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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
2 Institue of Modern Biopharmaceuticals, School of Life Science, Southwest University, Chongqing 400715, China
Correspondence
Ying Huang
huangy{at}im.ac.cn
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, belongs to the mycolic acid group of the actinomycetes and the suborder Corynebacterineae and forms a distinct phyletic line in the 16S rRNA gene phylogenetic tree (Stackebrandt et al., 1988, 1997). The genus encompasses 25 species with validly published names at the time of writing (November 2006), which can be distinguished from each other using a combination of morphological, physiological and chemical markers (Goodfellow et al., 1999).
Several members of this taxon were isolated from clinical samples and have been considered as opportunistic human pathogens (Tsukamura, 1971; Klatte et al., 1994; Iida et al., 2005; Kageyama et al., 2006). Nevertheless, most gordoniae have been isolated from other environments such as mangrove rhizosphere, soil, wastewater, oil-producing wells and activated sludge foam (Takeuchi & Hatano, 1998; Kummer et al., 1999; Kim et al., 2003; Maldonado et al., 2003; Xue et al., 2003; Shen et al., 2006; Soddell et al., 2006) and play important roles in pollutant biodegradation and bioremediation (Yoon et al., 2000) because of their abilities to metabolize macromolecules or contaminants, for example natural rubber, benzothiophene and hydrocarbons (Linos et al., 1999; Kim et al., 1999; Xue et al., 2003). It is therefore important for ecological reasons to unravel the species-richness of gordoniae in nature habitats. The aim of this study is to clarify the taxonomic position of a rod/coccus-like strain isolated from farmland soil.
Strain SD29T was isolated on ISP 2 agar (Shirling & Gottlieb, 1966), supplemented with 50 µg cycloheximide ml–1, which had been incubated for 5 days at 28 °C following inoculation with a suspension of soil sample collected from farmland in Shandong Province, China. The pure cells were stored in 20 % (v/v) glycerol at –20 °C. Biomass for chemotaxonomic study was harvested by centrifugation after 3 days at 28 °C in shake flasks of ISP 2 and TSB (trypticase soy broth), washed twice with distilled water and freeze-dried. Control type strains Gordonia hydrophobica DSM 44015T and Gordonia sihwensis DSM 44576T were incubated as described above.
Colonial characteristics of strain SD29T were observed on ISP 2 agar. Zeihl–Neelsen staining (Gordon, 1967) and morphological properties were recorded using a light microscope (Axioskop 20; Zeiss) and scanning electron microscope (FEI QUANTA) after 5 days on ISP 2 plates. The ability to utilize sole carbon sources for energy and growth and to decompose various substances was assessed according to the methods of Gordon & Mihm (1957). Standard chemotaxonomic analytical procedures were used as follows: Hasegawa et al. (1983) [isomers of diaminopimelic acid (A2pm)], Lechevalier & Lechevalier (1980) (whole-cell sugars), Collins (1985) and Wu et al. (1989) (menaquinones), Minnikin et al. (1984) (polar lipids), Sasser (1990) and Kämpfer & Kroppenstedt (1996) (fatty acids) and Minnikin et al. (1975) (mycolic acids).
Extraction of whole genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the PCR product were performed as described previously (Chun & Goodfellow, 1995; Huang et al., 2001). The almost-complete 16S rRNA gene sequence (1398 nt) of strain SD29T was aligned with corresponding sequences of all type strains within the genus Gordonia retrieved from GenBank using MEGA software version 3.1 (Kumar et al., 2004). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods. Evolutionary distances for the neighbour-joining algorithm were calculated with the Kimura two-parameter model (Kimura, 1980) and close-neighbour-interchange (search level = 2, random additions = 100) was applied in the maximum-parsimony analysis. The topology of the neighbour-joining tree was evaluated by bootstrap analysis on the basis of 1000 replications (Felsenstein, 1985).
The morphological and chemotaxonomic characteristics of strain SD29T were consistent with the diagnostic properties of the genus Gordonia (Stackebrandt et al., 1988). The organism was Gram-positive, aerobic, non-motile and slightly acid-fast, formed short elementary mycelia that separated into rod/coccus-shaped elements and produced light-yellow, rough colonies, different in appearance from the control type strains, on ISP 2 agar. It contained meso-A2pm, arabinose and galactose in whole-cell hydrolysates (cell-wall chemotype IV sensu Lechevalier & Lechevalier, 1970), MK-9(H2) as the predominant menaquinone and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides as diagnostic polar lipids (phospholipid type II sensu Lechevalier et al., 1977). The mycolic acid pattern detected by TLC was identical to that of the control strain G. sihwensis DSM 44576T. The fatty acid profile was composed mainly of C14 : 0 (3.5 %), C15 : 0 iso 2-OH (14.6 %), C16 : 0 (35.9 %), C18 : 1
9c (21.3 %) and TBSA (tuberculostearic acid, 10-methyl C18 : 0) (14.9 %) and differed from those of related type strains in the presence of a significant amount of C15 : 0 iso 2-OH (Table 1).
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), and shared the highest 16S rRNA gene sequence similarity (97.1 and 97.0 %, respectively) with these strains. Such low similarity values and the low bootstrap levels (37 and 40 %) supporting the clades indicated that the isolate was distinct from the type strains of G. sihwensis and G. hydrophobica. In the maximum-parsimony tree, the isolate formed an unstable clade (bootstrap level of 33 %) with G. sihwensis. The physiological properties listed in Table 2 also readily distinguished the isolate from the two type strains. It is apparent from the results of phenotypic and genotypic studies that strain SD29T represents a novel taxonomic unit in the genus Gordonia. It is therefore proposed that the strain should be recognized as the type strain of a novel species with the name Gordonia shandongensis sp. nov.
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Gram-positive, aerobic, non-motile, slightly acid-fast actinomycete that fragments into rod/coccus-like elements. Colonies are light yellow and rough with irregular margins on ISP 2 agar. L-proline is used as a sole carbon source for energy and growth but L-aspartate, L-rhamnose, sodium succinate, citric acid and sebacic acid are not. Aesculin is degraded but casein, starch, tyrosine and xanthine are not. Nitrate is reduced to nitrite. The organism grows well at 20–37 °C and at pH 5.5–9.0. Other physiological properties are shown in Table 2
. The cell wall chemotype is IV, and phospholipid type is PII. The principal menaquinone is MK-9(H2). The major fatty acids are C16 : 0 (35.9 %), C18 : 19c (21.3 %), 10-methyl C18 : 0 (TBSA) (14.9 %) and C15 : 0 iso 2-OH (14.6 %). Mycolic acids are present.
The type strain, SD29T (=CGMCC 4.3492T=JCM 13907T), was isolated from farmland soil collected in Shandong Province, China.
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ACKNOWLEDGEMENTS |
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