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1 Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi – 110 007, India
2 Center for Cellular and Molecular Biology, Hyderabad – 500 007, India
Correspondence
Rup Lal
duzdel{at}vsnl.com
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Tables detailing DNA–DNA relatedness values and the differential distribution of phospholipids of Amycolatopsis benzoatilytica sp. nov. DSM 43387T and the type strains of A. orientalis and A. albidoflavus are available as supplementary data in IJSEM Online.
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, comprises a large group of medically and commercially important amycolate, nocardioform actinomycetes. It belongs in the family Pseudonocardiaceae (Embley et al., 1988; Warwick et al., 1994), which is characterized by the presence of meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan (wall chemotype IV sensu Lechevalier & Lechevalier, 1970), N-acetylated muramic acid residues and fatty acids rich in iso- and anteiso-branched components, but its members lack mycolic acids (Takahashi, 2001). Currently, 26 species with validly published names constitute the genus Amycolatopsis.
Most members of the genus Amycolatopsis are commercially important because of their ability to produce bioactive compounds, such as antibiotics, and to degrade a wide range of aromatic compounds. Amycolatopsis orientalis DSM 43387 does not appear to produce antibiotics. Interestingly, it metabolizes m-hydroxybenzoate through a central intermediate protocatechuate, unlike other species of Amycolatopsis and Streptomyces that are not able to utilize this compound as a substrate for growth (Grund et al., 1990). In addition, it also contains a 29·6 kb conjugative plasmid, pA387 (Lal et al., 1991), and is thus one of three plasmid-bearing strains of Amycolatopsis so far reported (Dhingra et al., 2003). Strain DSM 43387 was isolated in Czechoslovakia from a patient suffering from submandibular mycetoma (Scharfen, 1971). The strain was initially identified as Nocardia brasiliensis (Scharfen, 1971) and deposited in IMRU under the accession number 1389. N. brasiliensis IMRU 1389 was reclassified as a member of Nocardia orientalis by Gordon et al. (1978), based on morphological and biochemical properties (acid production from erythritol and arabinose and decomposition of casein, hypoxanthine, tyrosine, urea, aesculin and starch) typical of N. orientalis (Mishra et al., 1980). However, the authors emphasized that the availability of more strains from different sources and a detailed study of their properties would be required to establish the status of the test strain as representing a distinct species (Gordon et al., 1978). At the time when the mycolic acid-lacking nocardioform actinomycetes were separated from the genus Nocardia, N. orientalis was renamed Amycolatopsis orientalis (Lechevalier et al., 1986).
It therefore appears that A. orientalis DSM 43387 (=IMRU 1389) has been misclassified in this species. The present investigation was designed to establish the taxonomic position of strain DSM 43387 on the basis of the 16S rRNA gene sequence, DNA–DNA hybridization, chemotaxonomy and morphological and biochemical properties. The genotypic and phenotypic data show that strain DSM 43387 should be reclassified as the type strain of a novel species of Amycolatopsis, for which the name Amycolatopsis benzoatilytica sp. nov. is proposed.
Genomic DNA was isolated from strain DSM 43387 by using the salting-out method described by Kieser et al. (2000). PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Reddy et al. (2000). An almost complete 16S rRNA gene sequence was determined for strain DSM 43387 (1417 nucleotides). The resultant sequence was first manually aligned with corresponding 16S rRNA gene sequences of 24 species of the genus Amycolatopsis with validly published names retrieved from GenBank (http://www.ncbi.nlm.nih.gov/), using the CLUSTAL_X program (version 1.8b; Thompson et al., 1997). Phylogenetic comparison of the 16S rRNA gene sequence of strain DSM 43387 with the corresponding sequences of representative members of the family Pseudonocardiaceae showed that the organism belongs to the genus Amycolatopsis. The levels of 16S rRNA gene sequence similarity between strain DSM 43387 and other members of species of the genus Amycolatopsis with validly published names ranged from 91·0 to 95·4 %. Strain DSM 43387 had a sequence similarity of 93·9 % with A. orientalis NBRC 12806T and it was also revealed that its closest relative was Amycolatopsis albidoflavus IMSNU 22139T, with a sequence similarity of 95·4 %. These values corresponded to 63 and 83 nucleotide differences out of 1372 nucleotides positions compared. For the construction of the phylogenetic tree, the sequence alignment was optimized manually and the terminal nucleotides were removed. The tree was constructed using the neighbour-joining method (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and parsimony analysis based on 1372 nucleotides present in all the strains, using the PHYLIP package (version 3.5c; Felsenstein, 1993). Prauserella rugosa DSM 43194T was used as an outgroup. The evolutionary distance matrix was calculated using the distance model of Jukes & Cantor (1969). The resultant tree topologies were evaluated by bootstrap analysis based on 1000 resamplings using the programs SEQBOOT and CONSENSE. It is clear from the phylogenetic tree that strain DSM 43387 forms a monophyletic clade with A. albidoflavus IMSNU 22139T (Fig. 1). The type strain, A. orientalis NBRC 12806T, forms a distinct phyletic clade distant from strain DSM 43387. Similar tree topologies were obtained using maximum-likelihood and parsimony analysis (data not shown). This clearly indicates that strain DSM 43387 forms a new centre of taxonomic variation in the genus Amycolatopsis and is quite distant from A. orientalis NBRC 12806T in the 16S rRNA gene-based phylogenetic tree. Thus phylogenetic clustering indicates that strain DSM 43387 represents a novel species of the genus Amycolatopsis.
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; Stackebrandt & Goebel, 1994). To further clarify the taxonomic status of strain DSM 43387, A. orientalis NBRC 12806T and its phylogenetic neighbour A. albidoflavus DSM 44639T (=IMSNU 22139T) were obtained from NBRC and DSMZ, respectively, and DNA–DNA hybridization was performed using the membrane filter method (Tourova & Antonov, 1987). First, genomic DNA was isolated from strain DSM 43387, A. orientalis NBRC 12806T and A. albidoflavus DSM 44639T by using the salting-out method described by Kieser et al. (2000). Experiments to determine DNA–DNA relatedness between the strains were performed as described by Bala et al. (2004). DNA (10 µg) of each strain was transferred onto a nylon membrane (Hybond-N; Amersham) using a dot-blot apparatus (Bio-Rad). The membrane was air-dried and UV cross-linked. The DNA probe of each strain was labelled with [
-32P] (BRIT) using a nick translation kit (Bangalore Genei). Hybridization was done overnight at 65 °C. After hybridization, the nylon membrane was washed twice, first with 0·5xSSC and 0·1 % SDS for 10 min at room temperature and then with 0·1xSSC and 0·5 % SDS for 20 min at 65 °C, to remove the unbound probe. The amount of bound probe DNA was estimated by using a scintillation counter (Beckman Instruments) and the hybridization values obtained were expressed as the percentage of the probe bound relative to the homologous reaction. In the experiments, DNA of each of the three strains was bound to the filters and hybridized with DNA probes prepared from each strain. The DNA–DNA hybridization data showed that strain DSM 43387 had DNA–DNA relatedness values of 24·1 and 45·7 %, respectively, with A. orientalis NBRC 12806T and A. albidoflavus DSM 44639T (=IMSNU 22139T) at the genome level. The hybridization value of below 70 %, which has been suggested as the threshold value for delineating a bacterial species (Wayne et al., 1987), clearly indicated that strain DSM 43387 represents a novel species of Amycolatopsis. The percentage relatedness calculated on the basis of the data obtained (mean of four replicates) by DNA–DNA hybridization is summarized in Supplementary Table S1 in IJSEM Online. These data support the 16S rRNA gene sequence results and lead to the conclusion that strain DSM 43387 represents a novel species of Amycolatopsis.
The polar lipids of strain DSM 43387 have already been reported (Yassin et al., 1993). Analysis of the polar lipids revealed that the phospholipid pattern of strain DSM 43387 is type PII (Lechevalier et al., 1977). The polar lipid profile contained the acidic lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, and the neutral lipids phosphatidylethanolamine and phosphatidylmethylethanolamine, as predominant phospholipids, together with several unidentified ninhydrin-positive phospholipids that were not present in A. orientalis NBRC 12806T. In contrast, several phospholipids possessing vicinal OH groups (designated P-OH) and reacting with periodate–Schiff's reagent were present in A. orientalis NBRC 12806T, in addition to the major components of the PII type phospholipid pattern, but phosphatidylinositol was absent. As shown in Supplementary Table S2 in IJSEM Online, phosphatidylglycerol, which is present as a major component in strain DSM 43387, was not present in A. albidoflavus IMSNU 22139T. Whereas the polar lipid composition underlies the integrity of the genus Amycolatopsis, it clearly differentiates strain DSM 43387 from A. orientalis ATCC 19795T (=NBRC 12806T) (Yassin et al., 1993) and A. albidoflavus IMSNU 22139T (Lee & Hah, 2001).
Because of the uneven distribution of fatty acids among different species of actinomycetes, they are a useful criterion for the delineation of species. Cellular fatty acid analysis of strain DSM 43387, A. orientalis NBRC 12806T and A. albidoflavus DSM 44639T was carried out by Microbial ID using the following procedure. Cultures of all three strains were grown on tryptic soy agar (TSBA; Himedia Laboratories Pvt. Ltd) under similar growth conditions and fatty acid methyl esters from 40 mg cells scraped from Petri plates were analysed by saponification, methylation and extraction, using the method of Sasser (1990). The fatty acid methyl ester mixtures were separated using the Sherlock Microbial Identification System (Microbial ID), which consisted of an Agilent model 5980 gas chromatograph fitted with a phenylmethyl silicon column (25 mx0·2 mm) and FID detector. Identification and comparison was carried out using the Aerobe (TSBA50, version 5) database of the Sherlock Microbial Identification System. The predominant fatty acids detected in the cell hydrolysate of strain DSM 43387 included 12-methyltridecanoic acid (iso-C14 : 0; 11·25 %), pentadecanoic acid (C15 : 0; 14·60 %), pentadecenoic acid (C15 : 1
6c; 12·81 %), 14-methylpentadecanoic acid (iso-C16 : 0; 12·80 %), heptadecanoic acid (C17 : 0; 11·31 %) and heptadecenoic acid (C17 : 18c; 11·31 %). The predominant fatty acids of A. orientalis NBRC 12806T and A. albidoflavus DSM 44639T were heptadecenoic acid (C17 : 18c; 17·08 %) and 14-methylpentadecanoic acid (iso-C16 : 0; 16·74 %), respectively. A comparison of the fatty acid compositions of strain DSM 43387, A. orientalis NBRC 12806T and A. albidoflavus DSM 44639T is given in Table 1. However, the fatty acid profiles of A. orientalis NBRC 12806T (=IMSNU 20058T) and A. albidoflavus DSM 44639T (=IMSNU 22139T) determined using TSBA varied from those obtained previously for these strains on YMG broth (Lee & Hah, 2001).
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The colour, shape, size and contour of colonies of strain DSM 43387 were observed on YM medium at 28 °C for 7 days. Cream-coloured, irregularly shaped, rough, dull and moderate-sized flat colonies with undulating margins were observed. The vegetative mycelia showed a tendency to fragment when the organism was grown in liquid culture. Similar to A. orientalis NBRC 12806T, strain DSM 43387 did not produce spores in any of the media tested. The strain was subjected to a battery of physiological tests. Growth at different temperatures and catalase tests were carried out as described by McCarthy & Cross (1984) using YM medium. Hydrolysis of Tween 80 and the ability of the strain to grow in the presence of NaCl were tested as described by Arden-Jones et al. (1979). Acid production from carbohydrates and degradation of xanthine and hypoxanthine were determined as described by Gordon et al. (1974). Urease activity was detected as described by Christensen (1946). Other physiological tests and methods used were as described by Collins et al. (1989). Strain DSM 43387 grew at a temperature range of 10–37 °C. The pH range for growth was 5–10. Similar to A. orientalis NBRC 12806T and A. albidoflavus IMSNU 22139T, strain DSM 43387 utilized adonitol, (+)-L-arabinose, (+)-D-cellobiose, meso-erythritol, (+)-D-fructose, (+)-D-galactose, myo-inositol, (+)-D-lactose, (+)-D-mannose, (+)-D-trehalose and (+)-D-xylose and degraded aesculin. However, differences in results from those for A. albidoflavus IMSNU 22139T and A. orientalis NBRC 12806T were observed in acid production from dextrin, (+)-D-glucose, (+)-D-maltose, (+)-L-rhamnose and sucrose and in decomposition of casein, gelatin, xanthine and hypoxanthine and hydrolysis of Tween 80. Differential properties of strain DSM 43387, A. orientalis NBRC 12806T and A. albidoflavus IMSNU 22139T are shown in Table 2. The differences in chemical and physiological data between strain DSM 43387 and the reference strains support the results of the 16S rRNA gene analysis, DNA–DNA hybridization and polar lipid and fatty acid analyses.
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Description of Amycolatopsis benzoatilytica sp. nov.
Amycolatopsis benzoatilytica (ben.zo.a.ti.ly'ti.ca. N.L. n. benzoas -atis benzoate; Gr. adj. lutikos able to loose, able to dissolve; N.L. adj. lyticus -a -um dissolving; N.L. fem. adj. benzoatilytica benzoate-degrading).
Cells are aerobic, non-motile, non acid-fast, Gram-positive, filamentous and differentiated into substrate and aerial mycelia. Vegetative mycelia have the tendency to fragment when the organism is grown in liquid culture. Cream-coloured aerial mycelium is produced on YM, nutrient agar, TSBA and Bennett's agar. Catalase- and urease-positive but amylase-negative. Soluble pigment is not produced. Acid is produced from adonitol, (+)-L-arabinose, (+)-D-cellobiose, dextrin, meso-erythritol, (+)-D-fructose, (+)-D-glucose, (+)-D-galactose, myo-inositol, (+)-D-lactose, (–)-D-mannitol, (+)-D-mannose, (+)-L-rhamnose, (–)-D-ribose, (+)-D-trehalose and (+)-D-xylose. Utilizes (+)-D-raffinose but not (–)-D-sorbitol or sucrose. Hydrolyses aesculin and decomposes allantoin. Tween 80, starch, casein, gelatin, xanthine and hypoxanthine are not hydrolysed or decomposed. Saturated, unsaturated and branched fatty acids are present. The predominant fatty acids are 12-methyltridecanoic acid (iso-C14 : 0), pentadecanoic acid (C15 : 0), pentadecenoic acid (C15 : 16c), 14-methylpentadecanoic acid (iso-C16 : 0), heptadecanoic acid (C17 : 0) and heptadecenoic acid (C17 : 18c). The phospholipid profile is type PII and contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and phosphatidylmethylethanolamine. Growth occurs at pH 5·0–10·0. Grows in the presence of 5 % NaCl. The temperature range for growth is 10–37 °C. Degrades m-hydroxybenzoate. Contains the 29·6 kb conjugative plasmid pA387.
The type strain, AK 16/65T (=DSM 43387T=ATCC 55165T=IMRU 1389T), was isolated in Czechoslovakia from a patient with submandibular mycetoma by Scharfen (1971) (Institute of Epidemiology and Microbiology, Prague).
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ACKNOWLEDGEMENTS |
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