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Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Correspondence
Linxian Ding
linxian{at}iam.u-tokyo.ac.jp
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ABSTRACT |
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and was assigned to the family Pseudonocardiaceae (Embley et al., 1988; Warwick et al., 1994). The principal characteristics of this genus are the presence of a type IV cell wall (meso-diaminopimelic acid and cell-wall sugars consisting of arabinose and galactose; Lechevalier & Lechevalier, 1970; Lechevalier et al., 1986) and MK-9(H4) menaquinones and the absence of mycolic acids. During the first 13 years of its existence, only 10 novel species were described for this genus, but, since 2000, many novel species have been described from soil, fresh water, human and animal clinical sources, natural caves, prairie soil and other natural areas. At the time of writing, the genus contains 34 species (as well as four subspecies). In this investigation, we describe two actinomycetes, LC2T and LC11T, which were isolated from a filtration substrate composed of volcanic soil. They are moderately thermophilic actinomycetes that can grow at 45 °C. Genotypic and phenotypic analyses indicated that these isolates should be classified within two novel species of the genus Amycolatopsis.
We used a filtration substrate as the substrate for the isolation of bacterial strains on NY medium (1.6 g nutrient broth, 0.5 g yeast extract, 1000 ml deionized water and 1.5 % agar, pH 7.0) with cycloheximide (0.05 g l–1) and kabicidin (0.1 g l–1). All of the isolates were maintained on NY medium or oatmeal agar and incubated at 27 °C.
Determination of the optimum temperature for growth and pH tolerance was carried out by incubating in both nutrient broth and nutrient agar. NaCl tolerance was determined in nutrient broth amended with 3, 5 and 7 % NaCl and incubated at 27 °C. Air-dried smears prepared at 48 h intervals were Gram-stained to determine the Gram reaction, and investigation of the cell morphology was done after incubating for 2 weeks. Biochemical tests were performed with API 50CH, API ZYM and API CORYNE strips (bioMérieux) according to the manufacturer's instructions.
Genomic DNA preparation and PCR amplification of the 16S rRNA gene were performed as described previously by Ding & Yokota (2002, 2004). Nucleotide sequences were obtained automatically using an Applied Biosystems DNA sequencer (model 377) and software provided by the manufacturer. Alignment was performed using CLUSTAL_X version 1.83 (Thompson et al., 1997). Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987) and the maximum-likelihood method (Felsenstein, 1981) and parsimony analysis based on 1348 nucleotides present in all strains. The evolutionary distance matrix was calculated using Kimura's two-parameter method (Kimura, 1980). The topology of the phylogenetic tree was evaluated by bootstrap analysis with 1000 sampling replications according to the method of Felsenstein (1985). The PHYLIP package (version 3.5c; Felsenstein, 1993) was used for all phylogenetic analyses.
Genomic DNA was prepared using a method described previously (Sambrook et al., 1989). The G+C content of the DNA was determined by HPLC (Mesbah et al., 1989). DNA–DNA relatedness experiments were performed by a modification of the microplate method of Ezaki et al. (1989), using photobiotin-labelled DNA and microdilution wells, as described by Willems et al. (2001). The DNA–DNA hybridization temperature was 55 °C.
The isomeric form of the diaminopimelic acid was determined by TLC from whole-cell hydrolysates using the method of Staneck & Roberts (1974). The whole-cell sugar composition was determined by TLC as described by Staneck & Roberts (1974), Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Analysis of menaquinones was carried out according to the methods of Minnikin et al. (1984) and Kroppenstedt (1985). Mycolic acids were examined by the acid methanolysis method of Minnikin et al. (1980) and Yano et al. (1972). The strains were incubated on TSBA medium for 48 h at 30 °C. Fatty acid methyl esters were obtained from the cells by saponification, methylation and extraction. Analysis by GC was controlled by MIS software (Microbial ID Inc.). The peaks were automatically integrated and identified by the Microbial Identification software package (Sasser, 1990).
The almost-complete 16S rRNA gene sequences of strains LC2T (1473 nt) and LC11T (1472 nt) were determined in this study. Database sequences for other members of the genus Amycolatopsis were obtained from DDBJ/EMBL/GenBank for use in comparative analyses, and the phylogenetic tree was constructed. 16S rRNA gene sequence comparisons showed clearly that isolates LC2T and LC11T are members of the genus Amycolatopsis. Fig. 1 shows that these two isolates, Amycolatopsis rubida, Amycolatopsis albidoflavus and Amycolatopsis benzoatilytica formed a single cluster, and the sequence similarity between strains LC2T and the type strains of A. rubida, A. albidoflavus, A. benzoatilytica and LC11T was 97.6, 98.1, 97.9 and 96.4 %, respectively, while the sequence similarity of strains LC11T and the type strains of A. rubida, A. albidoflavus and A. benzoatilytica was respectively 97.8, 98.2 and 96.1 %. These results suggest that strains LC2T and LC11T belong to two genetically distinct Amycolatopsis species, which are not closely related to A. rubida, A. albidoflavus and A. benzoatilytica. Furthermore, chromosomal DNA–DNA hybridization studies were performed to establish whether isolates LC2T and LC11T represent distinct species. Strain LC2T displayed low levels of DNA–DNA reassociation with A. albidoflavus JCM 11300T (44.7–48.3 %), A. rubida JCM 10871T (39.0–44.1 %) and strain LC11T (58.0–62.1 %); also, strain LC11T displayed low levels of DNA–DNA reassociation with A. albidoflavus JCM 11300T (32.8–34.4 %) and A. rubida JCM 10871T (28.3–35.3 %). These results are below the cut-off point recommended for the circumscription of bacterial genomic species by Wayne et al. (1987), and confirm the separation of isolates LC2T and LC11T from A. albidoflavus, A. rubida and A. benzoatilytica.
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. Both isolates are Gram-positive, aerobic, catalase-positive and oxidase-negative. Results of chemotaxonomic analysis of the isoprenoid menaquinone, fatty acid compositions and DNA G+C content of isolates LC2T and LC11T are shown in Table 2. The major isoprenoid menaquinone was MK-9(H4). The major cellular fatty acid was iso-C16 : 0 (31.9 and 40.0 % of the total in strains LC2T and LC11T), and other fatty acids (over 5 % of the total) included iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0, C15 : 0, anteiso-C17 : 0 and C17 : 1
6c. The isolates contained meso-diaminopimelic acid as the cell-wall diamino acid and arabinose and galactose as major whole-cell sugars (type IV cell wall, according to Lechevalier & Lechevalier, 1970). The strains did not contain mycolic acids. Other morphological and physiological characteristics are listed in the species descriptions.
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Description of Amycolatopsis echigonensis sp. nov.
Amycolatopsis echigonensis (e.chi.go.nen'sis. N.L. fem. adj. echigonensis referring to Echigo, the old name of Niigata Prefecture, Japan, from where the type strain was isolated).
Cells are aerobic, non-motile and Gram-positive and form a well-developed aerial and vegetative mycelium. The aerial mycelium is white and the vegetative mycelium is light yellow. The temperature range for growth is 5–45 °C and the optimum temperature for growth is 30 °C. Grows on medium containing 3–7 % NaCl. The pH range for growth is 6–11, and the optimum is pH 9. Catalase-positive and oxidase-negative. Acid is produced from D-xylose, D-galactose, L-rhamnose, inositol and D-lactose. Production of acid from erythritol, D-adonitol and D-glucose is weak. Using the API CORYNE and API ZYM systems, positive reactions are observed for nitrate reductase, pyrazinamidase, 
-glucosidase, 
-glucuronidase, -galactosidase, urease, gelatin hydrolysis, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, -galactosidase, alkaline phosphatase, N-acetyl--glucosaminidase, naphthol-AS-BI-phosphohydrolase and -mannosidase. No activity is detected for -glucosidase, lipase (C14) or -fucosidase; pyrrolidonyl arylamidase and -chymotrypsin activities are weak. The predominant fatty acid is iso-C16 : 0. The major menaquinone is MK-9(H4). The G+C content of the type strain is 72.7 mol%.
The type strain is strain LC2T (=IAM 15387T =CCTCC AB206019T), which was isolated from a filtration substrate made from volcanic soil, Niigata, Japan.
Description of Amycolatopsis niigatensis sp. nov.
Amycolatopsis niigatensis (ni.i.ga.ten'sis. N.L. fem. adj. niigatensis referring to Niigata Prefecture, Japan, from where the type strain was isolated).
Cells are aerobic, non-motile and Gram-positive and form a well-developed aerial and vegetative mycelium. The aerial mycelium is white or light yellow; the vegetative mycelium is purple or purple–brown. The temperature range for growth is 5–45 °C and the optimum temperature for growth is 30 °C. Grows in presence of 3–7 % NaCl. The pH range for growth is 6–11 and the optimum is pH 9. Catalase-positive and oxidase-negative. Acid is produced from D-galactose and L-rhamnose; no acid is produced from D-xylose, inositol or D-lactose; acid production for erythritol, D-adonitol and D-glucose is weak. Using the API CORYNE and API ZYM systems, positive reactions are observed for pyrazinamidase, pyrrolidonyl arylamidase, -glucosidase, -glucuronidase, -galactosidase, urease, esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, -glucosidase, alkaline phosphatase, N-acetyl--glucosaminidase, -chymotrypsin and -mannosidase. No activity is detected for nitrate reductase, esterase (C4), -galactosidase, lipase (C14) or -fucosidase; gelatin hydrolysis and acid phosphatase are weak. The predominant fatty acid is iso-C16 : 0. The major menaquinone is MK-9(H4). The G+C content of the DNA of the type strain is 72.4 mol%.
The type strain is strain LC11T (=IAM 15388T =CCTCC AB206020T), which was isolated from a filtration substrate made from volcanic soil, Niigata, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Ding, L. & Yokota, A. (2004). Proposals of Curvibacter gracilis gen. nov., sp. nov. and Herbaspirillum putei sp. nov. for bacterial strains isolated from well water and reclassification of [Pseudomonas] huttiensis, [Pseudomonas] lanceolata, [Aquaspirillum] delicatum and [Aquaspirillum] autotrophicum as Herbaspirillim huttiense comb. nov., Curvibacter lanceolatus comb. nov., Curvibacter delicatus comb. nov. and Herbaspirillum autotrophicum comb. nov. Int J Syst Evol Microbiol 54, 2223–2230.
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