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Int J Syst Evol Microbiol 55 (2005), 433-436; DOI  10.1099/ijs.0.63189-0
© 2005 International Union of Microbiological Societies

Nocardia takedensis sp. nov., isolated from moat sediment and scumming activated sludge

Hideki Yamamura1, Masayuki Hayakawa1, Youji Nakagawa1, Tomohiko Tamura2, Tetsuro Kohno3, Fumio Komatsu4 and Yuzuru Iimura1

1 Division of Applied Biological Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu 400-8511, Japan
2 NITE Biological Resource Center, Department of Biotechnology, National Institute of Technology and Evaluation, Kazusakamatari 2-5-8, Kisarazu 292-0818, Japan
3 Department of Civil and Environmental Engineering, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu 400-8511, Japan
4 Water Quality Control Laboratory, Nihon Hels Industry Corporation, Higashigoken-cho 3-25, Shinjuku-ku, Tokyo 162-0813, Japan

Correspondence
Masayuki Hayakawa
hayakawa{at}ab11.yamanashi.ac.jp


   ABSTRACT
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Chemotaxonomic and morphological characterization of two actinomycete strains, MS1-3T and AS4-2, respectively isolated from moat sediment and scumming activated sludge, was carried out. This characterization clearly demonstrated that strains MS1-3T and AS4-2 belong to the genus Nocardia. 16S rRNA gene sequencing studies showed that these isolates are most closely related to Nocardia beijingensis (98·1–98·3 % similarity), Nocardia brasiliensis (97·9–98·0 %) and Nocardia tenerifensis (97·8–97·9 %). However, the results of DNA–DNA hybridizations and physiological and biochemical tests showed that strains MS1-3T and AS4-2 could be differentiated from their closest phylogenetic relatives both genotypically and phenotypically. It is proposed that the two isolates be classified as representatives of a novel species of Nocardia, Nocardia takedensis sp. nov. The type strain is MS1-3T (=NBRC 100417T=DSM 44801T); AS4-2 (=NBRC 100418=DSM 44802) is a reference strain.

Abbreviations: A2pm, diaminopimelic acid

Published online ahead of print on 10 September 2004 as DOI 10.1099/ijs.0.63189-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Nocardia takedensis MS1-3T and AS4-2 are AB158277 and AB158278.

SEMs of cells of strain MS1-3T and an extended phylogenetic tree derived from 16S rRNA gene sequences are available as supplementary material in IJSEM Online.


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The genus Nocardia Trevisan 1889 belongs to the suborder Corynebacterineae (Stackebrandt et al., 1997Go) and currently encompasses about 40 species of mycolic-acid-containing actinomycetes, including the recently described species Nocardia puris, Nocardia inohanensis, Nocardia yamanashiensis, Nocardia tenerifensis and Nocardia neocaledoniensis (Yassin et al., 2003Go; Kageyama et al., 2004Go; Kämpfer et al., 2004Go; Saintpierre-Bonaccio et al., 2004Go).

The Nocardia selective isolation method utilizing sucrose density-gradient centrifugation was developed previously (Yamamura et al., 2003aGo). Using this technique, two Nocardia strains, MS1-3T and AS4-2, were isolated. MS1-3T was from a sediment sample taken from the moat surrounding Takeda Shrine in Yamanashi Prefecture, Japan, and AS4-2 was from a scumming activated sludge obtained from a sewage treatment plant in Saitama Prefecture, Japan (Yamamura et al., 2003bGo, 2004Go). These isolates could be differentiated from all known species of Nocardia in terms of their 16S rRNA gene restriction fragment length polymorphism (RFLP) patterns (Yamamura et al., 2003bGo). The aim of present study was to determine the taxonomic position of isolates MS1-3T and AS4-2 using a polyphasic approach.

Colony morphology and micromorphological properties of strains MS1-3T and AS4-2 were observed by light and scanning electron microscopy according to a previously described procedure (Hayakawa et al., 1996Go). Physiological and biochemical properties were recorded according to well-established procedures (Gordon et al., 1974Go; Isik et al., 1999Go). Diaminopimelic acid (A2pm) isomers and sugars in whole-cell hydrolysates were analysed based on the methods established by Hasegawa et al. (1983)Go and Schaal (1985)Go, respectively. Standard procedures were also used for extraction and analysis of mycolic acids (Schaal, 1985Go), fatty acids (Tamura et al., 1994Go) and isoprenoid quinones and polar lipids (Minnikin et al., 1984Go), and compared to the appropriate controls. Genomic DNA was prepared as described by Torres et al. (1996)Go. The G+C contents of DNA from the isolates were determined by HPLC, as described by Tamura et al. (1994)Go. DNA–DNA hybridization was carried out as described by Kusunoki et al. (1991)Go using biotinylated DNA. 16S rRNA genes were amplified by PCR and sequenced following procedures described previously (Yamamura et al., 2003aGo). The 16S rRNA gene sequences obtained in the present study were manually aligned with sequences of available from EMBL/GenBank/DDBJ. A phylogenetic tree was inferred using neighbour-joining tree-making algorithms (Saitou & Nei, 1987Go). The program CLUSTAL W (Thompson et al., 1994Go) was used to calculate evolutionary distances and similarity values. Topography of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein, 1985Go).

Chemotaxonomic and morphological characteristics of isolates MS1-3T and AS4-2 were consistent with their assignment to the genus Nocardia (Goodfellow, 1998Go; Goodfellow et al., 1999Go). Whole-cell hydrolysates of the test strains were rich in meso-A2pm, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970Go). The predominant menaquinone component was MK-8(H4, {omega}-cycl.). They also contained phosphatidylethanolamine (phospholipid type PII sensu Lechevalier et al., 1977Go). In addition, TLC revealed that the strains contained methyl mycolates, which are equivalent in mobility to nocardomycolic acid. C16 : 0, cis-9 C18 : 1 and 10-methyl C16 : 0 were present as major cellular fatty acids. Microscopic observation confirmed the formation of branched substrate hyphae, fragmenting into rod-shaped elements (Goodfellow & Lechevalier, 1989Go), and relatively short aerial hyphae with chains of arthrospores (see Supplementary Fig. A in IJSEM Online).

Almost complete 16S rRNA gene sequences (1345 nt) of isolates MS1-3T and AS4-2 were compared with sequences of recognized species of Nocardia. The 16S rRNA gene sequences of the test strains and those representative of the genus Nocardia had 95·3–98·3 % similarity. Highest sequence similarities were shown with Nocardia beijingensis (98·1–98·3 %), Nocardia brasiliensis (97·9–98·0 %) and N. tenerifensis (97·8–97·9 %). The 16S rRNA gene-based tree, constructed using the neighbour-joining method, showed that the test strains formed a monophyletic clade that is loosely associated with N. brasiliensis, N. tenerifensis and Nocardia transvalensis (Fig. 1Go). The taxonomic integrity of the test strains was supported by DNA relatedness data. Strain MS1-3T showed DNA relatedness values of 8·1–23·1 % to N. beijingensis, N. brasiliensis and N. tenerifensis, results that were well below the 70 % cut-off point recommended for assignment of bacterial strains to the same genomic species (Wayne et al., 1987Go). On the other hand, the 16S rRNA gene sequence similarity between MS1-3T and AS4-2 was 99·8 % and the DNA relatedness value was 91·0 %. The phenotypic properties of these two strains also clearly distinguished them from representatives of N. beijingensis, N. brasiliensis, N. tenerifensis and N. transvalensis (Table 1Go).



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  		Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences showing the relationship of strains MS1-3T and AS4-2 to the nearest Nocardia species. The tree was constructed using the neighbour-joining method and Knuc values (Saitou & Nei, 1987Go). Only values above 50 % significance are indicated. A tree based on available 16S rRNA gene sequences of all Nocardia species is available as Supplementary Fig. B in IJSEM Online.

 

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  		Table 1. Phenotypic properties that differentiate strains MS1-3T and AS4-2 from representatives of phylogenetically related Nocardia species

Strains: 1, MS1-3T and AS4-2; 2, N. beijingensis IFO (now NBRC) 16342T; 3, N. brasiliensis ATCC 19296T; 4, N. tenerifensis DSM 44704T; 5, N. transvalensis DSM 43405T. ND, Not determined; +, positive; –, negative; W, weak reaction. Data for reference strains were taken from Goodfellow & Lechevalier (1989)Go, Wang et al. (2001)Go, Yassin et al. (2003)Go and Kämpfer et al. (2004)Go.

 
Based on 16S rRNA gene sequence data, DNA–DNA hybridization studies and biochemical properties, as well as on RFLP patterns (Yamamura et al., 2003bGo), it is proposed that strains MS1-3T and AS4-2 represent a novel species in the genus Nocardia. The name Nocardia takedensis sp. nov. is proposed for these two strains.

Description of Nocardia takedensis sp. nov.
Nocardia takedensis (ta.ke.den'sis. N.L. fem. adj. takedensis pertaining to the Takeda Shrine, from where the organism was first isolated).

Aerobic, Gram-positive, non-motile actinomycete. Forms an orange substrate and white aerial mycelium, which fragments into rod-shaped elements. Diffusible pigments are not produced. Aesculin and urea are hydrolysed and nitrate is reduced. Does not degrade adenine, casein, elastin, hypoxanthine, tyrosine or xanthine, but does degrade uric acid. Grows well at 30 °C, but does not grow at 45 °C. Assimilates 2,3-butanediol, D-galactose, D-glucose, D-mannose, sucrose (all at 1 %, w/v) and acetate (at 0·1 %, w/v) as sole carbon sources, but not adonitol, L-arabinose, cellobiose, D-fructose, meso-erythritol, meso-inositol, isoamyl alcohol, D-mannitol, D-melezitose, D-melibiose, 1,2-propanediol, raffinose, L-rhamnose, D-sorbitol, D-trehalose, D-xylose (at 1 %, w/v), adipic acid, citrate, D-gluconate, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pimelic acid or sebacic acid (at 0·1 %, w/v). The major cellular fatty acids are C16 : 0 (45·0 %), cis-9 C18 : 1 (20·4 %) and 10-methyl C18 : 0 (10·4 %). The G+C content of the DNA is 68·6 mol%.

The type strain is MS1-3T (=NBRC 100417T=DSM 44801T), isolated from a sediment sample taken from the moat surrounding Takeda Shrine in Yamanashi Prefecture, Japan.


   ACKNOWLEDGEMENTS
 
We thank Professor Dr Hans G. Trüper, Institut für Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelm-Universität Bonn, for his helpful suggestions regarding the bacterial nomenclature. Appreciation is also given to Miss Y. Ishikawa for technical assistance.


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Goodfellow, M. & Lechevalier, M. P. (1989). Genus Nocardia Trevisan 1889. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2350–2361. Edited by S. T. Williams, M. E. Sharpe & J. P. Holt. Baltimore: Williams & Wilkins.

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