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1 Guangdong Provincial Microbial Culture Collection and Application Key Laboratory, Guangdong Institute of Microbiology, Guangzhou, Guangdong 510070, China
2 South China Agricultural University, Guangzhou, Guangdong 510642, China
3 Institute of Tropical Biology, Vietnamese Academy of Science and Technology, Ho Chi Minh City, Vietnam
4 CABI Bioscience UK Centre, Egham, Surrey, UK
Correspondence
Hong-hui Zhu
zhuhonghui66{at}yahoo.com.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces vietnamensis GIMV4.0001T is DQ311081.
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) and incubated for 5–7 days at 28 °C]. The strain produced large quantities of violet–blue diffusible pigment on Gause's synthetic agar medium.
International Streptomyces Project (ISP) media were prepared according to the methods of Shirling & Gottlieb (1966). Morphological and physiological characteristics were determined as recommended by Williams et al. (1989). Morphological observations of spores and mycelia were conducted via light microscopy (Leica DM RAR) and scanning electron microscopy (Phillip FEI-XL30). Physiological tests were carried out at 28 °C (unless otherwise indicated). All carbon sources for carbon-utilization tests were filter-sterilized. Melibiose, glucose, sorbinose, sucrose, D-fructose, xylose, D-galactose, rhamnose, arabinose and D-mannitol were tested as sole carbon source at concentrations of 0.1 % (w/v). Colour determination was referenced against Kornerup & Wanscher (1978).
Analysis of the isomer of diaminopimelic acid (DAP) and the whole-cell sugar composition followed the procedure described by Hasegawa et al. (1983) with the exception that dried cells were used instead of colonies from agar plates. Fatty acid methyl esters were prepared by using the trimethyl sulfonium hydroxide method (Butte, 1983). The base composition of the genomic DNA of strain GIMV4.0001T was determined in 0.1xSSC following the method of Mandel & Marmur (1968). Genomic DNA was extracted (Cui et al., 2001) and the 16S rRNA gene sequence was amplified by PCR by using universal bacterial 16S rRNA gene primers. The forward primer F27 (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1522R (5'-AAGGAGGTGATCCAGCCGCA-3') were adapted from primers pA and pH of Edwards et al. (1989). The 16S rRNA gene was sequenced using an automated capillary DNA sequencing system (ABI 3730) and a Bigdye Terminator cycle sequencing kit. DNA relatedness studies were conducted by using the fluorometric microdilution plate method (Ezaki et al., 1988; Sawabe et al., 1998).
Strain GIMV4.0001T grew well on yeast extract/malt extract agar (ISP2), oatmeal agar (ISP3) and Gause's synthetic agar. It exhibited moderate growth on inorganic salts/starch agar (ISP4) and glycerol–asparagine agar (ISP5), but poor growth on Czapek agar media (Atlas, 1993). Diffusible pigments of different colours were produced on the various test media (Table 1). The colour range was from bluish violet (18C8) to violet–blue (19C8).
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). Whole-cell hydrolysates contained mainly mannose and small quantities of ribose and galactose. Fatty acid analysis showed that strain GIMV4.0001T contained straight-chain, and iso- and anteiso-branched components and a high proportion of unsaturated components: iso-C14 : 0 (6.995 %), anteiso-C16 : 0 (20.1 %), C18 : 0 (5.8 %), C16 : 1 (17.8 %), C18 : 1 (33.5 %) and C18 : 2 (15.8 %). The G+C content of the genomic DNA was 73.9 mol%. Melanin was produced on tyrosine agar (ISP7).
A 1419-bp 16S rRNA gene sequence was determined for strain GIMV4.0001T. A BLAST search (Altschul et al., 1997) of the GenBank database using this sequence showed its similarity to that of many species of the genus Streptomyces. The 16S rRNA gene sequence of strain GIMV4.0001T showed levels of similarity of 99.4 % (over 1410 bases) to that of Streptomyces bikiniensis ATCC 11062T (GenBank accession no. X79851), 98.9 % (over 1404 bases) to that of Streptomyces showdoensis ATCC 15105T (GenBank accession no. AY999741), 99.1 % (over 1406 bases) to that of Streptomyces viridobrunneus ATCC 43698T (GenBank accession no. AJ781372) and <98 % to that of other Streptomyces species.
A phylogenetic tree based on 16S rRNA gene sequences of members of the genus Streptomyces was constructed according to the neighbour-joining method of Saitou & Nei (1987) with CLUSTAL W (version 1.81) and MEGA (version 3.1; Kumar et al., 2001) (Fig. 2). For the neighbour-joining analysis, a distance matrix was calculated according to Kimura's two-parameter correction model. This tree shows the close phylogenetic association of strain GIMV4.0001T with certain other Streptomyces species.
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Strain GIMV4.0001T produced reddish-brown substrate mycelium and grey spore mass, and developed cylindrical spores in flexuous chains. Melanin was produced. Bluish-violet or violet–blue diffusible pigments were produced. The pigment was pH-sensitive, and was stable at high temperature and under UV light. Comparison of the cultural characteristics of strain GIMV4.0001T and its closest phylogenetic neighbours (Table 2), however, revealed significant differences from those Streptomyces species showing >99 % 16S rRNA gene sequence similarity.
S. bikiniensis differed from strain GIMV4.0001T in that it did not produce a diffusible pigment on ISP media; S. showdoensis did not produce diffusible pigment on ISP media and S. viridobrunneus produced a green pigment. Despite the high 16S rRNA gene sequence similarity between strain GIMV4.0001T and S. bikiniensis, they could be differentiated based on morphological and cultural characteristics (Table 2), carbon-utilization patterns, fatty acid methyl esters and antibiotic resistance properties (Table 3), indicating that strain GIMV4.0001T does not belong to S. bikiniensis. DNA–DNA hybridization studies confirmed that strain GIMV4.0001T is unique. The level of DNA–DNA relatedness between strain GIMV4.0001T and S. bikiniensis ATCC 11062T was 50.3 %.
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, b, 1969); Streptomyces exfoliatus differed in producing pink aerial mycelium but no substrate mycelial pigment (Shirling & Gottlieb, 1968a); Streptomyces rubiginosohelvolus produced either a light-yellow diffusible pigment and light-yellow aerial mycelium or no pigment at all (Shirling & Gottlieb, 1968b); Streptomyces crystallinus produced a light-brown or dark-brown diffusible pigment (Williams et al., 1989). These results support the classification of strain GIMV4.0001T as representing a novel species of the genus Streptomyces, for which we propose the name Streptomyces vietnamensis sp. nov. Additional data from the phenotypic characterization of strain GIMV4.0001T are presented below.
Description of Streptomyces vietnamensis sp. nov.
Streptomyces vietnamensis (vi.et.nam.en'sis. N.L. masc. adj. vietnamensis pertaining to Vietnam, the geographical location from where the type strain was isolated).
Aerobic, Gram-positive, catalase-positive and forms a white aerial mycelium and a reddish-brown substrate mycelium. Verticils are not present. The mycelium does not fragment. Straight to flexuous chains of cylindrical spores are produced. Diffusible pigments are produced on ISP2, ISP3, ISP4 and ISP5 media and on Gause's synthetic agar, but not on Czapek solution agar. Melanin is produced on ISP7. Although growth on ISP4 is initially slow, very good growth with profuse sporulation is observed on this medium after 14 days. Very good growth occurs on ISP2, Gause's synthetic agar and ISP3. Moderate growth is observed on ISP5 but only poor growth on Czapek agar. The substrate mycelium is reddish brown on ISP2, ISP5, Gause's synthetic agar, ISP4 and ISP3, but greyish orange on Czapek medium. Cell wall contains LL-DAP (cell-wall type I). Whole-cell sugar pattern contains diagnostic sugars: mannose, small quantities of ribose and galactose. No antibiosis is exhibited against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 6538, Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231 or Penicillium citrinum AS3.2788. Utilizes melibiose, glucose, sorbinose, sucrose, D-fructose, xylose, D-galactose, rhamnose and arabinose. Positive for production of H2S, but pectin is not hydrolysed. The DNA G+C content of the type strain is 73.9 mol%.
The type strain, GIMV4.0001T (=CCTCC M 205143T=IAM 15340T), was isolated from a forest soil sample in Vietnam
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by L. C. Parks. Boca Raton, FL: CRC Press.
Butte, W. (1983). Rapid method for the determination of fatty acid profiles from fats and oils using trimethylsulphonium hydroxide for transesterification. J Chromatogr 261, 142–145.[CrossRef]
Cui, X. L., Mao, P. H., Zeng, M., Li, W. J., Zhang, L. P., Xu, L. H. & Jiang, C. L. (2001). Streptimonospora salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae. Int J Syst Evol Microbiol 51, 357–363.[Abstract]
Edwards, U., Rogall, T., Blocker, H., Emde, M. & Bottger, E. C. (1989). Isolation and direct complete nucleotide determination of entire genes, characterization of gene coding for 16S ribosomal RNA. Nucleic Acids Res 17, 7843–7853.
Ezaki, T., Hashimoto, Y., Takeuchi, N., Yamamoto, H., Liu, S.-L., Miura, H., Matsui, K. & Yabuuchi, E. (1988). Simple genetic method to identify viridans group streptococci by colorimetric dot hybridization and fluorometric hybridization in microdilution wells. J Clin Microbiol 26, 1708–1713.
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
Kornerup, A. & Wanscher, J. H. (1978). Methuen Handbook of Colour. London: Eyre Methuen.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis software. Tempe, AZ: Arizona State University http://www.megasoftware.net
Lechevalier, M. P. & Lechevalier, H. A. (1970). A critical evaluation of the genera of aerobic actinomycetes. In The Actinomycetales, pp. 393–405. Edited by H. Prauser. Jena: Gustav Fischer.
Mandel, M. & Marmur, J. (1968). Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol 12B, 195–206.[CrossRef]
Preobrazhenskaya, T. P., Sveshnikova, M. A., Terekhova, L. P. & Maximova, T. S. (1983). A Guide for the Determination of Actinomycetes. Genera Streptomyces, Streptoverticillium, and Chainia. Moscow: Nauka.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sawabe, T., Makino, H., Tatsumi, M., Nakano, K., Tajima, K., Iqbal, M. M., Yumoto, L., Ezura, Y. & Christen, R. (1998). Pseudomoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica. Int J Syst Bacteriol 48, 769–774.
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[Medline]
Shirling, E. B. & Gottlieb, D. (1968a). Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int J Syst Bacteriol 18, 69–189.
Shirling, E. B. & Gottlieb, D. (1968b). Cooperative description of type cultures of Streptomyces. III. Additional species descriptions from first and second studies. Int J Syst Bacteriol 18, 279–392.
Shirling, E. B. & Gottlieb, D. (1969). Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int J Syst Bacteriol 19, 391–512.
Shirling, E. B. & Gottlieb, D. (1972). Cooperative description of type strains of Streptomyces. V. Additional descriptions. Int J Syst Bacteriol 22, 265–394.
Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743–1813.
Williams, S. T., Goodfellow, M. & Alderson, G. (1989). Genus Streptomyces Waksman and Henrici 1943, 339AL. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2452–2492. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
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