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Actinobacteria

Nocardiopsis nikkonensis sp. nov., isolated from a compost sample

Hideki Yamamura, Shin-ya Ohkubo, Yuumi Ishida, Misa Otoguro, Tomohiko Tamura, Masayuki Hayakawa

  • 1Division of Applied Biological Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu 400-8511, Japan
  • 2NITE Biological Resource Center, National Institute of Technology and Evaluation, Kazusakamatari 2-5-8, Kisarazu, Chiba 292-0818, Japan
  • Correspondence
    Hideki Yamamura
    hyamamura{at}yamanashi.ac.jp

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2967–2971 · https://doi.org/10.1099/ijs.0.016956-0

    View at publisher PubMed

    Abstract

    An actinomycete strain, designated YU1183-22T, was isolated from a compost sample collected in Nikko, Japan. The isolate formed white aerial mycelium with relatively long aerial hyphae showing chains of arthrospores. Strain YU1183-22T grew with 0–10 % (w/v) NaCl, at pH 6–11 and at 10–37 °C (optimum 30 °C). Strain YU1183-22T contained meso-diaminopimelic acid and no diagnostic sugars. The predominant menaquinones were MK-10(H10) and MK-10(H8). The polar lipids were phosphatidylcholine and phosphatidylglycerol. The major cellular fatty acids were iso-C16 : 0, anteiso-C17 : 0 and tuberculostearic acid. The G+C content of the DNA was 72.3 mol%. Chemotaxonomic and morphological characterization clearly demonstrated that strain YU1183-22T belonged to the genus Nocardiopsis. Phylogenetic analysis using 16S rRNA gene sequences showed that the isolate was closely related to Nocardiopsis salina YIM 90010T (98.0 % 16S rRNA gene sequence similarity), Nocardiopsis xinjiangensis YIM 90004T (97.9 %) and Nocardiopsis kunsanensis HA-9T (97.3 %). However, DNA–DNA relatedness as well as physiological and biochemical analyses showed that strain YU1183-22T could be differentiated from its closest phylogenetic relatives. It is proposed that this strain be classified as a representative of a novel species of the genus Nocardiopsis, with the name Nocardiopsis nikkonensis sp. nov. The type strain is YU1183-22T (=NBRC 102170T =KCTC 19666T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YU1183-22T is AB491226.

    • A scanning electron micrograph of strain YU1183-22T is available with the online version of this paper.

    The genus Nocardiopsis is a member of the family Nocardiopsaceae (Meyer, 1976) and, at the time of writing, about 30 species have been described, including Nocardiopsis arabia, N. valliformis, N. quinghaiensis and N. ganjiahuensis (Hozzein & Goodfellow, 2008; Yang et al., 2008; Chen et al., 2008; Zhang et al., 2008). Many members of the genus Nocardiopsis have been isolated from hypersaline or alkaline environments. However, others have been isolated from non-hypersaline environments, including the sputum of a kidney transplant patient, the rhizosphere of Casuarina sp. and the atmosphere of a composting facility (Yassin et al., 1997; Evtushenko et al., 2000; Kämpfer et al., 2002). Nocardiopsis species are of value to industry as they are known to produce bioactive agents such as griseusin D, apoptolidin and methylpendolmycin (Li et al., 2007; Kim et al., 1997; Sun et al., 1991). Hence, the discovery of additional species of this genus will contribute both to an understanding of their ecological roles and to the provision of bioresources for industrial applications.

    Strain YU1183-22T was isolated from a matured compost sample collected in Nikko, Japan, by growth on HV agar (Hayakawa & Nonomura, 1987) containing (l−1) 20 mg nalidixic acid, 50 mg cycloheximide and 100 g NaCl. After incubation at 28 °C for 14 days, colonies of strain YU1183-22T showed a thin, flat morphology with sparse, white, aerial hyphae.

    Morphological features of cells of strain YU1183-22T grown on HV agar and oatmeal-YGG agar (Hayakawa et al., 1982) at 28 °C for 14 days were analysed by light and scanning electron microscopy. Cultural characteristics were determined after 2 weeks at 28 °C by using the methods adopted in the International Streptomyces Project (ISP) (Shirling & Gottlieb, 1966). All media were supplemented with 3 % (w/v) NaCl and the colours of both substrate and aerial mycelia were determined according to Japan Colour Research Institute (1954). The detailed results are shown in Table 1⇓. Spore motility in hanging drops was examined by light microscopy. Gram staining was examined by Hucker's method (Gerhardt, 1981). Carbon source utilization was examined using well-established procedures (Isik et al., 1999) with the addition of 3 % NaCl. Nitrate reduction was determined after 7 and 14 days of growth by the addition of 0.2 ml each of Griess-Ilosvay reagents I and II to stab cultures in sloppy nitrate medium supplemented with 0.2 % (w/v) KNO3 and 0.6 % Bacto agar (Difco) (Gordon & Mihm, 1962). Lipolysis was assessed on Sierra's medium (Sierra, 1957) using Tween 80 as a substrate. Hydrogen sulfide formation was detected by insertion of sterile lead acetate filter paper strips into the necks of the culture tubes containing ISP 6 medium (Küster & Williams, 1964). Melanin production was determined after 1–4 days of growth on ISP 6 and ISP 7 (Shirling & Gottlieb, 1966). Conditions for growth were tested using modified CM+YE medium (JCM medium no. 275) as follows. Growth at 15, 20, 25, 30, 37, 42 and 45 °C and pH 5, 6, 7, 8, 9, 10 and 11 was measured after 7 and 14 days; growth at 10 °C was also assayed after 6 weeks. Growth with 0–20 % (w/v) NaCl was determined after 14 and 21 days (Williams et al., 1983).

    Table 1.

    Cultural characteristics of strain YU1183-22T

    All media were adjusted to pH 7.0 and supplemented with 3 % (w/v) NaCl. The isolate formed white aerial mycelium on yeast extract/malt extract agar.

    Diaminopimelic acid isomers and sugars in whole-cell hydrolysates were analysed based on the methods established by Hasegawa et al. (1983) and Schaal (1985), respectively. Standard procedures were used for extraction and analysis of fatty acids (Tamura et al., 1994) and isoprenoid quinones and polar lipids (Minnikin et al., 1984) and the results were compared with the appropriate controls. Chromosomal DNA from strain YU1183-22T was isolated and purified by the method of Saito & Miura (1963) with a minor modification (Hatano et al., 2003). The G+C content of DNA from strain YU1183-22T was determined by HPLC, as described by Tamura et al. (1994). DNA–DNA hybridization was carried out as described by Kusunoki et al. (1991) using biotinylated DNA.

    PCR amplification of the 16S rRNA gene from strain YU1183-22T was carried out according to the procedures described by Tamura & Hatano (2001) and the amplification products were directly sequenced using an ABI Prism BigDye Terminator cycle sequencing kit (PE Applied Biosystems) and an automated DNA sequencer (model 3100 Genetic Analyzer; PE Applied Biosystems). The 16S rRNA gene sequence obtained was aligned with reference sequences of the genus Nocardiopsis available from public databases by using clustal x (Thompson et al., 1997). Phylogenetic trees were reconstructed by using mega version 4 (Tamura et al., 2007) and clustal x for the neighbour-joining method with Knuc values (Saitou & Nei, 1987) and minimum-evolution and maximum-parsimony methods (Takahashi & Nei, 2000) and phyml (Guindon et al., 2005) for the maximum-likelihood method. The topologies of the trees were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings (neighbour-joining, maximum-parsimony and minimum-evolution) and 500 resamplings (maximum-likelihood). 16S rRNA gene sequence similarity was calculated using the EzTaxon server (; Chun et al., 2007).

    Chemotaxonomic and morphological characteristics of strain YU1183-22T were consistent with its assignment to the genus Nocardiopsis (Li et al., 2004; Hozzein & Goodfellow, 2008). A whole-cell hydrolysate of strain YU1183-22T contained meso-diaminopimelic acid and displayed no diagnostic sugars (wall chemotype III sensu Lechevalier & Lechevalier, 1970). The major menaquinones were MK-10(H8) (28.1 %) and MK-10(H10) (24.1 %). The major polar lipids were phosphatidylcholine and phosphatidylglycerol (phospholipid type PIII sensu Lechevalier et al., 1977). The major cellular fatty acids were iso-C16 : 0 (26.0 %), anteiso-C17 : 0 (16.4 %) and 10-methyl C18 : 0 (16.3 %; tuberculostearic acid). Strain YU1183-22T formed branched substrate hyphae, which fragmented into rod-shaped elements, and relatively long aerial hyphae with chains of arthrospores (Supplementary Fig. S1, available in IJSEM Online).

    The almost-complete 16S rRNA gene sequence (1488 nt) of strain YU1183-22T was compared with sequences of members of the genus Nocardiopsis. The highest 16S rRNA gene sequence similarity was observed with Nocardiopsis lucentensis DSM 44048T (98.2 %), N. salina YIM 90010T (98.0 %), N. dassonvillei subsp. albirubida DSM 40465T (X97882) (98.0 %), N. xinjiangensis YIM 90004T (97.9 %), N. alba DSM 43377T (97.8 %), N. halotolerans DSM 44410T (97.8 %), N. synnemataformans IMMIB D-1215T (97.7 %), N. dassonvillei subsp. dassonvillei DSM 43111T (97.7 %), N. aegyptia DSM 44442T (97.5 %) and N. kunsanensis HA-9T (97.3 %). Neighbour-joining phylogenetic analysis revealed that strain YU1183-22T formed a monophyletic clade with N. salina YIM 90010T, N. kunsanensis HA-9T and N. xinjiangensis YIM 90004T and this cluster was also recovered with the other algorithms tested (Fig. 1⇓). N. lucentensis DSM 44048T did not cluster with strain YU1183-22T.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic tree derived from 16S rRNA gene sequences showing the relationship of strain YU1183-22T with the type strains of related species of the genus Nocardiopsis. Filled circles indicate that the corresponding nodes are also recovered in trees generated with the minimum-evolution, maximum-parsimony and maximum-likelihood methods. Bootstrap values (>50 %) based on 1000 resamplings are shown at the branch nodes. Bar, 0.01 substitutions per nucleotide position.

    DNA–DNA relatedness between strain YU1183-22T and N. lucentensis NBRC 15854T and its closest phylogenetic neighbours was 45.7–52.9 % (N. lucentensis, 51.7 %; N. salina JCM 13364T, 45.7 %; N. kunsanensis NBRC 100348T, 52.9 %; and N. xinjiangensis NBRC 104162T, 48.7 %). These values were well below the 70 % cut-off point recommended for the assignment of bacterial strains to the same genomic species (Wayne et al., 1987).

    Strain YU1183-22T could also be distinguished from its phylogenetic neighbours by a range of biochemical and phenotypic characteristics (Table 2⇓). The isolate could be differentiated from its closest phylogenetic neighbours and N. lucentensis NBRC 15854T by its ability to lyse Tween 80 and from its closest phylogenetic neighbours by its ability to grow without NaCl. N. salina, N. kunsanensis and N. xinjiangensis have been reported as being halophilic (Li et al., 2004; Chun et al., 2000; Li et al., 2003).

    Table 2.

    Phenotypic characteristics that distinguish strain YU1183-22T from the type strains of the most closely related species of the genus Nocardiopsis

    Strains: 1, Nocardiopsis nikkonensis sp. nov. YU1183-22T; 2, N. salina JCM 13364T; 3, N. kunsanensis NBRC 100348T; 4, N. xinjiangensis NBRC 104162T; 5, N. lucentensis NBRC 15854T; +, Positive; w, weakly positive; −, negative.

    Phenotypic, genotypic and chemotaxonomic characteristics show that strain YU1183-22T represents a novel species within the genus Nocardiopsis, for which the name Nocardiopsis nikkonensis sp. nov. is proposed.

    Description of Nocardiopsis nikkonensis sp. nov.

    Nocardiopsis nikkonensis (nik.ko.nen′sis. N.L. fem. adj. nikkonensis pertaining to Nikko City, Tochigi, Japan, where the type strain was isolated).

    Aerobic, Gram-positive, non-motile actinomycete that produces a white aerial mycelium. Substrate mycelium is pale yellow. Diffusible pigments are not produced. Melanoid pigments are not formed on ISP 6 or ISP 7. Short-branched substrate mycelium fragments into non-motile elements. Aerial mycelium shows a typical zig-zag formation prior to sporulation. Grows with 0–20 % (w/v) NaCl, at pH 6–11 and at 25 and 30 °C, but not at 42 °C. H2S is produced, starch is hydrolysed and Tween 80 is lysed. Nitrate is not reduced. Catalase-positive but oxidase-negative. As sole carbon sources, utilizes l-arabinose, d-fructose, d-galactose, d-glucose, l-rhamnose, sucrose and d-xylose, but not cellobiose, lactose, maltose, d-mannose, melibiose, raffinose, d-ribose, sodium acetate, glycerol, myo-inositol or d-mannitol. Contains meso-diaminopimelic acid but no diagnostic sugars. The predominant menaquinones are MK-10(H10) and MK-10(H8). The polar lipids are phosphatidylcholine and phosphatidylglycerol. The major cellular fatty acids are iso-C16 : 0, anteiso-C17 : 0 and tuberculostearic acid. The DNA G+C content of the type strain is 72.3 mol%.

    The type strain is YU1183-22T (=NBRC 102170T =KCTC 19666T), isolated from compost.

    Acknowledgments

    We are grateful to Miss Haruna Ashizawa for her excellent technical assistance.

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