SGM Journals
Actinobacteria

Microbacterium ginsengiterrae sp. nov., a β-glucosidase-producing bacterium isolated from soil of a ginseng field

Yeon-Ju Kim, Myung Kyum Kim, Thi Phuong Nam Bui, Ho-Bin Kim, Sathiyaraj Srinivasan, Deok-Chun Yang

  • Korean Ginseng Center and Ginseng Genetic Resource Bank, Kyung Hee University, 1 Seocheon-dong, Giheung-gu Yongin-si, Gyeonggi-do 449-701, Republic of Korea
  • Correspondence
    Deok-Chun
    Yangdeokchunyang{at}yahoo.co.kr

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2808–2812 · https://doi.org/10.1099/ijs.0.015784-0

    View at publisher PubMed

    Abstract

    Strain DCY37T was isolated from a soil sample of a ginseng field in the Republic of Korea and characterized in order to determine its taxonomic position. Cells were Gram-staining-positive, heterotrophic, strictly aerobic, non-motile short rods. 16S rRNA gene sequence analysis revealed that strain DCY37T belongs to the genus Microbacterium. According to 16S rRNA gene sequence analysis, it is closely related to Microbacterium aerolatum DSM 14217T (98.8 %), Microbacterium hydrocarbonoxydans DSM 16089T (98.5 %), Microbacterium natoriense JCM 12611T (98.5 %), Microbacterium foliorum (98.4 %) and Microbacterium phyllosphaerae (98.3 %). However, DNA–DNA hybridization studies showed reassociation values of less than 70 % between representative strains and DCY37T. The DNA G+C content was 64.5 mol%. Strain DCY37T possessed chemotaxonomic markers that were consistent with classification in the genus Microbacterium, i.e. MK-12 and MK-13 as the major menaquinones and anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 as the predominant cellular fatty acids. The major cell wall sugars were ribose, xylose and galactose. The diamino acid in cell-wall hydrolysates of strain DCY37T was ornithine and major cell-wall amino acids were alanine, glycine, d-glutamic acid and serine. The major polar lipids were glycolipid, phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids. Based on these data, DCY37T (=KCTC 19526T =JCM 15516T) should be classified as the type strain of a novel species of the genus Microbacterium, for which the name Microbacterium ginsengiterrae sp. nov. is proposed.

    • The GenBank/EMBL/DBBJ accession number for 16S rRNA gene sequence of strain DCY37T is EU873314.1.

    • A transmission electron micrograph of cells of strain DCY37T, and polar lipid and fatty acid profiles of strain DCY37T, are available with the online version of this paper.

    Members of the genus Microbacterium can be isolated from a wide range of different environmental habitats from soil to insects, human clinical specimens and marine environments. The genus Microbacterium (phylum Actinobacteria, class Actinobacteria, order Actinomycetales, family Microbacteriaceae) was first reported by Orla-Jensen and was emended by Collins et al.. More recently, the genus was emended again to unite the genera Microbacterium and Aureobacterium. At the time of writing, the genus Microbacterium consisted of 68 species with validly published names, including three species (Microbacterium soli, Microbacterium azadirachtae and Microbacterium agarici) whose descriptions were still in press. In this study, we characterized a new isolate belonging to the genus Microbacterium. Extensive physiological, chemotaxonomic and phylogenetic analyses were carried out to determine the precise taxonomic position of strain DCY37T.

    Cell morphology and motility were observed with a Nikon light microscope (×1000 magnification) using the hanging drop method, with cells grown on R2A agar for 2 days at 30 °C. Transmission electron microscopy was carried out as follows. Bacteria were grown on R2A plates at 30 °C for 24 h. Resuspended cells were placed on carbon- and Formvar-coated nickel grids for 30 s. Grids were floated on one drop of 0.1 % (w/v) aqueous uranyl acetate and then viewed with a Carl Zeiss LEO912AB electron microscope at 100 kV under standard operating conditions.

    The Gram reaction was performed according to the non-staining method (Buck, 1982). Oxidase activity was evaluated via the oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was revealed by bubble production after the application of 3 % (v/v) hydrogen peroxide solution. Growth on nutrient agar (NA), trypticase soy agar (TSA), R2A agar, LB agar and MacConkey agar was evaluated at 30 °C. Growth at different temperatures (4, 18, 25, 30, 37 and 42 °C) and various pH (5.0–10.0 at intervals of 0.5 pH units) was assessed after 5 days of incubation on R2A plates. Salt tolerance was tested in R2 broth supplemented with 0–10 % (w/v) NaCl after 5 days of incubation. Strain DCY37T and the type strains Microbacterium hydrocarbonoxydans DSM 16089T, Microbacterium natoriense JCM 12611T, Microbacterium aerolatum DSM 14217T and Microbacterium foliorum DSM 12966T were grown on TSA for 2 days at 30 °C for fatty acid analysis. The API 20NE, API ZYM and API ID32 GN microtest systems were employed following the recommendations of the manufacturer (bioMérieux). The API kits were incubated at 30 °C and results were checked after 24 h and 48 h, except for API ZYM where results were recorded after 10 h. Cells grown in LB for 6 days at 26 °C were used for determination of the respiratory quinone system. Differential phenotypic characteristics between strain DCY37T and the most closely related type strains of species of the genus Microbacterium are summarized in Table 1⇓.

    Table 1.

    Phenotypic characteristics differentiating strain DCY37T and related type strains of species of the genus Microbacterium

    Taxa: 1, Microbacterium ginsengiterrae sp. nov. DCY37T; 2, M. hydrocarbonoxydans DSM 16089T; 3, Microbacterium natoriense JCM 12611T; 4, Microbacterium aerolatum DSM 14217T; 5, Microbacterium foliorum DSM 12966T. Data from this study, except where indicated. All five strains were positive for activities of esterase (C4), esterase lipase (C8), arginine dihydrolase, urease, α-glucosidase, β-glucosidase and assimilation of sucrose. All five strains were negative for the reduction of nitrates to nitrites, indole production, alkaline phosphatase, valine arylamidase, cystine arylamidase, trypsin and α-fucosidase activities, assimilation of suberic acid, sodium malonate, potassium 5-ketogluconate, l-fucose, d-sorbitol, capric acid, adipate and phenylacetate, and for gelatin hydrolysis. +, Positive; −, negative; w, weakly positive; nd, no data available; py, pale yellow; y, yellow; dy, deep yellow.

    Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified by TLC and subsequently analysed by HPLC, as described by Collins & Jones (1981) and Shin et al. (1996). For fatty acid analysis, strain DCY37T and the other type strains (M. hydrocarbonoxydans DSM 16089T, M. natoriense JCM 12611T, M. aerolatum DSM 14217T and M. foliorum DSM 12966T) were grown on TSA for 48 h at 30 °C, and 50 mg of the biomass was harvested. Fatty acid methyl esters were prepared, separated and identified with the Sherlock Microbial Identification System (MIDI; Sasser, 1990).

    Polar lipids were extracted and examined by two-dimensional TLC (Minnikin et al., 1977; Bligh & Dyer, 1959). Amino-acid composition in the cell wall peptidoglycan was determined by using TLC after hydrolysis with 6 M HCl at 100 °C for 18 h (Busse et al., 1996; Schenkel et al., 1995; Komagata & Suzuki, 1987). Cell wall sugars were analysed as described by Staneck & Roberts (1974).

    Genomic DNA of the novel strain was extracted and purified, degraded by P1 nuclease into nucleosides, and the G+C content was determined as described by Mesbah et al. (1989) using reversed-phase HPLC.

    DNA–DNA hybridization was carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using a BioAssay reader for fluorescence measurement. The hybridization temperature was 37 °C. Five replications were performed for each sample; the highest and lowest values were excluded and the remaining three values were used for calculation of hybridization values. Reciprocal experiments were performed.

    Genomic DNA of strain DCY37T was extracted and purified by using a Genomic DNA Isolation kit (Core Bio System). The 16S rRNA gene was amplified and sequenced by using the universal bacterial primer sets 9F and 1512R, and 27F and 1492R, respectively (Kim et al., 2005). The purified PCR products were sequenced by Genotec, Daejeon, Korea. 16S rRNA gene sequences were compiled by using SeqMan software, and edited using the BioEdit program (Hall, 1999). Multiple alignments were performed with the clustal x program (Thompson et al., 1997), and the Kimura two-parameter model (Kimura, 1983) was used to calculate evolutionary distances. Neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony trees were reconstructed using the mega 3 (Felsenstein, 1985) and phylip programs (Choi et al., 2000), respectively.

    Strain DCY37T was an aerobic, Gram-positive, rod-shaped (0.5–0.7 μm in diameter and 1.5–2.0 μm in length; Supplementary Fig. S1, available in IJSEM online) and non-motile bacterium. Colonies on R2A agar were pale yellow, circular and slightly convex after 3 days of cultivation at 30 °C. Growth occurred between 15 and 45 °C and at pH 5 to 9; optimal growth occurred at 30 °C and pH 7.0. Poor growth was observed in the absence of NaCl and in the presence of >8 % (w/v) NaCl. The strain was slightly halophilic. The physiological characteristics of strain DCY37T are summarized in the species description and a comparison of selected characteristics with those of related type strains is shown in Table 1⇑.

    The main fatty acids detected in strain DCY37T were anteiso-C15 : 0 (44.9 %), anteiso-C17 : 0 (30.6 %) and iso-C16 : 0 (11.2 %) (Supplementary Table S1). The major menaquinones detected for this strain were MK-12 and MK-13. This pattern has been reported for other species of the genus Microbacterium (Schippers et al., 2005). The major polar lipids were glycolipid, phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids. The genus Microbacterium (and Aureobacterium) is characterized by diphosphatidylglycerol, phosphatidylglycerol and an unusual glycolipid, and these have been detected as the major polar lipids in numerous species of the genus (Young et al., 2010). The major cell-wall sugars were ribose, xylose and galactose. The diamino acid in cell-wall hydrolysates of strain DCY37T was ornithine. The major cell-wall amino acids were alanine, glycine, d-glutamic acid and serine. Analytical data suggested the presence of peptidoglycan type B2β in strain DCY37T.

    The 16S rRNA gene sequence of strain DCY37T, a continuous stretch of 1427 nt, was sequenced. The phylogenetic tree shows that strain DCY37T was clustered within the genus Microbacterium of the class Actinobacteria. It is closely related to M. aerolatum DSM 14217T (98.8 %), M. hydrocarbonoxydans DSM 16089T (98.5 %), M. natoriense JCM 12611T (98.5 %), M. foliorum DSM 12966T (98.4 %) and Microbacterium phyllosphaerae DSM 13468T (98.3 %) (Fig. 1⇓). The DNA G+C content of strain DCY37T was 64.5 mol%, a value within the range reported for members of the genus Microbacterium (Table 1⇑). However, the DNA–DNA relatedness values between strain DCY37T and strains M. aerolatum DSM 14217T, M. hydrocarbonoxydans DSM 16089T, M. natoriense JCM 12611T and M. foliorum DSM 12966T were 51.8±0.7, 41.8±0.4, 35.4±0.8 and 29.8±0.3 %, respectively. Reciprocal hybridization tests resulted in DNA–DNA relatedness values of 37.7 % between M. hydrocarbonoxydans DSM 16089T and strain DCY37T. These results show that strain DCY37T is an independent species at the genotype level according to the limits devised by Wayne et al. (1987).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of strain DCY37T and representative members of the genus Microbacterium. Rarobacter faecitabidus DSM 4813T was used as an outgroup. Bootstrap values >50 % based on 1000 replications are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the tree generated using the maximum-parsimony algorithm. Bar, 0.01 substitutions per nucleotide position.

    Based on the phylogenetic analysis and phenotypic characteristics, strain DCY37T is described as a novel species in the genus Microbacterium, for which the name Microbacterium ginsengiterrae sp. nov. (type strain DCY37T =KCTC 19526T =JCM 15516T) is proposed.

    Description of Microbacterium ginsengiterrae sp. nov.

    Microbacterium ginsengiterrae (gin.seng.i.ter′rae. N.L. n. ginsengum ginseng; L. n. terra soil; N.L. gen. n. ginsengiterrae of soil of a ginseng field, the source of the type strain).

    Cells are Gram-positive, heterotrophic, strictly aerobic, non-motile and rod-shaped (0.5–0.7 μm diameter and 1.5–2.0 μm length). Colonies on R2A agar are circular with regular edges, yellow and translucent with a diameter of 2–3 mm after 2 days of incubation on R2A agar. Grows between 15 and 45 °C and at pH 5 to 9. Optimal growth at 30 °C and pH 7.0. Poor growth in the absence of NaCl and in the presence of >8 % (w/v) NaCl. Slightly halophilic. Catalase, oxidase and DNase activities are present. In the API ZYM kit, α-glucosidase, β-glucosidase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase and α-mannosidase activities are present; alkaline phosphatase, lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and α-fucosidase activities are absent. In the API 20NE kit, arginine dihydrolase and urease reactions are positive; the following reactions are negative: reduction of nitrate to nitrite, reduction of nitrate to nitrogen, indole production, protease, β-galactosidase, glucose acidification, and assimilation of l-arabinose, d-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. In the API 32GN kit, the following substrates are assimilated: d-ribose, l-rhamnose, sucrose, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid; the following substrates are not assimilated: glucose, inositol, itaconic acid, suberic acid, sodium malonate, sodium acetate, l-alanine, glycogen, l-serine, salicin, melibiose, l-fucose, d-sorbitol, propionic acid, capric acid, valeric acid, l-histidine, 3-hydroxybutyric acid and l-proline. The predominant menaquinones are MK-12 and MK-13. The major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major cell-wall sugars are ribose, xylose and galactose. The cell-wall peptidoglycan is of the B2β type, (l-Hsr)–d-Glu→Gly→d-Orn. The major polar lipids are glycolipid, phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids. The DNA G+C content of the type strain is 64.5 mol%.

    The type strain, DCY37T (=KCTC 19526T =JCM 15516T), was isolated from soil of a ginseng field in the Republic of Korea.

    Acknowledgments

    This work was supported by a grant from the Plant Diversity Research Center of the 21st Century Frontier Research Program (code no. PF06222-00) funded by the Ministry of Science and Technology of the Korean government.

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