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Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
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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Strain WS-26T was subjected to morphological, cultural, physiological and chemotaxonomic characterization, in addition to phylogenetic analysis based on 16S rRNA gene sequence studies. Based on the phenotypic and phylogenetic data, it was shown that the organism was different from species of the genus Nocardia with validly published names.
Morphological characteristics were investigated by light microscopy. Cells were incubated on oatmeal agar for 14 days at 30 °C. A coverslip with growing cells attached was observed directly using a light microscope. The degree of growth and the level of pigmentation were investigated on ISP 2, ISP 3 and ISP 4 agar media (Shirling & Gottlieb, 1966
) and tryptic soy broth (Difco) supplemented with agar (TSBA) after incubation at 30 °C for 14 days. Strain WS-26T showed good growth on ISP 2 medium and TSBA, but only moderate growth on ISP 3 and ISP 4 media. Abundant, branched substrate mycelium, which was reddish orange in colour, was produced on all of the media tested and fragmented into irregular rod-shaped elements. The intensity of pigmentation was stronger on the nutrient-rich media, namely ISP 2 medium and TSBA. Diffusible pigments were not produced on most of the media tested. White, fragmenting aerial mycelium was produced to a moderate level on ISP 3 and ISP 4 media, but was sparse on ISP 2 medium and TSBA.
Genomic DNA was extracted and purified as described previously (Lee, 2006). The 16S rRNA gene of strain WS-26T was amplified by PCR as described previously (Lee et al., 2000a) and purified using the Wizard PCR Preps DNA purification system (Promega). The resultant 16S rRNA gene was sequenced directly using an ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 3730xl; Applied Biosystems). An almost complete 16S rRNA gene sequence of strain WS-26T was determined in this study and comprised a continuous stretch of 1397 nt. The sequence was subjected to a BLAST search (http://www.ncbi.nlm.nih.gov/), which indicated that the organism was a member of the family Nocardiaceae. Assignment to this family was also supported by the presence of a set of family-specific signature nucleotides in the 16S rRNA gene sequence (Stackebrandt et al., 1997).
CLUSTAL_X (Thompson et al., 1997) was used to align the sequence with available reference sequences retrieved from GenBank. Multiple alignments were optimized manually according to 16S rRNA secondary structure. Phylogenetic analyses were carried out using three treeing algorithms, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods. A total of 1257 unambiguous aligned positions present in all strains between nt 91 and 1392 (nucleotide numbering according to Escherichia coli positions; Brosius et al., 1978) was used for phylogenetic analyses. A phylogenetic tree was reconstructed by the neighbour-joining method from evolutionary distances calculated by the method of Jukes & Cantor (1969). Tsukamurella paurometabola DSM 20162T (GenBank accession no. X80628) was used as an outgroup. The reliability of the tree topology was evaluated by bootstrap analysis (Felsenstein, 1985).
The 16S rRNA gene sequence was compared with those of the 59 type strains of Nocardia species with validly published names at the time of writing. A neighbour-joining tree (Fig. 1), based on 16S rRNA gene sequence studies, showed that the organism formed the deepest branch outside the Nocardia carnea cluster, which included Nocardia carnea, N. flavorosea, N. pigrifrangens, N. sienata and N. testacea, with a bootstrap value of 41 %. This phylogenetic placement was also found in the tree constructed using the maximum-likelihood method, although strain WS-26T formed a cluster with Nocardia transvalensis when the maximum-parsimony method was employed (not shown). Levels of 16S rRNA gene sequence similarity between strain WS-26T and members of the genus Nocardia ranged from 94.6 to 97.9 %. The organism showed high 16S rRNA gene sequence similarity values to closely associated neighbours, namely N. carnea DSM 43398T (97.8 %), Nocardia cyriacigeorgica DSM 44484T (97.8 %), N. flavorosea JCM 3332T (97.7 %), N. pigrifrangens JCM 11884T (96.5 %), N. sienata IFM 10088T (97.7 %) and N. testacea IFM 0937T (97.8 %). Among loosely associated members, Nocardia abscessus DSM 44432T, Nocardia asiatica IFM 0245T, Nocardia brasiliensis ATCC 19296T and N. transvalensis DSM 43405T (sequence similarity values of 97.7–97.9 %) were equidistantly related to strain WS-26T, representing slightly higher values than those between the isolate and its phylogenetic neighbours. The unstable topology of the isolate in the tree (Fig. 1), as well as the somewhat low sequence similarities to the type strains of the genus Nocardia, indicated that strain WS-26T merits classification as a novel genomic species without the need to perform DNA–DNA hybridization experiments.
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), phospholipids (Minnikin et al., 1977), lipoquinones (Kroppenstedt, 1985), the isomer of diaminopimelic acid (Staneck & Roberts, 1974) and the acyl type (Uchida & Aida, 1984) of the peptidoglycan and the whole-cell sugar composition (Saddler et al., 1991). Cellular fatty acids were extracted from biomass obtained in trypticase soy broth (Difco) incubated at 30 °C for 3 days. Fatty acid methyl esters were prepared by alkaline methanolysis (Minnikin, 1988) and analysed using a gas chromatograph (model 6850; Agilent) as described previously (Lee et al., 2000b). The G+C content of the DNA was determined by HPLC as described previously (Mesbah et al., 1989). The results of chemical analyses, particularly the predominant lipoquinone, revealed that the organism could be assigned to the genus Nocardia (Collins et al., 1977). Whole-cell hydrolysates contained arabinose and galactose as characteristic sugars and meso-diaminopimelic acid as the principal diamino acid (type IV cell wall; Lechevalier & Lechevalier, 1970). The glycan moiety of the murein was N-acetylated. The predominant lipoquinone was a tetrahydrogenated menaquinone with eight isoprene units in which the two terminal isoprene moieties were cyclized [MK-8(H4, 
-cycl)]. This type of lipoquinone is found in all species of the genus Nocardia (Collins et al., 1977). Mycolic acids were present, with an Rf value of 0.47 on TLC. The phospholipids detected were phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol (phospholipid type PII pattern; Lechevalier et al., 1977). Cellular fatty acid profiles were characterized by the predominance of hexadecanoic (C16 : 0; 25.3 %), tuberculostearic (10-methyl-C18 : 0; 16.7 %) and cis-9-octadecenoic (cis9-C18 : 1; 12.7 %) acids, followed by 16-methylheptadecanoic (i-C18 : 0; 8.4 %), octadecanoic (C18 : 0; 6.3 %) and 2-hydroxytetradecanoic (2-OH-C14 : 0; 5.2 %) acids. Minor amounts of other saturated, iso- and anteiso-branched fatty acids were also detected in the whole-cell methanolysates.
The following physiological tests were performed using previously described methods (Lee, 2006): production of hydrogen sulfide, nitrate reduction, gelatin liquefaction, hydrolysis of casein, aesculin and starch and degradation of adenine, hypoxanthine, DL-tyrosine and xanthine. For hydrolysis and decomposition tests, strain WS-26T was grown on YE-SW agar as the basal medium. Gram staining and oxidase tests were carried out using the method of MacFaddin (1980). Catalase activity was determined with a 3 % (v/v) hydrogen peroxide solution. Urease activity was determined by a colour change in Bacto urea broth (Difco). NaCl tolerance was tested by growth on yeast extract/malt extract agar (ISP 2 medium) supplemented with NaCl at final concentrations of 0–9 % (w/v). Growth was tested at temperatures of 4, 10, 20, 30, 37 and 45 °C and at pH 4.1–10.1 (at intervals of 1.0) using YE-SW agar as the basal medium. Utilization of carbohydrates, alcohols and organic acids as sole carbon and energy sources was tested using ISP 9 medium (Shirling & Gottlieb, 1966) containing filter-sterilized carbon sources at final concentrations of 1 % (w/v) for carbohydrates and alcohols and 0.1 % (w/v) for organic acids. The results of morphological, cultural and physiological tests are given in the species description and in Table 1. Strain WS-26T utilized a limited range of carbohydrates and organic acids as the sole carbon source. Interestingly, strain WS-26T showed growth over a broad pH range of 4.1–10.1. The ability to grow at pH 4.1 may be a key characteristic for identification of strain WS-26T, as most nocardial strains do not grow under acidic conditions.
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), could be differentiated from the isolate on the basis of cultural and physiological features, as well as the origin of isolation (Lechevalier, 1989; Chun et al., 1998; Yassin et al., 2000, 2003; Kageyama et al., 2004a, b; Wang et al., 2004). The phenotypic properties that differentiated the isolate from its phylogenetic relatives are given in Table 1. In particular, strain WS-26T was readily distinguished from N. transvalensis, which formed a phylogenetic cluster with the isolate in the tree constructed using the maximum-parsimony method (data not shown), by pigmentation of the substrate mycelium (Lechevalier, 1989), in the utilization of D-xylose, meso-erythritol, D-sorbitol and acetate, in the decomposition of hypoxanthine and in nitrate reduction (Table 1). The results of polyphasic taxonomic characterization strongly suggest that the organism be classified as a novel species of the genus Nocardia. The name Nocardia harenae sp. nov. is proposed, with WS-26T as the type strain.
Description of Nocardia harenae sp. nov.
Nocardia harenae (ha.re'nae. L. gen. n. harenae of sand, referring to the isolation of the type strain from beach sand).
Forms a well-developed and branched substrate mycelium that fragments into irregular rod-shaped elements. The colour of the substrate mycelium is reddish orange. White aerial mycelium is produced to a moderate level on ISP 3 and ISP 4 media, but is sparse on ISP 2 and TSBA. Aerobic, Gram-positive, acid–alcohol-fast, catalase-positive, urease-positive and non-motile. H2S is not produced. Nitrate is not reduced to nitrite. Gelatin liquefaction does not occur. Aesculin is hydrolysed, but not starch or casein. Growth occurs between 10 and 40 °C. The pH range for growth is 4.1–10.1. Growth occurs in the presence of 0–5 % NaCl, but not 6–9 % NaCl. Hypoxanthine, DL-tyrosine, elastin and xanthine are not degraded. Dextrin, D-glucose, sucrose, D-trehalose, malate, succinate and D-xylose are used as sole carbon and energy sources, but not adonitol, D-arabinose, L-arabinose, 2,3-butanediol, meso-erythritol, myo-inositol, inulin, D-lactose, D-mannose, D-melezitose, melibiose, methyl 
-D-glucoside, methyl -D-mannoside, 1,2-propanediol, D-raffinose, L-rhamnose, L-ribose, salicin, D-sorbitol, L-sorbose or D-xylitol. Utilization of D-cellobiose, D-dulcitol, D-fructose, glycerol, maltose and D-mannitol shows a weak response. Assimilation of malate and succinate is observed, but not of acetate, benzoate, citrate, formate or D-tartrate. The predominant cellular fatty acids are C16 : 0, 10-methyl-C18 : 0, cis9-C18 : 1, i-C18 : 0, C18 : 0 and 2-OH-C14 : 0 acids. The G+C content of the DNA is 68.9 mol%.
The type strain is strain WS-26T (=KCCM 42317T=NRRL B-24459T), isolated from beach sand on the coast of Jeju Island, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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