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1 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
2 Department of Microbiology, Chung-Ang University College of Medicine, 221 Huksuk-dong, Dongjak-gu, Seoul 156-756, Republic of Korea
3 School of Biological Sciences, Seoul National University, 56-1 Shilim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Michael Goodfellow
m.goodfellow{at}ncl.ac.uk
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ABSTRACT |
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for organisms previously classified as Corynebacterium paurometabolum (Steinhaus, 1941) and ‘Gordona aurantiaca’ (Tsukamura & Mizuno, 1971). The taxon is well defined and currently encompasses six species with validly published names, namely Tsukamurella inchonensis Yassin et al. 1995, Tsukamurella pulmonis Yassin et al. 1996, Tsukamurella paurometabola (Steinhaus 1941) Collins et al. 1988, Tsukamurella spumae Nam et al. 2003, Tsukamurella strandjordii Kattar et al. 2001 and Tsukamurella tyrosinosolvens Yassin et al. 1997. These species form a distinct clade within the evolutionary radiation occupied by mycolic-acid-containing actinomycetes, i.e. by organisms classified in the suborder Corynebacterineae Stackebrandt et al. 1997. Members of these taxa share very high 16S rRNA gene nucleotide similarity values but can be distinguished by DNA–DNA relatedness and phenotypic data (Kattar et al., 2001; Nam et al., 2003). Tsukamurellae have been described in association with clinical disease (Yassin et al., 1995, 1996, 1997; Kattar et al., 2001) and as agents of foaming in activated sludge plants (Goodfellow et al., 1996, 1998; Seong et al., 1999). Activated sludge foams cause operational problems and may represent a public health hazard because of the potential spread of pathogens by aerosols (Goodfellow et al., 1998).
The aim of the present study was to establish the taxonomic position of two organisms, represented by strains JC85 and N1176T, isolated from activated sludge foam and provisionally labelled ‘Tsukamurella spumae’ (Goodfellow et al., 1998). It was evident from this study that the two isolates were closely related on the basis of whole-organism pyrolysis mass spectrometric data. In a recent polyphasic study, strain N1176T was distinguished from strains of T. spumae and considered to represent a prospective novel species of the genus Tsukamurella (Nam et al., 2003).
The two organisms were maintained as glycerol suspensions (20 %, v/v) at –20 °C and as glucose/yeast extract agar slopes (Gordon & Mihm, 1962) at room temperature, as were the type strains of Tsukamurella species (Table 1). Strain JC85 was examined for a combination of phenotypic tests, using standard procedures (Chun, 1995; Nam et al., 2003). Extraction of chromosomal DNA, PCR amplification and the isolation, cloning and sequencing of the amplified 16S rDNA of strain JC85 was carried out following established procedures (Chun & Goodfellow, 1995). The resultant 16S rRNA gene sequence was compared to corresponding sequences of representatives of the suborder Corynebacterineae as described previously (Nam et al., 2003).
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), as modified by Goris et al. (1998). Photobiotin-labelled DNA from isolate N1176T was individually hybridized with single-stranded unlabelled DNA samples of the remaining strains, non-covalently bound to microtitre wells. The hybridization experiments were conducted under stringent conditions in 50 % formamide at 43 °C. Fluorescent intensities were measured using a Fluoroskan CF fluorimeter (Thermo Lab Systems Inc.) at a wavelength of 360 mm for excitation and 450 mm for emission. Mean percentage DNA–DNA relatedness values were calculated from triplicate hybridization experiments.
The morphological, degradative, nutritional and physiological properties recorded for isolate JC85 were virtually identical to those reported for strain N1176T (Nam et al., 2003), a result that underpins the close relationship found between these organisms based on Curie-point pyrolysis mass spectrometry (Goodfellow et al., 1998). Strain JC85 was also found to contain meso-A2pm, arabinose and galactose in whole-organism hydrolysates (wall chemotype IV sensu Lechevalier et al., 1971), N-glycolyl residues in the glycan moiety of the cell wall, unsaturated menaquinones with nine isoprene units as the predominant isoprenologue, mycolic acids and major proportions of hexadecanoic (23·3 % of total fatty acids), oleic (29·9 %) and tuberculostearic (15·2 %) acids. All of these chemical properties are consistent with the classification of strain JC85 in the genus Tsukamurella (Collins et al., 1988; Yassin et al., 1995, 1996, 1997; Kattar et al., 2001).
It is evident from Fig. 1 that strains JC85 and N1176T have identical 16S rRNA gene sequences. The mean DNA–DNA relatedness values and standard deviations found between strain N1176T and T. inchonensis N1238T, T. paurometabola JC7T, T. pulmonis N1240T, T. strandjordii N1275T, T. tyrosinosolvens N1274T and T. spumae N1173 were 35±0·6, 15±0·8, 42±0·7, 34±1·1, 35±0·7 and 46±0·7 %, respectively, values well below the 70 % cut-off point recommended by Wayne et al. (1987) for the delineation of genomic species. In contrast, strains N1176T and JC85 shared a mean DNA–DNA relatedness value of 82±2·0 % and hence are considered to belong to the same genomic species. Strain JC85 shared mean DNA–DNA relatedness values with T. inchonensis N663, T. paurometabola JC7T and T. spumae N1173 of 22±0·1, 14±0·4 and 58±1·4 %, respectively. It can be seen from Table 2 that strains JC85 and N1176T share several phenotypic properties that readily distinguish them from representatives of Tsukamurella species with validly published names.
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Description of Tsukamurella pseudospumae sp. nov.
Tsukamurella pseudospumae (pseu.do.spu'mae. Gr. adj. pseudes false; L. gen. n. spumae of foam and specific epithet of a bacterial species; N.L. n. pseudospumae the false spumae, referring to the close relationship to Tsukamurella spumae).
The description is based on data taken from this and earlier studies (Chun, 1995; Goodfellow et al., 1998; Nam et al., 2003). Aerobic, Gram-positive, partially acid–alcohol-fast, non-motile, non-spore-forming actinomycete, which forms straight to slightly curved rods and a few long filaments, but which does not differentiate into substrate or aerial hyphae. Colonies on glucose/yeast extract agar are large (<5 mm), orange to red with irregular margins and elevation. Tween 80 and uric acid are degraded but xanthine and xylan are not. Grows at 10, 25 and 37 °C, but not at 45 °C. D(+)-Galactose, D(–)-gentiobiose, D(+)-glucose, meso-inositol, D(–)-lactose, D(+)-mannose, 
-L-rhamnose, D(+)-sucrose, D(+)-trehalose, D(+)-turanose, D(–)-xylitol (1 %, w/v), amyl alcohol, butane-2,3-diol (1 %, v/v), sodium citrate and sodium pyruvate (0·1 %, w/v) are used as sole sources of carbon for energy and growth, but adonitol (1 %, w/v), butane-1,4-diol, ethanolamine, D(–)-glucuronic acid, methanol (1 %, v/v), resorcinol, sodium benzoate and sodium tartrate (0·1 %, w/v) are not. L-Asparagine, L-phenylalanine and L-serine are used as sole carbon and nitrogen sources, but L-histidine, L-lysine, succinamide and L-valine are not. Resistant to crystal violet (0·001 %, w/v), 5-fluorouracil (20 µg ml–1), bekanamycin (16, 32 and 64 µg ml–1), clindamycin (2 µg ml–1), colistin (25 µg ml–1), fusidic acid (10 µg ml–1), gentamicin sulphate (16 and 32 µg ml–1), kanamycin sulphate (4, 8, 16 and 32 µg ml–1), nalidixic acid (5 µg ml–1), neomycin sulphate (4, 8, 16 and 32 µg ml–1), novobiocin (16 µg ml–1), oleandomycin phosphate (16, 32 and 64 µg ml–1), rifampicin (0·5 and 2 µg ml–1), tetracycline hydrochloride (10 µg ml–1) and vancomycin hydrochloride (1, 2 and 4 µg ml–1), but susceptible to chlortetracycline hydrochloride (2 and 8 µg ml–1), ciprofloxacin (5 µg ml–1), erythromycin (2, 4 and 8 µg ml–1), novobiocin (64 µg ml–1), penicillin G (16, 32 and 64 µg ml–1) and rifampicin (8 and 16 µg ml–1). Other phenotypic properties are shown in Table 2
. Contains mycolic acids with 68–76 carbon atoms and up to seven double bonds; the major products from pyrolysis gas chromatography of methyl mycolates are straight-chain fatty acids C20 : 1 and C22 : 1. Additional chemical markers are typical of members of the genus Tsukamurella.
The type strain is N1176T (=DSM 44118T=NCIMB 13963T). Strains JC85 and N1176T were isolated from activated sludge foam collected from Stoke Bardolph Water Reclamation Works near Nottingham, UK.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.[CrossRef][Medline]
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