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1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
2 Thailand Institute of Scientific and Technological Research (TISTR), 35 Moo 3, Technopolis, Khlong 5, Khlong Luang, Pathum Thani 12120, Thailand
3 Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand
Correspondence
Shoichi Hosoya
shouichi.hosoya{at}mbio.jp
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ABSTRACT |
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6c and 16 : 0. On the basis of the data from DNA–DNA hybridization, physiological and chemotaxonomic analyses and 16S rRNA gene sequence comparisons, strain 59SAT represents a novel species of the genus Aureispira, for which the name Aureispira maritima sp. nov. is proposed. The type strain is 59SAT (=IAM 15439T=TISTR 1726T).
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), Plesiocystis pacifica (Iizuka et al., 2003), Krokinobacter eikastus, Krokinobacter diaphorus (Khan et al., 2006), Flammeovirga aprica, Flammeovirga arenaria and Flammeovirga yaeyamensis (Takahashi et al., 2006). The highest content of arachidonic acid amongst these was found in F. aprica NBRC 15941T (24.2 % of total fatty acids). Recently, the arachidonic acid-producing marine genus Aureispira was proposed by Hosoya et al. (2006). This genus has the unusual property of containing a large amount of arachidonic acid (about 40 %). At present, however, this genus consists of only one species, Aureispira marina. In this study, we have characterized a novel arachidonic acid-containing bacterium, designated strain 59SAT, isolated from marine barnacle debris from the southern coastline of Thailand. Our results suggest that strain 59SAT should be classified as a novel species within the genus Aureispira.
The sample was collected from marine barnacle debris in Krabi Province, Thailand. Isolation was carried out according to methods described by Hosoya et al. (2006) and Sangkhobol & Skerman (1981). Purification of the gliding bacteria was performed by using the Skerman micromanipulation technique (Skerman, 1968) as described in Sly & Arunpairojana (1987). Strain 59SAT was then cultured and maintained at 30 °C on trypticase soy agar (BBL), supplemented with 0.5x artificial seawater (1x artificial seawater consists of 3 % NaCl, 0.07 % KCl, 1.08 % MgCl2 . 6H2O, 0.54 % MgSO4 . 7H2O and 0.1 % CaCl2 . 2H2O).
The 16S rRNA gene sequence was obtained by direct sequencing of PCR amplicons as described by Hosoya et al. (2006). Sequences were edited and assembled using the BioEdit program (Hall, 1999) and were compared with sequences by using BLAST to search the GenBank database (Altschul et al., 1990). Nucleotide substitution rates (Knuc; Kimura, 1980) were determined and a distance matrix tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the CLUSTAL_X program (version 1.83; Thompson et al., 1997). Alignment gaps and unidentified base positions were not taken into consideration in the calculation. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 replicate trials. The results of the phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate fell within the genus Aureispira in the family ‘Saprospiraceae’ (Fig. 1). Strain 59SAT showed 94 % sequence similarity with respect to A. marina 24T. To analyse genetic relatedness, DNA–DNA hybridization was carried out at 40 °C and measured fluorometrically using the method of Ezaki et al. (1989). The DNA–DNA relatedness between strain 59SAT and A. marina 24T was less than 5 %, which is significantly lower than the threshold value accepted for the definition of a species (Wayne et al., 1987).
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and was investigated using the method of Mesbah et al. (1989). The DNA G+C contents of strain 59SAT and A. marina 24T were 38.7 and 39.4 mol%, respectively. Flexirubin pigments were investigated using the bathochromatic shift test with 20 % (w/v) KOH, as described by McCammon & Bowman (2000). No flexirubin pigments were detected in strain 59SAT or in A. marina 24T.
The following physiological features were investigated: respiratory quinones (as described by Komagata & Suzuki, 1987); growth at different temperatures (8–37 °C), salt tolerance, growth at different pH values, oxidase activity, catalase activity, degradation of DNA and alginate, hydrolysis of agar and carboxymethylcellulose (Hosoya et al., 2006); degradation of casein and starch, acid production from carbon sources (Smibert & Krieg, 1994) and degradation of Tweens 20, 40, 60 and 80 and L-tyrosine (Barrow & Feltham, 1993). Tests with the commercial API ZYM and API 20E systems (bioMérieux) were generally performed according to the manufacturer's instructions. The API ZYM tests were read after 4 h incubation at 30 °C and the API 20E tests were read after 48 h at 30 °C. Cell movement at colony edges was verified by using phase-contrast microscopy. For analysis of the cellular fatty acids, cells were grown for 48 h at 30 °C on trypticase soy agar supplemented with 0.5x artificial seawater and analysed by using the GC-based Microbial Identification System (MIDI).
Strain 59SAT was found to be a Gram-negative, aerobic, non-sporulating, non-fruiting, gliding bacterium. The isolate formed yellow colonies and the cells were 0.7–0.8x3–6 µm in size. Helical cells were formed that were 1.5–2.0 µm wide and 20–90 µm long, with a twist occurring every 4–5 µm (Fig. 2). Phenotypic characteristics are given in the species description and are shown in Table 1. The phenotypic differences between strain 59SAT and A. marina 24T related to features including esterase, valine arylamidase, cystine arylamidase and trypsin activities and degradation of carboxymethylcellulose and DNA. The major cellular fatty acids in strain 59SAT were 20 : 46c (arachidonic acid) and 16 : 0 (Table 2).
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Although bacteria belonging to the genus Aureispira were originally described as being positive for cytochrome oxidase and catalase activities, A. marina 24T and strain 59SAT were found to be variable for these properties. For this reason, an emended description of the genus Aureispira is also provided.
Emended description of the genus Aureispira Hosoya et al. 2006
The description is as given by Hosoya et al. (2006), with the following changes. Cytochrome oxidase and catalase activities are variable. The type species is Aureispira marina.
Description of Aureispira maritima sp. nov.
Aureispira maritima (ma.ri'ti.ma. L. fem. adj. maritima inhabiting marine environments).
Cells are 0.7–0.8x3–6 µm. Helical cells form that are 1.5–2.0 µm wide and 20–90 µm long, with a twist occurring every 4–5 µm. The optimal growth temperature is 30 °C; no growth occurs at 17 or 37 °C. The pH range for growth is 6.0–8.0. Growth occurs at seawater concentrations of 20–150 % (w/v). Flexirubin pigments are not detected. Positive for activities of esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin and naphthol-AS-BI-phosphohydrolase and for the degradation of casein, carboxymethylcellulose, DNA, gelatin and Tweens 20, 40, 60 and 80. Tyrosine is degraded. Agar, alginate, citrate and starch are not decomposed. Nitrate is not reduced. Acetoin, H2S and indole are not produced. Negative in tests for esterase (C4), lipase (C4), chymotrypsin, 
-galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosamidase, -mannosidase and -fucosidase. Does not produce acid from arabinose, cellobiose, dulcitol, fructose, galactose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, raffinose, rhamnose, sorbitol, sucrose, trehalose or xylose. The DNA G+C content is 38.7 mol%.
The type strain, 59SAT (=IAM 15439T=TISTR 1726T), was isolated from marine barnacle debris in Krabi Province, Thailand.
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REFERENCES |
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Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press.
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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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Hosoya, S., Arunpairojana, V., Suwannachart, C., Kanjana-Opas, A. & Yokota, A. (2006). Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand. Int J Syst Evol Microbiol 56, 2931–2935.
Iizuka, T., Jojima, Y., Fudou, R., Hiraishi, A., Ahn, J.-W. & Yamanaka, S. (2003). Plesiocystis pacifica gen. nov., sp. nov., a marine myxobacterium that contains dehydrogenated menaquinone, isolated from the Pacific coasts of Japan. Int J Syst Evol Microbiol 53, 189–195.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
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Takahashi, M., Suzuki, K.-i. & Nakagawa, Y. (2006). Emendation of the genus Flammeovirga and Flammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. and Flammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol 56, 2095–2100.
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Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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