SGM Journals
Actinobacteria

Pseudonocardia babensis sp. nov., isolated from plant litter

Yayoi Sakiyama, Nguyen K. N. Thao, Hoang V. Vinh, Nguyen M. Giang, Shinji Miyadoh, Duong V. Hop, Katsuhiko Ando

  • 1NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), Kazusakamatari 2-5-8, Kisarazu, Chiba, 292-0818, Japan
  • 2Institute of Microbiology and Biotechnology (IMBT), Vietnam National University, Hanoi (VNUH), Japan
  • Correspondence
    Yayoi Sakiyama
    sakiyama-yayoi{at}nite.go.jp

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(10):2336–2340 · https://doi.org/10.1099/ijs.0.018127-0

    View at publisher PubMed

    Abstract

    A novel actinomycete, designated strain VN05A0561T, was isolated from plant litter collected at Ba Be National Park, Vietnam. The substrate mycelia and spore chains fragmented in a manner similar to nocardioform actinomycetes; the spores had smooth surfaces and were rod-shaped. Strain VN05A0561T had the following chemotaxonomic characteristics: meso-diaminopimelic acid in the cell-wall peptidoglycan, arabinose and galactose as characteristic sugars, MK-8(H4) as the major isoprenoid quinone, phosphatidylcholine as the diagnostic phospholipid and iso-C16 : 0 as the major cellular fatty acid. Strain VN05A0561T shared low levels of 16S rRNA gene sequence similarity (<97 %) with the type strains of recognized species of the genus Pseudonocardia and could be differentiated from its closest phylogenetic relatives based on phenotypic characteristics. These results suggested that strain VN05A0561T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia babensis sp. nov. is proposed. The type strain is VN05A0561T (=VTCC-A-1757T=NBRC 105793T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain VN05A0561T is AB514449.

    The genus Pseudonocardia was established as a nocardioform actinomycete by Henssen (1957), and its description has been emended based on both chemotaxonomic and morphological variations (Reichert et al., 1998; Huang et al., 2002; Park et al., 2008). On the basis of the lack of mycolic acids and the presence of cell-wall type IV, the genera Amycolata (Lechevalier et al., 1986) and Pseudoamycolata (Akimov et al., 1989) were also proposed as nocardioform actinomycetes. However, the latter two genera were found to have chemotaxonomic properties similar to those of the genus Pseudonocardia (Kothe et al., 1989; Takeuchi et al., 1992), and were incorporated in the genus Pseudonocardia on the basis of 16S rRNA gene sequence analysis (Warwick et al., 1994; McVeigh et al., 1994). Subsequently, members of the genus Actinobispora (Jiang et al., 1991) were also transferred to the genus Pseudonocardia based on identification with the use of specific PCR primers, and re-analysis of 16S rRNA gene sequences and menaquinones of the type species (Huang et al., 2002).

    At the time of writing, the genus Pseudonocardia comprises 30 recognized species. Strains of this genus have been isolated from various environmental samples, such as active sludge soil, including those polluted by industrial chemicals (Lee et al., 2004; Kämpfer & Kroppenstedt, 2004; Mahendra & Alvarez-Cohen, 2005; Liu et al., 2006; Kämpfer et al., 2006; Park et al., 2008) and plant samples, including stems, leaves, root nodules, tree-bark compost and a traditional Chinese medicinal plant (Evtushenko et al., 1989; Reichert et al., 1998; Gu et al., 2006; Chen et al., 2009).

    In the present study, a Pseudonocardia-like strain isolated from a plant litter sample collected during the course of a study on the diversity of actinomycetes inhabiting Vietnam (Sakiyama et al., 2009) was studied by using a polyphasic taxonomic approach.

    Plant litter samples were collected at the Ba Be National Park, Bac Kan, in the mountainous area of northern Vietnam. Samples were dried at room temperature for 5–7 days and subsequently used for isolation. Rehydration and centrifugation methods (Hayakawa et al., 2000) were employed for isolation by using humic acid-vitamin agar (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l−1) and kabicidine (0.75 mg l−1). Strain VN05A0561T was isolated after incubation for more than 10 days at room temperature.

    Strain VN05A0561T was cultured on yeast extract-soluble starch medium (YS medium; per litre distilled water: 2 g yeast extract, 10 g soluble starch and 15 g agar; pH 7.3) at 28 °C for 10–14 days. Pale yellow colonies with white aerial mycelia appeared on the YS medium. The substrate mycelia of the strain fragmented as per those of a nocardioform actinomycete. The spore chains sometimes formed zig-zag-shaped and intercalary swellings. The spores were rod-shaped (width, 0.3–0.5 μm; length, 1.0–2.0 μm) with a smooth surface (Fig. 1⇓). Spores were non-motile. The cultural characteristics of strain VN05A0561T were observed on ISP media 2–7 (Shirling & Gottlieb, 1966) and YS medium at 28 °C for 3 weeks. Moderate growth was observed on ISP 2–7 and YS medium. Raised and wrinkled colonies with moderate yellow colour appeared on ISP 2, 6 and 7. White aerial mycelia developed on ISP 2 and YS medium.

    Figure image not available in archive
    Fig. 1.

    Scanning electron micrograph of cells of strain VN05A0561T grown on ISP 2 medium for 14 days at 28 °C. Bar, 5 μm.

    For chemotaxonomic analysis, biomass of strain VN05A0561T was obtained by centrifugation and lyophilization after incubation in yeast extract-glucose broth (per litre distilled water: 10 g yeast extract and 10 g glucose; pH 7.3) for 10 days at 28 °C. Diaminopimelic acids and whole-cell sugars were analysed according to the method described by Staneck & Roberts (1974), isoprenoid quinones and phospholipids according to Minnikin et al. (1984), and cellular fatty acids according to Tamura et al. (1994) and by using the MIDI Sherlock Microbial Identification system (Microbial ID). Strain VN05A0561T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and arabinose and galactose as characteristic sugars. The cell wall was of type IV (Lechevalier & Lechevalier, 1970). The major isoprenoid quinone was MK-8(H4). The cellular fatty acid profile of strain VN05A0561T contained iso-C15 : 0 (7.9 %), iso-C16 : 1 H (10.5 %), iso-C16 : 0 (54.4 %) and anteiso-C17 : 0 (14.1 %) as major components, with minor amounts of iso-C14 : 0 (3.4 %), anteiso-C15 : 0 (1.3 %), C15 : 1 B (1.7 %), C16 : 0 9? methyl (1.8 %), iso-C17 : 0 (1.3 %) and anteiso-C17 : 0 (3.7 %). The phospholipid was of type PIII, and the polar lipid profile consisted of phosphatidyl-N-methylethanolamine, phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol and three unknown glycolipids. The chemotaxonomic properties of strain VN05A0561T were consistent with the characteristics described for the genus Pseudonocardia.

    For 16S rRNA gene sequence analysis, the DNA of strain VN05A0561T was extracted and amplified by using the procedures described by Sakiyama et al. (2009). The 16S rRNA gene sequence of strain VN05A0561T was aligned via the clustal_x program (Thompson et al., 1997) by using the corresponding sequences of the type strains of all recognized species of the genus Pseudonocardia available in the DDBJ/EMBL/GenBank databases. Phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) and maximum-likelihood (Felsenstein, 1981) methods. The topology of the phylogenetic trees was evaluated by bootstrap analysis of 1000 replicates (Felsenstein, 1985). The DNA G+C content of strain VN05A0561T was determined by using the method described by Mesbah et al. (1989).

    The 16S rRNA gene sequence of strain VN05A0561T determined herein was 1454 nt long and showed 93.9–96.9 % nucleotide sequence similarity with those of the type strains of all recognized species of the genus Pseudonocardia. Strain VN05A0561T was most closely related to the type strains of Pseudonocardia xinjiangensis (96.9 % 16S rRNA gene sequence similarity), Pseudonocardia chloroethenivorans, Pseudonocardia hydrocarbonoxydans, Pseudonocardia alaniniphila, Pseudonocardia aurantiaca (all 96.6 %) and Pseudonocardia asaccharolytica (96.4 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain VN05A0561T formed a cluster with the type strains of P. asaccharolytica and Pseudonocardia acaciae (Fig. 2⇓), but did not form any definite clusters with other closely related Pseudonocardia strains. 16S rRNA gene sequence analysis thus suggested that strain VN05A0561T represents a novel species of the genus Pseudonocardia (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers, 2006). The DNA G+C content of strain VN05A0561T was 73 mol%.

    Figure image not available in archive
    Fig. 2.

    Neighbour-joining phylogenetic tree reconstructed based on 16S rRNA gene sequences showing the position of strain VN05A0561T among members of the genus Pseudonocardia. Numbers on branches are confidence limits estimated by bootstrap analysis of 1000 replicates (only values above 500 are presented). Closed circles indicate branches that were also recovered with both the maximum-parsimony and the maximum-likelihood method; open circles indicate branches that were also recovered with the maximum-likelihood method. Bar, 0.005 sequence divergence (Knuc).

    The physiological and biochemical characteristics of strain VN05A0561T were examined after incubation at 28 °C for 3 weeks. YS medium containing 0, 1, 2, 3, 4 and 5 % NaCl (w/v) was used for tests of NaCl tolerance. Urease production, degradation of casein and other compounds, utilization of citrate, other organic acids and carbohydrates, and acid production from carbohydrates were examined according to the methods of Sakiyama et al. (2009).

    Strain VN05A0561T could be differentiated from P. xinjiangensis based on NaCl tolerance, urease production, hypoxanthine decomposition and acid production from d-sorbitol and adonitol (Table 1⇓).

    Table 1.

    Differential physiological and biochemical characteristics between strain VN05A0561T and closely related species of the genus Pseudonocardia

    Taxa: 1, strain VN05A0561T; 2, P. xinjiangensis CCTCC AA97020T; 3, P. chloroethenivorans SL −1T; 4, P. hydrocarbonoxydans DSM 43281T; 5, P. alaniniphila CCTCC AA97001T; 6, P. aurantiaca CCTCC AA97002T; 7, P. asaccharolytica; 8, P. acaciae GMKU 095T. Data for reference strains were taken from Lee et al. (2004) and Duangmal et al. (2009). +, Positive; −, negative; w, weakly positive; nd, no data.

    On the basis of data from the present taxonomic study, we suggest that strain VN05A0561T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia babensis sp. nov. is proposed.

    Description of Pseudonocardia babensis sp. nov.

    Pseudonocardia babensis (ba.ben′sis. N.L. fem. adj. babensis referring to Ba Be National Park, Vietnam, from where the type strain was isolated).

    Aerobic, Gram-positive, non-motile actinomycete. Colonies on YS medium are pale yellow and on ISP media 2, 6 and 7 are moderate yellow. Substrate mycelium fragments. White aerial mycelia and rod-shaped spores (width, 0.3–0.5 μm; length, 1.0–2.0 μm) with a smooth surface form on ISP 2 and YS medium. Grows at 10–45 °C and in the presence of 0–3 % (w/v) NaCl. Decomposes aesculin, casein and testosterone, but not adenine, hypoxanthine, xanthine, l-tyrosine or urea. Utilizes carbohydrates such as l-arabinose, d-arabitol, cellobiose, dulcitol, d-fructose, d-galactose, d-glucose, myo-inositol, lactose, maltose, d-mannitol, melezitose, melibiose, methyl α-d-glucoside, raffinose, l-rhamnose, salicin, d-sorbitol, l-sorbose, starch, sucrose, trehalose, xylitol and d-xylose, but not adonitol. Utilizes organic acids such as fumarate, malate and succinate, but not benzoate, mucate, oxalate, or l-tartrate. Produces acid from l-arabinose, cellobiose, d-fructose, d-galactose, d-glucose, melezitose, d-sorbitol, sucrose and d-xylose, but not from adonitol or i-erythritol . The cell-wall diamino acid is meso-diaminopimelic acid. Whole-cell sugars are arabinose, mannose, glucose and galactose. The major menaquinone is MK-8(H4). Major fatty acids are iso-C16 : 0, iso-C16 : 1 and anteiso-C17 : 0. Phospholipids are phosphatidyl-N-methylethanolamine, phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol and three unknown glycolipids. The DNA G+C content of the type strain is 73 mol%.

    The type strain, VN05A0561T (=VTCC-A-1757T=NBRC 105793T), was isolated from plant litter collected in Vietnam.

    Acknowledgments

    This work was conducted as a joint research project between the Department of Biotechnology, National Institute of Technology and Evaluation, Japan, and the Institute of Microbiology and Biotechnology (IMBT), Vietnam National University, Hanoi. We are grateful to Dr Tomohiko Tamura, Ms Kozue Anzai, Mr Nobuyuki Goto, Dr Misa Otoguro, Mr Shinpei Ino, Ms Ayako Hashimoto and all IMBT members for their technical help and advice.

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