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 lÀ1), Table 1. Culture characteristics of isolate 3-54(41)T nystatin (50 mg lÀ1) and nalidixic acid (20 mg lÀ1). 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 Æ medium 2)] and maintained as working cultures on yeast- mycelium are scored as: (++), good; (+), moderate; ( ), doubtful/poor; (À), no growth and no spores formed; Ã,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)/Æ 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 Æ/Light amber (3ic)/+ sucrose-nitrate agar at 30C for 21 days. Spore suspensions Yeast extract-malt extract ++/Amber (3lc)/À were obtained by gentle scraping of the agar surface and Oatmeal ++/Mustard gold (2ne)/À mixed with 10 ml of sterile tap water. The spore suspension Bennett ++/Mustard gold (2ne)/À was incubated at 30C for about 1 h. The suspension was Glucose-yeast extract ++/Melon yellow (3ga)/À 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 Æ/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)/À 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)/À were grown for 21 days at 30C on various agarView media ISP medium 6 +/Cinnamon (3le)/À as described by Waksman23,24); Shirling & Gottlieb25), and ISP medium 1 +/Mustard gold (2ne)/+ Asano & Kawamoto9) (Table 1). The Colour Harmony 1/20 V8 juiceà +/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
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