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ciak2
1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
2 Laboratory of Medical Microbiology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland
3 Institute of Space Medico-Engineering, Beijing 100094, PR China
Correspondence
Mariola Paciak
pasciak{at}immuno.iitd.pan.wroc.pl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of A. palatopharyngis sp. nov. 1BDZT is AF479268.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and was assigned to the family Pseudonocardiaceae (Embley et al., 1988; Warwick et al., 1994), which also contains the genera Actinopolyspora, Kibdelosporangium, Prauserella, Pseudonocardia, Saccharomonospora, Saccharopolyspora and Thermocrispum (Kim & Goodfellow, 1999; Labeda & Kroppenstedt, 2000; Huang et al., 2002). Recently, increasing interest has been shown in Amycolatopsis, as it contains strains that produce commercially significant bioactive compounds. Eight novel species with validly published names have been described during the last 3 years (Goodfellow et al., 2001; Huang et al., 2001; Lee & Hah, 2001; Kim et al., 2002; Al-Musallam et al., 2003; Labeda et al., 2003). The 18 species of this genus phylogenetically form two groups, the Amycolatopsis methanolica and Amycolatopsis orientalis subclades (Kim et al., 2002). The A. orientalis subclade comprises 11 mesophilic species, Amycolatopsis alba, Amycolatopsis albidoflavus, Amycolatopsis azurea, Amycolatopsis coloradensis, Amycolatopsis japonica, Amycolatopsis keratiniphila, Amycolatopsis mediterranei, A. orientalis, Amycolatopsis pretoriensis, Amycolatopsis rubida and Amycolatopsis sulphurea (Huang et al., 2001; Lee & Hah, 2001; Kim et al., 2002; Al-Musallam et al., 2003; Labeda et al., 2003), and three moderately thermophilic species, Amycolatopsis kentuckyensis, Amycolatopsis lexingtonensis and Amycolatopsis sacchari, which grow at 45 °C (Goodfellow et al., 2001; Labeda et al., 2003). The A. methanolica subclade comprises three thermophilic species, Amycolatopsis eurytherma, A. methanolica and Amycolatopsis thermoflava, which grow at temperatures above 50 °C (de Boer et al., 1990; Chun et al., 1999; Kim et al., 2002). The remaining thermophilic species, Amycolatopsis fastidiosa, which grows over a wide temperature range (10–60 °C) (Henssen et al., 1987), is loosely associated with the others and forms a distinct phyletic line at the foot of the Amycolatopsis phylogenetic tree (Goodfellow et al., 2001; Huang et al., 2001; Lee & Hah, 2001; Kim et al., 2002). In addition, Barreiro et al. (2000) also proposed that the cephamycin producer ‘Nocardia lactamdurans’ should be incorporated into the genus Amycolatopsis as ‘Amycolatopsis lactamdurans’, which falls into the A. orientalis subclade (Kim et al., 2002). Members of the genus Amycolatopsis can be distinguished from all other genera belonging to the family Pseudonocardiaceae by a combination of chemical and morphological properties (Kim & Goodfellow, 1999). In the present investigation, a polyphasic study was undertaken to determine the taxonomic position of a Gram-positive, facultatively anaerobic strain, 1BDZT, which was isolated from a clinical sample of the palatopharyngeal mucosa of an elderly human patient. Genotypic and phenotypic data indicated that this strain should be classified as a novel species of the genus Amycolatopsis, for which we propose the name Amycolatopsis palatopharyngis sp. nov.
Strain 1BDZT was isolated on a brain heart infusion agar plate that had been seeded using swabs and incubated at 37 °C for 5 days under microaerophilic conditions. The clinical sample was collected from the infected palatopharyngeal mucosa of a 70-year-old male patient. The isolate was maintained on modified Bennett's agar (Jones, 1949) slants at 4 °C and as suspensions of mycelial fragments in glycerol (20 %, v/v) at -20 °C. Biomass for chemical and molecular systematic analyses was prepared as described previously (Huang et al., 2002) with the modification that the strain was grown at 37 °C for 3–5 days and cells were then killed by shaking with formalin (1 %, v/v).
Cultural and morphological properties were observed using a Zeiss Axioskop 20 light microscope and a Hitachi S-570 scanning electron microscope after growth on modified Bennett's, glucose/yeast extract/malt extract (International Streptomyces Project medium 2; Shirling & Gottlieb, 1966) and inorganic salt/starch agars (International Streptomyces Project medium 4; Shirling & Gottlieb, 1966) for 7–14 days at 28 °C.
Phenotypic tests were carried out by following the procedures of Goodfellow et al. (1997) and Gordon et al. (1974). Antibiotic-resistance tests were performed using Bennett's medium and the method described previously (Huang et al., 2001).
Isomers of diaminopimelic acid and whole-cell sugars were analysed according to the procedures developed by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Mycolic acids were checked by the acid methanolysis method of Minnikin et al. (1980). Polar lipids were examined by two-dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted and purified as described by Collins (1985) and were analysed by HPLC (Wu et al., 1989). Cellular fatty acids were determined by treating dry cell mass (2 mg) with 1 M methanolic HCl (0·5 ml) at 80 °C for 1 h and then extracting the fatty acid methyl esters liberated and analysing them directly using GLC–MS (Paciak et al., 2002). The G+C content of the DNA was determined by using the thermal denaturation (Tm) method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as the standard.
Genomic DNA preparation and PCR amplification of 16S rDNA were performed by employing the method of Chun & Goodfellow (1995). The amplified product was sequenced as described previously (Huang et al., 2001). The nucleotide sequence was obtained automatically by using an Applied Biosystems DNA sequencer (model 377) and software provided by the manufacturer. The 16S rDNA sequence of strain 1BDZT was aligned manually against corresponding sequences retrieved from the GenBank database using the CLUSTAL X 1.8 program (Thompson et al., 1997). Phylogenetic trees were inferred by using the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) treeing algorithms. Evolutionary distance matrices were generated as described by Kimura (1980). The resultant unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method, based on 1000 resamplings. The PHYLIP package (Felsenstein, 1993) was used for all phylogenetic analyses.
An almost complete 16S rDNA sequence was obtained for strain 1BDZT (1427 nt). Phylogenetic analysis in which this sequence was compared with corresponding sequences of representatives of the suborder Pseudonocardineae indicated that this strain belongs to the family Pseudonocardiaceae (data not shown). Further analysis, supported by a high bootstrap value of 100 %, showed that strain 1BDZT is a member of the genus Amycolatopsis (Fig. 1); a bootstrap value of 89 % supports the assessment that it forms a distinct line with the A. methanolica subclade. The 16S rDNA sequence similarities between strain 1BDZT and its nearest neighbours, the three thermophilic species A. methanolica, A. thermoflava and A. eurytherma, were respectively 96·40, 96·32 and 96·16 %. Similarity values with other members of the genus Amycolatopsis were in the range 93·86–95·99 %. Thus, the unique phylogenetic position and moderately low 16S rDNA sequence similarities indicate that strain 1BDZT represents a novel taxon within the genus Amycolatopsis.
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); tetrahydrogenated menaquinones with nine isoprene units were the predominant isoprenoid quinones. The phospholipid pattern consisted of major amounts of phosphatidylethanolamine (a taxonomically significant phospholipid) and minor amounts of phosphatidylglycerol and phosphatidylinositol. The cellular fatty acid profiles included a large amount of 14-methylpentadecanoic acid (i-C16 : 0; 50·3 % of total). Considerable amounts of 13-methylpentadecanoic acid (ai-C16 : 0; 3·6 %), hexadecanoic acid (C16 : 0; 21·7 %), 14-methylhexadecanoic acid (ai-C17 : 0; 12·4 %), heptadecanoic acid (C17 : 0; 8·5 %) and octadecanoic acid (C18 : 0; 3·7 %) were also present. The organism did not contain mycolic acids.
The classification of strain 1BDZT in the genus Amycolatopsis is also supported by phenotypic and morphological characteristics. The organism is a non-motile, Gram-positive, not acid–alcohol-fast, catalase-positive actinomycete; it forms branched substrate mycelium that fragments into rod-like elements and carries sparse to moderate, white aerial hyphae that differentiate into long chains of spore-like structures. A combination of phenotypic properties easily distinguishes this strain from representatives of its nearest neighbours, the A. methanolica subclade (Table 1), and also from those of all Amycolatopsis species with validly published names. It is mesophilic, growing well in both aerobic and anaerobic conditions over a wide pH range (pH 6·0–10·0) and over a wide range of NaCl concentrations (0·5–7 %, w/v). Weak growth also occurs on 10 % NaCl. Additional physiological characteristics are shown in Table 1.
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Description of Amycolatopsis palatopharyngis sp. nov.
Amycolatopsis palatopharyngis (pa.la.to.pha.ryn'gis. N.L. gen. n. palatopharyngis of the palatopharynx).
The organism forms branched yellow to yellow-brown substrate mycelium that fragments into rod-like elements. White aerial mycelium is produced, sparsely on Bennett's, glucose/yeast extract/malt extract and brain heart infusion agars and moderately on inorganic salts/starch agar. The aerial mycelium differentiates into long chains of spore-like structures. Diffusible pigments are not produced. Facultatively anaerobic and catalase-positive. Growth occurs between pH 6 and 10, between 0·5 and 10 % NaCl and between 10 and 40 °C, but not at 45 °C. Acid is produced from adonitol, (+)-L-arabinose, meso-erythritol, (+)-D-fructose, (+)-D-galactose, meso-inositol, (+)-D-raffinose, (+)-D-trehalose and (+)-D-xylose, but not from (+)-D-cellobiose, dextrin, (+)-D-lactose, (+)-D-maltose, (-)-D-mannitol, (+)-D-melezitose, (+)-D-melibiose, methyl 
-D-glucoside, (+)-L-rhamnose, salicin, (-)-D-sorbitol or (-)-D-sucrose. Casein, gelatin, hypoxanthine, L-tyrosine and xanthine are degraded, but aesculin, allantoin and starch are not. Exhibits nitrate reductase and weak urease activity. Resistant to ampicillin (10 µg), carbenicillin (100 µg) and cephalothin (30 µg), but not to acetylspiramycin (15 µg), chloramphenicol (30 µg), clarithromycin (15 µg), doxycycline (30 µg), gentamicin (10 µg), kanamycin (30 µg), midecamycin (15 µg), minocyline (30 µg), novobiocin (5 µg), penicillin (10 IU), rifampicin (5 µg), streptomycin (10 µg) or tobramycin (10 µg). Chemotaxonomic characteristics are typical of those for the genus Amycolatopsis. The G+C content of the DNA is 65·8 mol% (Tm method).
The type strain, 1BDZT [=AS 4.1729T (Chinese General Microbiological Collection Centre)=PCM 2600T (Polish Collection of Microorganisms)], was isolated from the infected palatopharyngeal mucosa of an elderly human patient.
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ACKNOWLEDGEMENTS |
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