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1 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
2 Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
3 Discovery Research Laboratories, Tanabe Seiyaku Co. Ltd, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
4 First Department of Internal Medicine, Kurume University School of Medicine, 67 Asahimachi, Kurume 830-0011, Japan
5 Laboratory of Clinical Microbiology, Toranomon Hospital, Toranomon 2-2-2, Minato-ku, Tokyo 105-8470, Japan
6 National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
7 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
Correspondence
Yuzuru Mikami
mikami{at}faculty.chiba-u.jp
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The GenBank/EMBL/DDBJ accession number accession numbers for the 16S rRNA gene sequences of Gordonia araii IFM 10211T and Gordonia effusa IFM 10200T are AB162800 and AB162799, respectively.
A table giving detailed physiological characteristics of members of the genus Gordonia is available as supplementary material in IJSEM Online.
Present address: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan. 
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). This suborder also contains the genera Corynebacterium, Dietzia, Mycobacterium, Nocardia, Skermania, Rhodococcus, Tsukamurella, Turicella and Williamsia (Goodfellow, 1998; Goodfellow et al., 1999; Kämpfer et al., 1999). These genera are mutually distinguishable using a combination of biochemical, chemotaxonomic and morphological characteristics. The genus Gordonia was erected by Stackebrandt et al. (1988), and at the time of writing comprises 19 recognized species. Among them, Gordonia aichiensis, G. bronchialis, G. otitidis, G. sputi and G. terrae have been isolated from clinical specimens (Iida et al., 2005). G. bronchialis, Gordonia rubropertincta and G. sputi were specifically isolated from the sputum of patients with pulmonary disease (Klatte et al., 1994; Tsukamura, 1978). Two hundred and thirty-five strains of pathogenic actinomycetes isolated from clinical specimens were referred to our research centre for identification during 2000–2003. Among these clinical specimens, strains IFM 10211T (in 2001) and IFM 10200T (in 2002) were isolated from sputum specimens of Japanese patients with kidney dysfunction and pulmonary disease, respectively. These organisms were found to be related to the genus Gordonia based on their phenotypic characteristics and TLC profile of mycolic acids. Furthermore, the two strains were found to cluster with the type strains of Gordonia amarae, G. hydrophobica, G. sihwensis and G. hirsuta based on 16S rRNA gene sequence analysis, but differences in several phenotypic characteristics and their genetic distinctiveness render them distinguishable. Comparative DNA–DNA relatedness data indicated that the strains belong to different species. Based on the phenotypic and genotypic data, we therefore suggest that the two clinical isolates represent two novel species of the genus Gordonia.
Strains IFM 10211T and IFM 10200T were isolated respectively from the sputum of a 48-year-old male Japanese patient with bacterial pneumonia, and of a 74-year-old Japanese male patient with kidney dysfunction, and referred to our research centre for identification.
Strains IFM 10211T and IFM 10200T and reference strains G. amarae IFM 0210T, G. hirsuta IFM 10287T and G. hydrophobica IFM 10286T were cultured on Mueller–Hinton II (MH II; Difco Laboratories) agar slants with 1 % glucose and 1 % glycerol for 1 week at 30 °C. For extraction of DNA and sequencing, bacterial strains were cultured in brain heart infusion (BHI; Difco Laboratories) broth containing 1 % glucose for 4 days at 30 °C. For DNA–DNA hybridization experiments, strains were cultured in BHI broth containing 2 % glucose and 2 % glycine for 3 days at 30 °C. Micromorphological properties were observed by using electron microscopy according to a previously described method (Kageyama et al., 2004a). TLC analysis of mycolic acids for the differentiation of actinomycete genera containing these acids was performed according to the method of Miyadoh (2001). Carbon utilization tests were performed as described by Takeuchi & Hatano (1998). The modified Ziehl–Neelsen method was used for the acid-fast staining test. Whole-cell hydrolysates were analysed for diaminopimelic acid (DAP) isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared as described by Lechevalier & Lechevalier (1980) and analysed by TLC (Miyadoh, 2001). Methyl esters of cellular fatty acids mycolic acid trimethylsilyl esters were prepared and analysed as described by Klatte et al. (1994) using the standard Microbial Identification System (Microbial ID Inc.). Isoprenoid quinones were extracted by the method of Collins et al. (1977) and were analysed by HPLC with a Cosmosil 5C18 column (4.6 mmx150 mm; Nacalai Tesque Inc.). A mixture of methanol and 2-propanol (2 : 1, v/v) was used as the elution solvent.
Preparation of genomic DNA samples for sequencing was performed using the guanidine thiocyanate method (Kageyama et al., 2004a, b).
The 16S rRNA gene was amplified and sequenced by PCR by employing six universal primers for the prokaryotic 16S rRNA gene. We performed PCR with a DNA thermal cycler (TaKaRa) using 35 cycles consisting of denaturation at 94 °C for 60 s, primer annealing at 60 °C for 60 s and primer extension at 72 °C for 120 s. The PCR products were purified using a Centri-Sep column (Princeton Separations). DNA sequences were analysed with an automatic sequence analyser (ABI PRISM 3100; PE Applied Biosystems) by using a dye terminator cycle sequencing kit (PE Applied Biosystems).
A BLAST comparison of the 16S rRNA gene sequences of the two new isolates indicated that their closest neighbours were G. amarae, G. hydrophobica, G. sihwensis and G. hirsuta. Sequence data of related species were retrieved from GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972) and phylogenetic trees were constructed based on the neighbour-joining method (Saitou & Nei, 1987). Tree topology was evaluated using a bootstrap analysis of sequence data with CLUSTAL W software (Thompson et al., 1994).
DNA was isolated using a modified method of that described by Saito & Miura (1983). Levels of DNA–DNA relatedness were determined using the method of Ezaki et al. (1989) using photobiotin and microplates.
Whole-cell hydrolysates of strains IFM 10211T and IFM 10200T contained meso-DAP as the only diamino acid of the peptidoglycan and arabinose plus galactose as major whole-cell sugars (wall chemotype IV sensu Lechevalier & Lechevalier, 1980). The fatty acid profiles of strains IFM 10200T and IFM 10211T consisted of straight-chain saturated and unsaturated fatty acids plus tuberculostearic acid, patterns similar to those of all genera in the suborder Corynebacterineae. The predominant menaquinone was MK-9(H2) for strain IFM 10211T; strain IFM 10200T had MK-9(H8) and MK-9(H6), but also a small amount of MK-9(H4). These results were consistent with strain IFM 10211T belonging to the genus Gordonia, but no such conclusion could be made for strain IFM 10200T on this basis.
Strains IFM 10200T and IFM 10211T had mycolic acids with chain lengths of 62–70 and 58–64 carbons, respectively. The mycolic acid pattern of strain IFM 10211 T thus differs from those of recognized Gordonia species, more closely resembling that of the genus Tsukamurella in the suborder Corynebacterineae. The pattern for strain IFM 10200T matched that of other members of the genus Gordonia.
Strains IFM 10211T and IFM 10200T produced short elementary branching hyphae that disintegrated into rod-like and cocci-like elements. Growth of strain IFM 10200T was faster than that of IFM 10211T; no soluble pigment was produced in either strain. Almost complete 16S rRNA gene sequences of strains IFM 10211T (1499 bp) and IFM 10200T (1509 bp) were determined. Phylogenetic analyses demonstrated that the two strains formed a monophyletic clade associated with G. amarae, G. hirsuta and G. hydrophobica (Fig. 1). On the basis of phylogenetic analysis, strains IFM 10211T and IFM 10200T were considered to belong to the genus Gordonia, but differences in the mycolic acid pattern of strain IFM 10211T and menaquinone composition of strain IFM 10200T distinguished them from recognized species of the genus Gordonia. 16S rRNA gene sequence similarity between strains IFM 10211T and IFM 10200T was 97.9 %; similarity values between strains IFM 10211T and IFM 10200T and their closest neighbour, G. amarae DSM 43392T, were 97.4 and 97.7 %, respectively. Lower levels of sequence similarity (<97.6 %) were observed with the type strains of other neighbouring species, including G. hydrophobica and G. hirsuta (Table 1). The genomic delineation among strains IFM 10211T and IFM 10200T and the type strains of G. amarae, G. hydrophobica and G. hirsuta was supported by the DNA–DNA relatedness data (Table 1). The two new isolates showed levels of DNA–DNA relatedness of 6.2–6.9 % to each other, and values in the range 3.2–10.9 % to G. amarae, G. hydrophobica, G. hirsuta and G. sihwensis. These values are well below the 70 % cut-off point recommended for the delineation of genomic species (Wayne et al., 1987).
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). Both strains were readily distinguishable from these species based on a combination of physiological, biochemical and chemotaxonomic characteristics (see also Supplementary Table S1 available in IJSEM Online). They also differed phylogenetically from other Gordonia species (Fig. 1
). Both phenotypic and genotypic data indicated that strains IFM 10211T and IFM 10200T represent two novel species of the genus Gordonia, for which we propose the names Gordonia araii sp. nov. and Gordonia effusa sp. nov., respectively.
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Aerobic, Gram-positive, slightly acid-fast, non-motile rods (0.3–0.5x0.5–1.1 µm). Colonies are white, becoming beige. Colonies are rough with irregular margins. No soluble pigment is produced. Colonies are 1.5–3.5 mm in diameter after 7 days at 30 °C on MH II medium. Utilizes glucose, but not adonitol, arabinose, erythritol, galactose, myo-inositol, maltose, mannose or sorbitol. Adenine, hypoxanthine, tyrosine, urea and xanthine are not hydrolysed. Contains meso-DAP, arabinose and galactose (cell wall chemotype IV sensu Lechevalier & Lechevalier, 1980). Predominant menaquinone is MK-9(H2). Major fatty acids are C14 : 0, C16 : 1cis-9, C16 : 0, C18 : 1cis-9, C18 : 0 and 10-methyl C18 : 0. Mycolic acids are present with a range of total carbon number from 64 to 70.
The type strain, IFM 10211T (=DSM 44811T=NBRC 100433T=JCM 12131T), was isolated from a clinical specimen.
Description of Gordonia effusa sp. nov.
Gordonia effusa (ef.fu'sa. L. fem. adj. effusa poured out, extensive, vast, broad, wide, referring to the spreading colonial growth).
Aerobic, Gram-positive, slightly acid-fast, non-motile rods (0.3–0.6x0.5–2.1 µm). Colonies are white, becoming beige. Colonies are rough with irregular margins. No soluble pigment is produced. Colonial growth is fast; colony diameter is 3.0–5.0 mm after 7 days at 30 °C on MH II medium. Utilizes sucrose, D-turanose, N-acetyl-D-glucosamine, citrate, 4-aminobutyrate, succinate, L-alanine, L-aspartate, L-valine and putrescine, but not D-galactose, L-rhamnose, D-ribose, D-arabitol or myo-inositol. Contains meso-DAP, arabinose and galactose (cell wall chemotype IV sensu Lechevalier & Lechevalier, 1980). Predominant menaquinones are MK-9(H8) and MK-9(H6); a small amount of MK-9(H4) is also present. Major fatty acids are C14 : 0, C15 : 0, C16 : 0, C17 : 0, C18 : 0 and 10-methyl C18 : 0. Mycolic acids are present with a range of total carbon number from 60 to 64.
The type strain, IFM 10200T (=DSM 44810T=NBRC 100432T=JCM 12130T), was isolated from a clinical specimen.
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