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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305-600, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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A supplementary table detailing the phenotypic characteristics that differentiate species of the genus Agromyces is available in IJSEM Online.
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and emended by Zgurskaya et al. (1992), currently includes the following species: Agromyces ramosus (Gledhill & Casida, 1969), Agromyces cerinus (Zgurskaya et al., 1992), Agromyces mediolanus (Suzuki et al., 1996), Agromyces luteolus, Agromyces rhizospherae and Agromyces bracchium (Takeuchi & Hatano, 2001), Agromyces albus (Dorofeeva et al., 2003), Agromyces aurantiacus (Li et al., 2003), Agromyces fucosus and Agromyces hippuratus (Zgurskaya et al., 1992; Ortiz-Martinez et al., 2004), Agromyces ulmi (Rivas et al., 2004), Agromyces neolithicus and Agromyces salentinus (Jurado et al., 2005a), Agromyces humatus, Agromyces italicus and Agromyces lapidis (Jurado et al., 2005b) and Agromyces subbeticus (Jurado et al., 2005c). Species of this genus are characterized chemotaxonomically by having MK-12 as the common predominant menaquinone, anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 as the major fatty acids and by DNA G+C contents of 65.3–73.3 mol% (Gledhill & Casida, 1969; Zgurskaya et al., 1992; Suzuki et al., 1996; Sasaki et al., 1998; Takeuchi & Hatano, 2001; Dorofeeva et al., 2003; Li et al., 2003; Jurado et al., 2005a, b, c). Here, we report the detailed taxonomic characterization of two Agromyces-like bacterial strains, UMS-62T and UMS-101, which were isolated from the rhizosphere of Allium victorialis var. platyphyllum, a type of wild edible greens grown on Ulleung island, Korea.
Strains UMS-62T and UMS-101 were isolated by a standard dilution plating technique at 30 °C on nutrient agar (NA; Difco). To investigate their morphological, physiological and biochemical characteristics, the strains were routinely cultivated at 30 °C on NA. Growth at various temperatures from 4 to 40 °C was measured on NA and tolerance to various NaCl concentrations was measured in nutrient broth (NB; Difco). The optimal pH and pH range for growth were determined in NB that was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on NA and on NA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Cell morphology and presence of flagella were examined by light microscopy (E600; Nikon) and transmission electron microscopy by using cells cultivated on NA. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Catalase and oxidase activities and hydrolysis of casein, starch, hypoxanthine, xanthine, tyrosine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were studied as described by Lanyi (1987). Acid production from carbohydrates was determined according to Leifson (1963). Utilization of various substrates for growth was investigated as described by Yurkov et al. (1994). The API ZYM system (bioMérieux) was used to characterize enzyme activity. The API 20E system (bioMérieux) was used to examine other physiological and biochemical properties. Susceptibility to antibiotics was tested on NA plates using antibiotic discs containing the following concentrations of antibiotic; ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), chloramphenicol (100 µg), gentamicin (30 µg), lincomycin (15 µg), kanamycin (30 µg), neomycin (30 µg), novobiocin (5 µg), oleandomycin (15 µg), penicillin G (20 U), polymyxin B (100 IU), streptomycin (50 µg) and tetracycline (30 µg).
Strains UMS-62T and UMS-101 were cultivated for 3 days in NB at 30 °C to obtain the cell biomass required for DNA extraction and analyses of isoprenoid quinones, polar lipids and whole-cell sugars. Chromosomal DNA was extracted and purified according to a previously described procedure (Yoon et al., 1996). 16S rRNA gene amplification was conducted according to the method described previously using two universal primers (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). A phylogenetic analysis was conducted using the CLUSTAL W software package (Thompson et al., 1994). Isoprenoid quinones were analysed as described previously (Komagata & Suzuki, 1987) using reverse-phase HPLC. For fatty acid methyl ester analysis, cell mass of strains UMS-62T and UMS-101 was harvested from NA plates after cultivation for 3 days at 30 °C. Fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to Minnikin et al. (1984) and were identified using two dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). Whole-cell sugars were analysed as described previously (Staneck & Roberts, 1974). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA–DNA relatedness was determined by the microplate hybridization method (Ezaki et al., 1989) using photobiotin-labelled DNA probes. The type strains of five Agromyces species were used as reference strains for DNA–DNA hybridization. Samples of A. salentinus DSM 16198T, A. lapidis DSM 16390T, A. italicus DSM 16388T, A. ramosus DSM 43045T and A. neolithicus DSM 16197T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany.
Morphological, cultural, physiological and biochemical characteristics of strains UMS-62T and UMS-101 are given in the species description (see below) or are shown in Table 1 and Supplementary Table S1 in IJSEM Online. The almost complete 16S rRNA gene sequences of strains UMS-62T and UMS-101 determined in this study each comprised 1483 nt. The 16S rRNA gene sequences of the two novel strains were 99.9 % similar, showing only two nucleotide differences. Comparative 16S rRNA gene sequence analyses showed that strains UMS-62T and UMS-101 fell within the radiation of the cluster comprising the recognized Agromyces species (Fig. 1).
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, together with those of some phylogenetically related Agromyces species. Strains UMS-62T and UMS-101 contained large amounts of straight-chain and branched fatty acids; the major components (>10 % of total fatty acids) were anteiso-C15 : 0 (34.3–39.1 %), iso-C16 : 0 (11.8–28.7 %) and anteiso-C17 : 0 (17.0–18.3 %). C16 : 0 (6.4–24.1 %) was detected as a major component only in strain UMS-101. The fatty acid profiles of the two novel strains were similar to those of recognized Agromyces species, although there were differences in the proportions of some fatty acids, perhaps because of differences in cultivation conditions and extraction procedures (Takeuchi & Hatano, 2001; Li et al., 2003; Jurado et al., 2005a, b, c). The major polar lipids detected in strains UMS-62T and UMS-101 were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The whole-cell sugars detected in the novel strains were rhamnose, ribose and galactose; xylose was detected only in strain UMS-62T. Minor amounts of mannose and glucose were also detected. The DNA G+C contents of strains UMS-62T and UMS-101 were 71.1 and 71.3 mol%, respectively.
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). Strains UMS-62T and UMS-101 exhibited a mean DNA–DNA relatedness value of 91.1 % when their DNAs were used individually as labelled DNA probes for cross-hybridization. This value indicates that strains UMS-62T and UMS-101 are members of the same genomic species (Wayne et al., 1987). Levels of DNA–DNA relatedness between strains UMS-62T and UMS-101 and the type strains of A. salentinus, A. lapidis, A. italicus, A. ramosus and A. neolithicus, which showed 16S rRNA gene sequence similarity values of >97.0 % to the two novel strains, were in the range 13.4–54.2 %, indicating that the two novel strains represent a different genomic species to the five recognized Agromyces species tested (Wayne et al., 1987). Strains UMS-62T and UMS-101 also differed from the recognized Agromyces species in several phenotypic characteristics (Table 1; Supplementary Table S1 in IJSEM Online). Phylogenetic distinctiveness, DNA–DNA relatedness data and differential phenotypic properties suggest that strains UMS-62T and UMS-101 represent a species that is distinct from the recognized Agromyces species. Therefore, on the basis of the data presented, strains UMS-62T and UMS-101 should be placed in the genus Agromyces as a novel species, for which the name Agromyces allii sp. nov. is proposed.
Description of Agromyces allii sp. nov.
Agromyces allii (al.li'i. N.L. gen. n. allii of Allium, referring to the source of isolation of the micro-organisms, the rhizosphere of Allium victorialis var. platyphyllum).
Cells are Gram-positive, slightly curved, rod-shaped and 0.2–0.4x0.5–1.5 µm. Non-motile. Strictly aerobic. Colonies are circular, convex, smooth and yellow. Growth occurs at 4 and 33 °C, with an optimum temperature of 30 °C. Optimal pH for growth is between 6.5 and 7.5; growth occurs at pH 5.5, but not at 5.0. Growth occurs at <3 % (w/v) NaCl. Aesculin is hydrolysed, but starch and Tweens 20, 40 and 60 are not. Arginine decarboxylase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. D-Glucose, D-galactose, D-cellobiose, D-mannose, D-trehalose, D-xylose, L-arabinose, sucrose, maltose and salicin are utilized as sole carbon and energy sources, but D-fructose, pyruvate, formate and L-glutamate are not. Acid is produced from melibiose, but not from D-sorbitol, myo-inositol and D-melezitose. Acid production from D-fructose is variable (positive for type strain). Using the API ZYM system, esterase(C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI phosphohydrolase, 
-glucosidase, 
-glucosidase and N-acetyl--glucosaminidase activities are present, but alkaline phosphatase, lipase (C14), trypsin, -mannosidase and -fucosidase activities are absent. Susceptible to streptomycin, chloramphenicol, cephalothin, gentamicin, tetracycline, lincomycin, oleandomycin, neomycin and carbenicillin. The predominant menaquinones are MK-11 and MK-12. The major cellular fatty acids are anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. Cell-wall sugars are rhamnose, ribose and galactose; xylose is detected only in the type strain as the major component. The DNA G+C content is 71.1–71.3 mol%.
The type strain, strain UMS-62T (=KCTC 19181T=JCM 13584T), was isolated from the rhizosphere of Allium victorialis var. platyphyllum, a type of wild edible greens grown on Ulleung island, Korea.
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ACKNOWLEDGEMENTS |
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