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1 College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung, 402, Taiwan, Republic of China
2 Division of Biology, King George VIth Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Chiu-Chung Young
ccyoung{at}mail.nchu.edu.tw
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ABSTRACT |
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MAIN TEXT |
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. The taxon is well-defined and can be distinguished readily from the nine other genera that constitute the suborder, notably by using chemotaxonomic, morphological and 16S rRNA gene sequence data (Soddell et al., 2006a). At the time of writing the genus comprises 20 recognized species, most of which have been described in the last 10 years using polyphasic taxonomic approaches (Kim et al., 1999; Iida et al., 2005). All but one of these taxa have been isolated from natural and artificial habitats, including activated sludge foam (Soddell et al., 2006b), industrial wastewater (Kim et al., 2003), mangrove rhizosphere (Takeuchi & Hatano, 1998), tar-contaminated soil (Kummer et al., 1999) and an oil-producing well (Xue et al., 2003). The exception, Gordonia otitidis Iida et al. 2005, belongs to the growing number of Gordonia species associated with human and animal diseases (Goodfellow & Maldonado, 2006). However, in general, Gordonia strains are widely distributed in soil and aquatic habitats, including marine sediments (Goodfellow & Maldonado, 2006). The aim of the present study was to determine the taxonomic position of an actinomycete, designated strain CC-AB07T, that formed a pale orange colony on a trypticase soy agar (TSA) plate that had been incubated at 30 °C for 3 days following inoculation with a suspension of a soil sample collected from the campus of National Chung Hsing University, Taichung, Taiwan. The organism was subjected to a polyphasic taxonomic study, which showed that it was a novel species of the genus Gordonia.
Extraction of genomic DNA, PCR amplification and sequencing of 16S rRNA genes was carried out as described by Shen et al. (2005). Sequence analysis was performed using a DNA sequencer (ABI PRISM 310; Applied Biosystems) and sequence assembly by using the Fragment Assembly System program from the Wisconsin Package 9.1 supplied by the National Health Research Institute of Taiwan. The resultant sequence was compared with corresponding results taken from the RDP II database for representatives of the genera classified in the suborder Corynebacterineae. Phylogenetic trees were inferred using the maximum-likelihood, maximum-parsimony and neighbour-joining algorithms, as explained by Soddell et al. (2006a). The unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining dataset using the SEQBOOT and CONSENSE options from the PHYLIP package (Felsenstein, 1989). A nearly complete 16S rRNA gene sequence (1485 nt) that corresponded to Escherichia coli positions 26–1511 (Brosius et al., 1978) was obtained for strain CC-AB07T. A comparison of the sequence with those of representatives of the genera classified in the suborder Corynebacterineae showed that the organism fell within the evolutionary radiation occupied by the genus Gordonia (data not shown). 16S rRNA gene sequence similarity between strain CC-AB07T and the type strains of members of the genus Gordonia ranged from 94.0 to 98.2 %.
Chemosystematic studies were carried out to establish whether strain CC-AB07T had a chemical profile consistent with its assignment to the genus Gordonia. Biomass for chemical studies was grown in shake flasks of glucose–yeast extract broth (Gordon & Mihm, 1962) for 5 days at 28 °C, checked for purity, harvested by centrifugation, washed twice in distilled water and freeze-dried. Standard methods were used for analysis of the isomers of diaminopimelic acid, fatty acids, isoprenoid quinones, muramic acid type, mycolic acids, polar lipids and sugars, as described by Soddell et al. (2006b). The isolate contained meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970); N-glycolated muramic acid; dihydrogenated menaquinones with nine isoprene units as the predominant isoprenologue; phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977) and a fatty acid profile rich in palmitic (C16 : 0; 33.6 % of the total fatty acid composition), palmitoleic (C16 : 1; 10.4 %), oleic (C18 : 1; 11.7 %) and tuberculostearic (29.4 %) acids. It was also characterized by the presence of mycolic acids that co-migrated (RF value 0.47) with those extracted from Gordonia bronchialis DSM 43247T. All of these properties are typical of representatives of the genus Gordonia (Goodfellow & Maldonado, 2006).
It is evident from the phylogenetic tree in Fig. 1 that strain CC-AB07T forms a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Gordonia. The organism was most closely related to G. bronchialis DSM 43247T, sharing a 16S rRNA gene sequence similarity of 98.2 %, a value that corresponds to 26 nt differences at 1468 locations. Strain CC-AB07T also shares comparatively high 16S rRNA gene sequence similarities with the type strains of Gordonia polyisoprenivorans (98.1 %), Gordonia rhizosphera (97.7 %), Gordonia amicalis (97.6 %), Gordonia desulfuricans (97.6 %), Gordonia terrae (97.4 %), Gordonia westfalica (97.4 %), Gordonia alkanivorans (97.3 %) and Gordonia rubripertincta (97.3 %). DNA–DNA relatedness experiments were not carried out between strain CC-AB07T and its closest phylogenetic neighbours as the type strains of several Gordonia species share higher 16S rRNA gene sequence similarities, but have levels of DNA–DNA relatedness well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987). The type strains of G. alkanivorans and G. rubripertincta, for example, have a 16S rRNA gene sequence similarity of 99.1 % (which corresponds to a 13-nt difference), but a DNA–DNA relatedness value of only 52 % (Kummer et al., 1999).
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. Degenerate PCR primers GYRB1 and GYRB2 were designed and used to amplify a gyrB fragment of approximately 1.2 kb from the genomic DNA extracted from the type strains of recognized Gordonia species. Sequencing primers were designed from the flanking regions of gyrB conserved sequences with two forward primers, GYRBF1 and GYRBF2, and the reverse primer GYRBR1. The resultant sequence was compared with corresponding gyrB sequences of available Gordonia type strains drawn from the EMBL and ICB database (http://seasquirt.mbio.co.jp/icb/) using the CLUSTAL_X program (Thompson et al., 1997). A phylogenetic tree was inferred with the neighbour-joining algorithm (Saitou & Nei, 1987) using MEGA version 2.1 software (Kumar et al., 2001). An evolutionary distance matrix was generated as described by Kimura (1980). The resultant unrooted tree was evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings.
It can be seen from Fig. 2 that strain CC-AB07T forms a distinct phyletic line in the Gordonia gyrB gene sequence tree. The strain is most closely related to G. amicalis DSM 44461T (84.5 % gyrB gene sequence similarity), G. desulfuricans (81.3 %), G. polyisoprenivorans (79.8 %), G. rhizosphera DSM 44383T (78.8 %), Gordonia sputi DSM 43896T (78.0 %), G. bronchialis DSM 43247T (77.9 %) and Gordonia aichiensis DSM 43978T (77.5 %). These data provide further evidence that isolate CC-AB07T is not particularly closely related to representatives of recognized Gordonia species, especially as classifications based on gyrB gene nucleotide sequence data correlate well with those based on corresponding DNA–DNA relatedness data (Shen et al., 2006). These workers determined gyrB gene sequence similarities between the type strains of 12 recognized Gordonia species and reported similarity values in the range 79.3–97.2 % (41–270 nt difference); the corresponding 16S rRNA gene sequences differed within the range 0.3–3.8 %.
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; Soddell et al., 2006b). Cultural characteristics were examined on a TSA plate that had been incubated for 2 days at 30 °C. Smears taken from the plate were Gram-stained (Shen et al., 2005) and examined for micromorphological properties. Additional smears were stained using a modification of the Ziehl-Neelsen method (Gordon, 1967) to determine whether the organism was acid–alcohol-fast. Standard biochemical and degradation tests were carried out using well-established procedures (Kim et al., 1999). The enzymic profile of strain CC-AB07T was determined using API Coryne (bioMérieux) and API ZYM (bioMérieux) kits according to the manufacturer's instructions. Similarly, sole carbon source tests were performed using a Biolog GP-II kit. The ability of the organism to grow at a range of temperatures was determined on TSA plates and motility was tested as described by Shen et al. (2005). Oxidase activity was determined using oxidase reagent (bioMérieux). It is apparent from the results (Table 1) that strain CC-AB07T can be distinguished from representatives of its close phylogenetic relatives using a combination of phenotypic attributes.
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Description of Gordonia soli sp. nov.
Gordonia soli (so.li. L. gen. n. soli of/from the soil).
Aerobic, Gram-positive, partially acid–alcohol-fast, non-motile actinomycete that forms elementary branching hyphae that fragment into rods and cocci. Pale orange-coloured, circular colonies (about 2 mm in diameter) with filamentous margins are formed on TSA after incubation for 2 days at 30 °C. Neither aerial hyphae nor diffusible pigments are produced. Grows at pH 5.5–10.0 and at 28–35 °C, but not at 4 or 40 °C. Degrades starch, produces catalase and oxidase, but does not hydrolyse allantoin or arbutin. Acid and alkaline phosphatase, butyrate esterase, caprylate esterase, 
-glucosidase, 
-glucosidase, naphthol-AS-BI-phosphohydrolase and pyrazinamidase are produced, but myristate lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -mannosidase, -fucosidase, N-acetyl--glucosaminidase, pyrrolidonylarylamidase and trypsin are not. Does not ferment D-glucose, glycogen, D-lactose, D-maltose, D-mannitol, D-ribose, sucrose or D-xylose. Utilizes N-acetyl-L-glutamic acid, D-alanine (weak), L-alanine, L-alanyl glycine (weak), L-alaninamide, -cyclodextrin, -cyclodextrin, D-fructose 6-phosphate (weak), -D-glucose, L-glutamic acid, glycogen (weak), -hydroxybutyric acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, DL-lactic acid (weak), D-malic acid (weak), mannan, D-mannose, 3-methyl D-glucose (weak), D-psicose, L-pyroglutamic acid, pyruvic acid (weak), putrescine, D-sorbitol (weak), succinic acid (weak), sucrose (weak), Tweens 20 and 40 and uridine 5-monophosphate as sole carbon sources. Additional phenotypic properties are given in Table 1
. Chemotaxonomic properties are typical of the genus Gordonia.
The type strain, CC-AB07T (=BCRC 16810T=DSM 44995T), was isolated from a soil sample collected from the campus of National Chung Hsing University, Taichung, Taiwan.
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ACKNOWLEDGEMENTS |
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