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1 Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
2 Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA
Correspondence
Soon Dong Lee
sdlee{at}cheju.ac.kr
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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The genus Amycolatopsis, which was proposed by Lechevalier et al. (1986)
for aerobic, amycolate, nocardioform actinomycetes, is well defined as a result of chemotaxonomic characterization (Lechevalier et al., 1986; Henssen et al., 1987; Mertz & Yao, 1993; Yassin et al., 1993) and phylogenetic analyses based on the comparison of 16S rRNA gene sequences (Embley et al., 1988; Warwick et al., 1994) and currently contains 26 species with validly published names. Members of the genus Amycolatopsis, which belongs to the family Pseudonocardiaceae (Embley et al., 1988; Warwick et al., 1994), are represented chemotaxonomically by the following features: wall chemotype IV (meso-diaminopimelic acid, arabinose and galactose in cell-wall hydrolysates); a tetrahydrogenated menaquinone with nine isoprene units (MK-9(H4)] as the major menaquinone; a phospholipid pattern of type PII (phosphatidylethanolamine as a diagnostic phospholipid); fatty acid profiles that include complex mixtures of saturated and branched-chain acids; and the absence of mycolic acids.
It is evident from our study that the two isolates could be readily differentiated from all Amycolatopsis species with validly published names on the basis of a battery of phenotypic and genetic data and merit recognition as a novel species.
Strains 32U-2T and 32U-4 were isolated from a prairie soil by using the dilution plating method with oatmeal agar and were maintained as 20 % (v/v) glycerol suspensions at –70 and –20 °C. Morphological and cultural characteristics were determined by cultivating the isolates at 30 °C for 7–14 days on several media, such as yeast extract/malt extract agar (ISP 2 medium), oatmeal agar (ISP 3 medium) and ISP 4 medium (Shirling & Gottlieb, 1966). Specimens with growth were critical-point-dried, coated with gold and then observed under a scanning electron microscope (model JSM 5410LV; JEOL). The organisms produced well-developed, branched vegetative mycelium bearing white-coloured aerial mycelium. The reverse sides of colonies were light yellow in colour. Both types of hyphae fragmented into rod-shaped cells.
Chromosomal DNA used for genetic characterization was extracted and purified with the Wizard genomic DNA purification kit (Promega) according to the instructions of the manufacturer. The 16S rRNA gene of the chromosomal DNA was amplified by a PCR and cloned into Escherichia coli JM109 (Promega) as described previously (Lee et al., 2000). The resultant inserts were sequenced using an ABI PRISM BigDye terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 3730xl; Applied Biosystems). These experiments were performed in the first author's laboratory in Korea. The sequences determined in this study were aligned and compared with the corresponding sequences of all Amycolatopsis species with validly published names by using the CLUSTAL X program (Thompson et al., 1997) and then manually optimized according to the secondary structure of the E. coli sequence (Brosius et al., 1978). A phylogenetic analysis was performed using the neighbour-joining method (Saitou & Nei, 1987). Evolutionary distances for the neighbour-joining method were computed by using the method of Jukes & Cantor (1969). The confidence level of the tree topology was evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining data, using 1000 replications.
The almost-compete 16S rRNA gene sequences for the isolates (32U-2T and 32U-4) each comprised continuous stretches of 1510 nt. A total of 1347 unambiguous aligned positions present in all strains between E. coli positions 72 and 1452 were used for phylogenetic analysis. Pseudonocardia thermophila was used as an outgroup for tree construction. A phylogenetic tree (Fig. 1) based on 16S rRNA gene sequence comparisons showed that the isolates form a distinct lineage within the radius of the genus Amycolatopsis, this being supported by a bootstrap value of 100 %. The organisms showed 16S rRNA gene sequence similarity of 99·7 % to each other. The levels of 16S rRNA gene sequence similarity between the isolates and the type strains of recognized Amycolatopsis species ranged from 94·1 to 97·9 %. Of these species, Amycolatopsis coloradensis (97·6–97·9 %), Amycolatopsis orientalis and Amycolatopsis alba (97·3–97·6 %) and Amycolatopsis lurida (97·2–97·5 %) showed the highest levels of 16S rRNA gene sequence similarity with respect to the isolates.
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; Uchida & Aida, 1984; Minnikin et al., 1977, 1980, 1984; Mesbah et al., 1989; Saddler et al., 1991). Cellular fatty acid methyl esters were prepared by alkaline methanolysis (Minnikin, 1988) and analysed by GC with an Agilent mode 6850 gas chromatograph as described previously (Lee & Hah, 2001).
The chemotaxonomic properties supported the phylogenetic assignment of the isolates within the radiation of the genus Amycolatopsis, as follows. The organisms were characterized by a type IV cell wall (meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates), MK-9(H4) as a major menaquinone and muramic acid of the N-acetylated type. Mycolic acids were not detected. The DNA G+C contents of strains 32U-2T and 32U-4 were 69·5 and 70·2 mol%, respectively. The polar lipid profiles were characterized by the presence of considerable amounts of phosphatidylmethylethanolamine, diphosphatidylglycerol and phosphatidylinositol, but the organisms showed minor differences in the relative amounts of phosphatidylethanolamine. The cellular fatty acids comprised a mixture of saturated and branched-chain acids, and were characterized by the predominance of iso-hexadecanoic acid (i-C16 : 0). Small amounts of unsaturated and branched hydroxy fatty acids were also detected. The cellular fatty acid profiles of the isolates are shown in Table 1.
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). Catalase activity was determined with a 3 % (v/v) hydrogen peroxide solution. The temperature for growth was tested at 10, 30, 37 and 45 °C. The production of hydrogen sulfide was detected on trypticase soy broth by using lead acetate strips. Urease activity was determined by means of a colour change in Bacto urea broth (Difco). Hydrolysis of casein, gelatin and starch, and nitrate reduction, were examined by using previously described methods (MacFaddin, 1981). NaCl tolerance was studied on ISP 2 medium containing NaCl at final concentrations of 2, 3, 5, 7 and 10 % (w/v).
The strains, whilst showing slight differences in their 16S rRNA gene sequences and some chemical properties, showed the same results for a broad range of physiological characteristics. They produced acid from D-arabinose, D-cellobiose, D-fructose, methyl 
-D-mannoside, L-ribose, salicin, D-trehalose, D-xylose, D-xylose, glycerol, myo-inositol and D-mannitol but did not produce acid from inulin, D-melezitose, D-raffinose, L-sorbose, 2,3-butanediol, dulcitol or 1,2-propanediol. H2S production and gelatin liquefaction were observed. Both isolates degraded casein, hypoxanthine and DL-tyrosine. Other physiological properties that differentiate the isolates from their phylogenetic neighbours are listed in Table 2.
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Description of Amycolatopsis minnesotensis sp. nov.
Amycolatopsis minnesotensis (min.ne.sot.en'sis. N.L. fem. adj. minnesotensis of Minnesota, the origin of the soil sample from which the type strain was isolated).
Forms well-developed aerial and vegetative mycelia that fragment into rod-shaped elements. The aerial mycelium is white and the vegetative mycelium is yellow. Aerobic, Gram-positive, not acid–alcohol-fast. Catalase-positive. H2S production is observed. Nitrate is reduced to nitrite. Growth occurs between 10 and 30 °C. Growth does not occur at 37 °C. Acid is produced from D-arabinose, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, D-lactose, maltose, D-mannose, methyl -D-mannoside, L-rhamnose, L-ribose, salicin, sucrose, D-trehalose, D-xylose, L-xylose, adonitol, meso-erythritol, glycerol, myo-inositol, D-mannitol, D-sorbitol and D-xylitol. No acid is produced from dextran, inulin, D-melezitose, melibiose, methyl -D-glucoside, D-raffinose, L-sorbose, 2,3-butanediol, dulcitol or 1,2-propanediol. Casein, gelatin, hypoxanthine, DL-tyrosine and urea are decomposed but starch and xanthine are not. Growth occurs in the presence of 5 % NaCl but not 7 % NaCl. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol and an unknown phospholipid (a phospholipid type PII pattern). Mycolic acids are not present. The major cellular fatty acids are i-C16 : 0 (23·1 %), C16 : 0 (13·8 %), i-C15 : 0 (12·3 %) and C17 : 0 (10·7 %). The DNA G+C content is 69·5 mol%.
The type strain is 32U-2T (=KCCM 42246T=NRRL B-24435T), isolated from a prairie soil in Minnesota, USA. Strain 32U-4 is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Embley, T. M., Smida, J. & Stackebrandt, E. (1988). The phylogeny of mycolate-less wall chemotype IV actinomycetes and the description of Pseudonocardiaceae fam. nov. Syst Appl Microbiol 11, 44–52.
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