SGM Journals
Actinobacteria

Amycolatopsis ultiminotia sp. nov., isolated from rhizosphere soil, and emended description of the genus Amycolatopsis

Soon Dong Lee

  • Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
  • Correspondence
    Soon Dong Lee
    sdlee{at}cheju.ac.kr

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(6):1401–1404 · https://doi.org/10.1099/ijs.0.006577-0

    View at publisher PubMed

    Abstract

    A novel actinomycete, designated strain RP-AC36T, was isolated from a cliff-associated plant (Peucedanum japonicum Thunb.) in the Republic of Korea and its taxonomic status was determined by using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequence analysis showed that the organism formed a distinct clade within the radiation of the genus Amycolatopsis. The chemotaxonomic properties supported the assignment of the isolate to the genus Amycolatopsis. High levels of 16S rRNA gene sequence similarity were found with Amycolatopsis sulphurea (98.2 %), Amycolatopsis halotolerans (97.5 %) and Amycolatopsis jejuensis (97.1 %). DNA–DNA relatedness data, together with phenotypic differences, clearly distinguished the isolate from its closest relative A. sulphurea. Based on the phenotypic and genotypic evidence, it is suggested that the organism be assigned as representing a novel species of the genus Amycolatopsis, for which the name Amycolatopsis ultiminotia sp. nov. is proposed. The type strain is RP-AC36T (=NRRL B-24662T=DSM 45180T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RP-AC36T is FM177516.

    • The phospholipid profile of cells of strain RP-AC36T and fatty acid compositions of strain RP-AC36T and its phylogenetic neighbours are available as supplementary material with the online version of this paper.

    The genus Amycolatopsis is one of the well-defined taxa in the family Pseudonocardiaceae, based on chemotaxonomic characteristics (Lechevalier et al., 1986; Kim & Goodfellow, 1999; Yassin et al., 1993; Groth et al., 2007) and phylogenetic evidence based on the comparison of 16S rRNA gene sequences (Lee et al., 2000; Tan et al., 2006). In the recently emended genus description (Groth et al., 2007), some species contain MK-11(H4), instead of MK-9(H4), as the predominant menaquinone. At the time of writing, the genus consists of 38 recognized species and many novel species have been described from diverse environments such as soil, vegetation, human and animal clinical sources, fresh water, rock and subterranean sites. The present polyphasic study was designed to determine the taxonomic status of an Amycolatopsis strain that was isolated from a rhizosphere soil sample.

    Strain RP-AC36T was isolated from a cliff-associated plant (Peucedanum japonicum Thunb.) on Mara Island, Jeju, Republic of Korea. For bacterial isolation, 10-fold dilutions of rhizosphere soil samples with sterile distilled water were inoculated on starch-casein agar and incubated at 30 °C for 14 days, as described by Lee (2006). The organism was maintained as mycelial fragments in 20 % (v/v) glycerol at −80 °C.

    Morphological and cultural characteristics of strain RP-AC36T were examined on various media: ISP 2, ISP 3, ISP 4, ISP 5, ISP 6 and ISP 7 (Shirling & Gottlieb, 1966), oatmeal-nitrate agar (Prauser & Bergholz, 1974) and HV agar (Nonomura & Ohara, 1969). The results were recorded after incubation for 14 days at 30 °C. For electron microscopy, cells were prepared as described previously (Lee, 2006) and observed with a scanning electron microscope (JSM 5410LV; JEOL). For genetic comparison, Amycolatopsis sulphurea IMSNU 20060T was grown on ISP 2 medium at 30 °C.

    Strain RP-AC36T grew well on all media tested. Substrate mycelium was well-developed, branched and white to yellow in colour on the different media. Whitish aerial mycelium was abundant on ISP 4 medium, oatmeal-nitrate agar and HV agar but scanty or absent on ISP 2, ISP 3, ISP 5, ISP 6 and ISP 7 media. Both aerial and substrate mycelia fragmented into rod-shaped cells. Soluble pigments were not produced on any of the media tested.

    NaCl tolerances (0–9 %, w/v) and temperatures (4, 10, 20, 30, 37, 42 and 45 °C) for growth were tested on ISP 2 medium as the basal medium. The initial pH for growth was examined over the range pH 4.1–12.1 (with intervals of 1.0 unit). Acid production from carbohydrates, degradation of casein, hypoxanthine, starch, dl-tyrosine, urea and xanthine, nitrate reduction, gelatin liquefaction and catalase activity were examined as described previously (Lee et al., 2006). Hydrolysis of chitin (0.5 %, w/v), CM-cellulose (0.5 %, w/v; Sigma) and elastin (0.4 %, w/v) was examined on ISP 2 medium. DNA hydrolysis was determined by using DNase test agar (Difco). Data for physiological properties are shown in the species description and Table 1⇓.

    Table 1.

    Phenotypic characteristics that differentiate strain RP-AC36T from its phylogenetic neighbours

    Strains: 1, strain RP-AC36T (A. ultiminotia sp. nov.); 2, A. jejuensis N7-3T; 3, A. sulphurea IMSNU 20060T. Data are from this study, Lee & Hah (2001) and Lee (2006). All strains produced acid from d-galactose, d-mannitol and d-mannose but not from l-arabinose, meso-erythritol, melezitose, melibiose, methyl α-d-glucoside, raffinose, salicin, d-sorbitol, l-sorbose or d-xylitol, grew in the presence of 2 % (w/v) NaCl and were positive for casein hydrolysis, gelatin liquefaction and dl-tyrosine degradation. +, Positive; −, negative; w, weak; nd, no data available.

    Biomass for chemotaxonomic characterization was obtained from cultures grown in trypticase soy broth (Difco) for 3 days at 30 °C with shaking. Analyses of the isomer of diaminopimelic acid (Staneck & Roberts, 1974) and sugars (Saddler et al., 1991) in whole-cell hydrolysates, mycolic acids (Minnikin et al., 1980) and polar lipids (Minnikin et al., 1975; 1984) were performed as described previously (Lee et al., 2006). Menaquinones were extracted by using the method of Minnikin et al. (1984) and analysed by HPLC (Kroppenstedt, 1985). Cellular fatty acid methyl esters were prepared by alkaline methanolysis and analysed by GC with an Agilent model 6850 gas chromatograph, as described previously (Lee & Hah, 2001).

    The chemotaxonomic characteristics of strain RP-AC36T were typical of the genus Amycolatopsis in having a type IV cell-wall composition (meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates), MK-9(H4) as the major menaquinone and a polar lipid profile including phosphatidylmethylethanolamine and an unknown ninhydrin-positive phospholipid (see Supplementary Fig. S1, available in IJSEM Online). Mycolic acids were not present. The cellular fatty acids of the isolate were represented by considerable amounts of saturated and branched-chain acids, with small amounts of unsaturated or hydroxy fatty acids. The major fatty acids were C17 : 0, C15 : 0, i-C16 : 0 and i-C15 : 0. The cellular fatty acid profiles of strain RP-AC36T and its phylogenetic neighbours, Amycolatopsis jejuensis N7-3T and A. sulphurea IMSNU 20060T, are given in Supplementary Table S1 (in IJSEM Online). Strain RP-AC36T showed significant differences from A. jejuensis N7-3T and A. sulphurea IMSNU 20060T in the relative amounts of C15 : 0, C16 : 0, C17 : 0, C18 : 0, i-C14 : 0, i-C17 : 0 and ai-C15 : 0 fatty acids. The DNA G+C content was analysed by HPLC (Mesbah et al., 1989) and measured to be 67.5 mol%.

    Extraction of chromosomal DNA and the amplification of the 16S rRNA gene by PCR were performed as described previously (Lee, 2006). The resultant PCR product was purified using a Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's instructions and subjected to direct sequencing as described previously (Lee, 2006). The clustal_x program (Thompson et al., 1997) was used for multiple sequence alignments. Phylogenetic analyses, tree construction and bootstrap analysis were carried out by using several programs contained in the phylip package (Felsenstein, 1993).

    A partial 16S rRNA gene sequence (1413 nt) of strain RP-AC36T was compared with the corresponding sequences of representatives of the genus Amycolatopsis. The neighbour-joining tree (Fig. 1⇓) revealed that the organism belongs to the genus Amycolatopsis and forms a branch between A. sulphurea and A. jejuensis. The phylogenetic position was supported by a high bootstrap value (82 %) and was also found in trees constructed using maximum-parsimony and maximum-likelihood algorithms (not shown). The closest relatives were A. sulphurea IMSNU 20060T (98.2 % 16S rRNA gene sequence similarity) followed by Amycolatopsis halotolerans NRRL B-24428T (97.5 %) and A. jejuensis NRRL B-24427T (97.1 %). Strain RP-AC36T shared 16S rRNA gene sequence similarities between 94.8 and 97.0 % with the type strains of other representatives of the genus Amycolatopsis.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining (Saitou & Nei, 1987) tree showing the phylogenetic relationships between strain RP-AC36T and related taxa of the genus Amycolatopsis. Evolutionary distances were calculated by using the model of Jukes & Cantor (1969). Asterisks indicate branches that were also found in the maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) trees. Bootstrap values greater than 50 % are indicated at nodes. Bar, 0.01 substitutions per nucleotide position.

    DNA–DNA hybridization of strain RP-AC36T was performed against the closest relative, A. sulphurea IMSNU 20060T. Genomic DNA was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described previously (Cashion et al., 1977). DNA–DNA hybridization was carried out using the spectrophotometric method as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983). Strain RP-AC36T had a DNA–DNA relatedness of 26.6 % (31.0 % in the duplicate measurement) with A. sulphurea IMSNU 20060T. According to the criterion recommended by Wayne et al. (1987), a DNA–DNA hybridization value less than 70 % indicates that the isolate can be assigned to a different species. Differential phenotypic characteristics of the isolate from its phylogenetic neighbours, A. jejuensis and A. sulphurea, are given in Table 1⇑. Strain RP-AC36T differed from both strains in nitrate reduction, xanthine degradation and in acid production from cellobiose, d-fructose, lactose, l-rhamnose and d-xylose.

    The phenotypic and DNA–DNA hybridization data presented here suggest that the isolate can be assigned as a novel species of the genus Amycolatopsis, for which the name Amycolatopsis ultiminotia sp. nov. is proposed.

    Emended description of the genus AmycolatopsisLechevalier et al. 1986

    Amycolatopsis (A.my.co.la.top′sis. M.L. fem. n. Amycolata genus belonging to the order Actinomycetales; Gr. n. opsis appearance; M.L. fem. n. Amycolatopsis that which appears similar to Amycolata).

    The description of the genus Amycolatopsis Lechevalier et al. 1986 is emended as follows. The diagnostic phospholipid is phosphatidylethanolamine (with or without phosphatidylmethylethanolamine) or phosphatidylmethylethanolamine (type II phospholipid pattern).

    Description of Amycolatopsis ultiminotia sp. nov.

    Amycolatopsis ultiminotia (ul.ti.mi.no′ti.a. L. sup. adj. ultimus farthest, extreme; L. fem. adj. notia southern; N.L. fem. adj. ultiminotia farthest southern, implying that the type strain was isolated from the southernmost parts of the Republic of Korea).

    Aerobic, Gram-positive, non-acid–alcohol-fast, catalase-positive. The aerial mycelium is white and the vegetative mycelium is cream to yellow. Both aerial and substrate hyphae fragment into rod-shaped elements. Growth occurs between 10 and 37 °C, with optimal growth at 20–37 °C. Growth does not occur at 42 °C. Initial pH for growth is pH 5.1–12.1, with optimal growth at pH 5.1–9.1. Growth occurs in the presence of up to 5 % (w/v) NaCl. Acid is produced from dulcitol but not from d-arabinose, inulin, methyl α-d-mannoside or d-ribose. DNA and elastin are hydrolysed but chitin or CM-cellulose are not hydrolysed. Other phenotypic features are given in Table 1⇑. Major fatty acids are C17 : 0 (23.7 %), C15 : 0 (19.8 %), i-C16 : 0 (13.2 %) and i-C15 : 0 (10.0 %). The DNA G+C content of the type strain is 67.5 mol%.

    The type strain is strain RP-AC36T (=NRRL B-24662T =DSM 45180T), isolated from the rhizosphere of a cliff-associated plant (Peucedanum japonicum Thunb.).

    Acknowledgments

    This work was supported by the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science & Technology, Republic of Korea.

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