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1 School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, People's Republic of China
2 Institute of Space Medico-Engineering, Beijing 100094, People's Republic of China
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
4 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Jianli Zhang
zhangjianli{at}bit.edu.cn
Zhiheng Liu
zhliu{at}sun.im.ac.cn
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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to accommodate aerobic, Gram-positive actinomycetes that form an extensively branched substrate mycelium and scant aerial hyphae, and contain meso-diaminopimelic acid in whole-organism hydrolysates (wall chemotype III sensu Lechevalier & Lechevalier, 1970), galactose, mannose and rhamnose as diagnostic sugars, tetrahydrogenated menaquinones as the predominant isoprenologue, phosphatidylethanolamine as the key polar lipid (phospholipid type II sensu Lechevalier et al., 1977) and fatty acids rich in saturated and monounsaturated iso- and anteiso-components. The genus encompasses two species, Lechevalieria aerocolonigenes, the type species, and Lechevalieria flava; these taxa form a distinct branch within the evolutionary radiation occupied by the family Actinosynnemataceae Labeda and Kroppenstedt 2000. This family also contains the genera Actinosynnema Hasegawa et al. 1978, Lentzea Labeda et al. 2001 and Saccharothrix Labeda et al. 1984. The present investigation was designed to establish the taxonomic position of a soil organism, isolate Z6T, which had been considered by Xunchu Yan in 1978 (unpublished) to be a strain of ‘Streptomyces fradiae’ on the basis of a few biochemical, morphological and physiological properties. Here, a polyphasic taxonomic approach was used to study this isolate; the data showed that it should be recognized as a representative of a novel species of Lechevalieria.
Strain Z6T was isolated on a glucose-yeast extract-malt extract agar plate, which had been seeded with a soil suspension and incubated at 28 °C for 2 weeks. The soil sample was collected from Wutaishan Mountain in Shanxi Province, China. It was maintained on modified Sauton's agar (Mordarska et al., 1972) at 4 °C and as suspensions of mycelial fragments in 20 % (v/v) glycerol at –20 °C. Biomass for the chemotaxonomic and molecular systematic studies was prepared as described by Zhang et al. (2002).
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and purification of the PCR product from isolate Z6T were carried out after Rainey et al. (1996) and the PCR product was sequenced directly following the procedure described by Lu et al. (2001). The resultant 16S rRNA gene sequence was aligned manually with corresponding sequences of representatives of genera classified in the suborder Pseudonocardineae retrieved from the DDBJ/EMBL/GenBank databases using CLUSTAL_X 1.8 software (Thompson et al., 1997). Phylogenetic trees were generated using the least-squares, maximum-likelihood and neighbour-joining algorithms from the PHYLIP suite of programs (Felsenstein, 1993); evolutionary distance matrices for the least-squares and neighbour-joining methods were produced after Kimura (1980). Topologies of the resultant unrooted trees were evaluated by bootstrap analyses of the neighbour-joining dataset based on 1000 resamplings using the SEQBOOT and CONSENSE options from the PHYLIP package. In a corresponding second analysis, the sequence of the tested strain was compared with those of members of genera classified in the family Actinosynnemataceae.
It is apparent from Fig. 1 that strain Z6T forms a distinct phyletic line that lies towards the periphery of the Lechevalieria 16S rRNA clade. It shared 16S rRNA gene similarities of 98.4 and 98.0 % with the type strains of L. aerocolonigenes and L. flava, respectively, values that correspond to 22 and 28 nt differences at 1407 positions. It is also significant that the 16S rRNA gene sequence of the isolate includes signature nucleotides that are characteristic of members of the genus Lechevalieria (Labeda et al., 2001), the family Actinosynnemataceae (Labeda & Kroppenstedt, 2000) and the suborder Pseudonocardineae (Stackebrandt et al., 1997).
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). Standard chromatographic procedures were used for the extraction and analysis of the diagnostic isomers of diaminopimelic acid (A2pm; Hasegawa et al., 1983), menaquinones (Collins et al., 1987; Wu et al., 1989), mycolic acids (Minnikin et al., 1975), polar lipids (Minnikin et al., 1984) and whole-organism sugars (Lechevalier & Lechevalier, 1980), using appropriate controls. In turn, fatty acids were extracted, purified, methylated and quantified by GC using the standard Microbial Identification System (MIDI; Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The base composition of the genomic DNA of the isolate was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as the control. The organism contained: meso-A2pm as the wall diamino acid; galactose, mannose and rhamnose as diagnostic sugars; tetrahydrogenated menaquinones with nine isoprene units as the predominant isoprenologue and dihydrogenated menaquinones with nine isoprene units as the minor isoprenologue (peak area MK-9(H4) : MK-9(H2) ratio of 27 : 10); phosphatidylethanolamine and diphosphatidylglycerol as the key polar lipids; and fatty acids rich in saturated and mono-unsaturated iso- and anteiso-components. However, strain Z6T lacked mycolic acids. The G+C content of the DNA of the isolate was 68.0 mol%. All of these properties are consistent with the classification of the isolate in the genus Lechevalieria.
Colonial and micromorphological properties were examined on glucose-yeast extract-malt extract agar, modified Sauton's agar (Mordarska et al., 1972) and on standard media used in the International Streptomyces Project (ISP; Shirling & Gottlieb, 1966). Micromorphological features were observed using the coverslip technique of Kawato & Shinobu (1959) following the procedure described by Zhou et al. (1998). Peptone-yeast extract-iron agar and tyrosine agar plates (Shirling & Gottlieb, 1966) were examined for the production of melanin pigments following incubation at 28 °C for 14 days. Additional phenotypic properties were determined using standard media and methods (Gordon et al., 1974; Zhang et al., 2003). DNA–DNA relatedness studies were carried out between the isolate and the type strains of L. aerocolonigenes and L. flava using a thermal denaturation procedure (De Ley et al., 1970; Huß et al., 1983) with a UV-1206 spectrophotometer (Shimadzu) fitted with a TB-85 thermobath and standard software (Jahnke, 1992); the results were expressed as the mean of two determinations.
Strain Z6T exhibited phenotypic properties typical of members of the genus Lechevalieria (Labeda et al., 2001). It was an aerobic, Gram-positive, non-motile, catalase-positive actinomycete, which formed an extensively branched substrate mycelium that fragmented into coccoid- and rod-shaped elements. The mean DNA–DNA hybridization values found between the isolate and the type strains of L. aerocolonigenes and L. flava were 45 and 37 %, respectively; values well below the 70 % cut-off point recommended by Wayne et al. (1987) for the delineation of genomic species. The isolate could also be distinguished from the two type strains using a combination of phenotypic properties (Table 1).
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Description of Lechevalieria fradiae sp. nov.
Lechevalieria fradiae (fra'di.ae. N.L. gen. n. fradiae of Fradia, a patronymic).
Aerobic, Gram-positive, catalase-positive, non-motile actinomycete. Forms an extensively branched substrate mycelium that fragments in situ into coccoid- and rod-shaped elements. A yellow to orange mycelium is formed on ISP media 2, 3 and 4, glucose-yeast extract-malt extract agar and modified Sauton's agar. Soluble pigments are not produced nor are melanin pigments formed on glucose-yeast extract-iron or tyrosine agars. Colony elevation is convex to irregular and colony margins are filamentous. Grows at 15–45 °C and pH 5–10. Aesculin, arbutin, gelatin and urea are hydrolysed and nitrate is reduced. H2S is not produced. Degrades casein, starch and tyrosine, but not adenine, guanine, hypoxanthine or xanthine. Acid is formed from (+)-L-arabinose, meso-erythritol, methyl 
-D-glucoside, (+)-D-raffinose, (+)-D-trehalose and (+)-D-xylose, but not from (+)-D-cellobiose, dextrin, (+)-D-fructose, (+)-D-glucose, glycerol, (+)-D-maltose, (+)-D-mannitol, (+)-D-mannose, (+)-D-melezitose, (+)-D-melibiose, -L-rhamnose, (+)-D-sorbitol or (+)-D-sucrose. Sodium acetate, sodium lactate, sodium malate, sodium malonate and sodium succinate are used as sole carbon and energy sources, but not sodium benzoate, sodium citrate, sodium oxalate or sodium tartrate. Growth occurs in the presence of crystal violet (0.001 %, w/v) and phenol at 0.05 % (w/v), but not in the presence of phenol at 0.1 % (w/v). Resistant to (in µg ml–1 unless otherwise stated): lysozyme (0.005 %, w/v); amoxycillin plus clavulanic acid (10); ampicillin (10); aztreonam (30); clindamycin hydrochloride (2); mezlocillin (75); and penicillin G (10 international units). Susceptible to (in µg ml–1): amikacin (30); cefotaxime (30); chloramphenicol (30); ciprofloxacin (5); erythromycin (15); gentamicin sulfate (10); kanamycin sulfate (30); ofloxacin (5); rifampicin (5); streptomycin sulfate (10); tetracycline hydrochloride (30); and tobramycin sulfate (10). Additional phenotypic properties are shown in Table 1
. Chemotaxonomic properties are typical of the genus Lechevalieria. The fatty acid profile is composed mainly of iso-C16 : 0 (56.8 %), iso-C14 : 0 (13.8 %), H-iso-C16 : 1 (12.3 %), C17 : 1
6c (3.5 %), iso-C15 : 0 (3.3 %) and C15 : 16c (3.0 %).
The type strain is Z6T (=CGMCC 4.3506T=JCM 14205T), isolated from a soil sample collected from Wutaishan Mountain, Shanxi Province, China. The DNA G+C content of strain Z6T is 68.0 mol%. The species description is based on a single strain and hence serves as a description of the species.
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ACKNOWLEDGEMENTS |
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