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Department of Molecular and Cell Biology, University of Cape Town, Private Bag 1, Rondebosch, 7701 Cape Town, South Africa
Correspondence
Paul R. Meyers
pmeyers{at}science.uct.ac.za
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ABSTRACT |
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The GenBank accession number for the 16S rRNA gene sequence of Streptomyces speibonae DSM 41797T (=ATCC BAA-411T) is AF452714.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Streptomycetes may represent a considerable proportion of the actinomycete communities in soils (Elander, 1987
). Despite the fact that streptomycetes have been studied extensively for many decades and many species have been described, from time to time, a novel streptomycete is isolated.
Strain PK-BlueT was isolated by J. M. Pule and T. Kwetane on the Upper Campus of the University of Cape Town (South Africa) in August 2000, as part of their 3rd year BSc project on actinomycetes.
Morphological and physiological characteristics were determined as recommended by Williams et al. (1989), with the exception that antibiotic resistance was determined by incorporation of the antibiotics into Bennett's medium agar plates (Atlas, 1993) at the concentrations recommended instead of using antibiotic-impregnated filter discs. Non-standard antibiotics were tested at the following concentrations: capreomycin (20 µg ml-1), cefotaxime (100 µg ml-1), D-cycloserine (50 µg ml-1), kanamycin (10 µg ml-1) and viomycin (8 µg ml-1). Antimicrobial activity against Enterococcus faecium, Escherichia coli and Pseudomonas aeruginosa was determined using 5-day-old colonies of strain PK-BlueT grown on nutrient agar (Williams et al., 1989).
International Streptomyces Project (ISP) media were prepared according to the methods of Shirling & Gottlieb (1966). Tests for physiological characteristics were carried out at 28 °C (unless otherwise indicated) and results were read after the recommended incubation periods. All carbon sources for carbon-utilization tests were filter-sterilized. meso-Erythritol, glycerol, maltose, methyl 
-D-glucoside, D(-)-ribose and L(-)-sorbose were tested as sole carbon sources at concentrations of 1 % (w/v). Sodium benzoate, sodium butyrate, sodium formate, sodium DL-malate, sodium maleate, sodium oxalate, sodium salicylate, sodium succinate and sodium L(+)-tartrate were tested as sole carbon sources at 0·1 % (w/v). DL-Citrulline, DL-ornithine and 4-amino-n-butyric acid were tested as sole nitrogen sources at 0·1 % (w/v).
Determination of the isomer of diaminopimelic acid (DAP) and the whole-cell sugar pattern were carried out as 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 the trimethylsulphonium hydroxide method (Butte, 1983). The base composition of genomic DNA of strain PK-BlueT was determined in 0·1x SSC by the method of Mandel & Marmur (1968).
A 16S rRNA gene sequence of strain PK-BlueT was amplified by PCR using universal bacterial 16S rDNA primers (forward primer adapted from primer fD1 of Weisburg et al., 1991; reverse primer adapted from primer p1525r of Chun & Goodfellow, 1995). The 16S rDNA was sequenced using an ALFexpress DNA automated sequencer (Applied Biosystems) and an Amersham Pharmacia Biotech Cy5 Thermo Sequenase dye terminator kit.
Strain PK-BlueT was Gram-positive by Gram stain, did not grow under anaerobic conditions and gave a positive reaction in the catalase test. Light microscopy showed a branched mycelium without verticils. Scanning electron microscopy revealed Spirales-type spore chains with hairy spore sheaths (Fig. 1). Chemotaxonomic tests showed that the cell wall contained LL-DAP, indicating that it has cell-wall type I; no diagnostic sugars were detected in hydrolysates of whole cells. Fatty acid analysis showed that strain PK-BlueT contained a high proportion of saturated straight-chain and iso- and anteiso-branched fatty acids: iso-15 : 0 (12·0 %), iso-16 : 0 (35·9 %), 16 : 0 (14·9 %) and anteiso-17 : 0 (9·4 %). The G+C content of the genomic DNA was 73·4 mol%.
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) against the GenBank database using this sequence showed that it was most similar to the 16S rDNA sequences of ‘Nocardioides thermolilacinus’ strains IFO 14336 and IFO 14335 (both 98 % similar over 1471 nt) and many species of Streptomyces. Although it was surprising that the highest similarity of the PK-BlueT 16S rDNA was to ‘N. thermolilacinus’ (strain PK-BlueT does not have a Nocardioides-type life cycle), these organisms are considered to have been misidentified and are believed to be streptomycetes (Prauser, 1989).
A phylogenetic tree of Streptomyces 16S rDNA sequences was constructed by the neighbour-joining method of Saitou & Nei (1987) using CLUSTAL W (version 1.81) and MEGA (version 2.1; Kumar et al., 2001) (Fig. 2).
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Strain PK-BlueT differs from other streptomycetes that produce a blue substrate mycelium in several respects. Some strains of Streptomyces griseoflavus produce a blue substrate mycelium (Williams et al., 1989), but this species differs from PK-BlueT in producing Retinaculiaperti spore chains and a green or yellow spore mass and by its ability to hydrolyse pectin and degrade adenine. Most strains of S. griseoflavus do not produce melanin. S. griseoflavus does not degrade xanthine, is sensitive to vancomycin and does not grow at 45 °C or in the presence of 10 % NaCl. PK-BlueT grows on L-valine as a sole nitrogen source and on sodium malonate as a sole carbon source. S. griseoflavus does not use either of these.
There are no full-length or almost-complete 16S rDNA sequences for S. griseoflavus (or any of its subjective synonyms: Streptomyces cyanoalbus, Streptomyces hirsutus, Streptomyces pilosus, Streptomyces prasinopilosus and Streptomyces prasinus) in the GenBank database. However, 119–121 bp sequences, from the variable region of the 16S rRNA molecule, have been determined for each of these species (Kataoka et al., 1997). These single sequences correspond to nucleotide positions 158–277 of the Streptomyces ambofaciens 16S rRNA sequence of the rrnD rRNA gene cluster (Pernodet et al., 1989). The similarity of these sequences to the PK-BlueT 16S rDNA sequence was determined by pairwise alignment using the DNAMAN software (Lynnon BioSoft; version 4.13). The range of similarity is 83·2 % (S. prasinopilosus JCM 4404T; accession no. D44119) to 96·7 % (S. griseoflavus JCM 4479T; D44174). These differences eliminate the possibility that PK-BlueT is a member of the S. griseoflavus species group.
Streptomyces anthocyanicus, Streptomyces caeruleus, Streptomyces tricolor and Streptomyces violaceoruber produce blue substrate mycelia (Williams et al., 1989), but differ from PK-BlueT in producing smooth spores and in the production of diffusible pigments. Strain PK-BlueT produces Spirales-type spore chains, whereas S. anthocyanicus has Retinaculiaperti-type spore chains and S. caeruleus has Rectiflexibiles-type spore chains. S. anthocyanicus, S. caeruleus and S. violaceoruber do not produce melanin. Strain PK-BlueT uses, as sole carbon sources, L(+)-arabinose, L(+)-rhamnose, D(+)-xylose (S. caeruleus does not use any of these) and D-mannitol (not used by S. anthocyanicus). Strain PK-BlueT does not use salicin (S. violaceoruber is able to use this glucoside as a sole carbon source).
The single available 121 bp sequences for S. anthocyanicus JCM 5058T, S. tricolor JCM 5065T and S. violaceoruber JCM 4423T, from the variable region (Kataoka et al., 1997), all show 89·3 % similarity to the PK-BlueT 16S rDNA sequence by pairwise alignment (accession nos D44427, D44434 and D44135, respectively). A 451 bp 16S rDNA sequence of S. violaceoruber (AF434717) shows only 35·8 % similarity to the PK-BlueT 16S rDNA sequence by pairwise alignment. These differences support the other evidence that strain PK-BlueT is not a strain of S. anthocyanicus, S. caeruleus, S. tricolor or S. violaceoruber.
Strain PK-BlueT also differs from other streptomycetes that produce hairy spores. Streptomyces acrimycini produces a green spore mass and no melanin (Williams et al., 1989). Streptomyces bambergiensis has Retinaculiaperti-type spore chains and a green spore mass. It produces a pH-sensitive red–orange substrate mycelium and pH-sensitive red–orange diffusible pigments, but no melanin. This species is also sensitive to lincomycin (100 µg ml-1) (Williams et al., 1989).
Streptomyces capillispiralis differs from strain PK-BlueT in producing a light brownish-grey spore mass and yellow–brown to brownish-black substrate mycelium. A brown diffusible pigment may be produced, but melanin is not produced. This species produces urease, but does not degrade starch or reduce nitrate (Williams et al., 1989).
Streptomyces chromofuscus produces a yellow–brown substrate mycelium and is sensitive to oleandomycin (100 µg ml-1). Most strains produce smooth spores. Streptomyces flaveolus and Streptomyces pactum produce yellow–brown substrate mycelia and yellow–brown diffusible pigments, but no melanin. S. pactum is also unable to use D(+)-xylose as a sole carbon source (Williams et al., 1989).
Streptomyces finlayi differs from PK-BlueT in producing Rectiflexibiles-type spore chains, a green substrate mycelium and no melanin. This species is sensitive to lincomycin (100 µg ml-1) (Williams et al., 1989).
Streptomyces geysiriensis differs from PK-BlueT in not producing melanin. Streptomyces glaucescens produces a blue spore mass, a red–orange substrate mycelium and red–orange diffusible pigments. This species also lacks proteolytic activity (Williams et al., 1989).
Streptomyces griseostramineus produces a green spore mass and greyish-yellow or greyish-yellow–green substrate mycelium. Streptomyces prasinosporus produces a green spore mass, yellow–brown substrate mycelium and no melanin on tyrosine agar. It is also unable to degrade tyrosine or to use L-arginine as a sole nitrogen source (Williams et al., 1989).
Streptomyces viridoviolaceus differs from strain PK-BlueT in producing a light brownish-grey to greyish-yellow–brown spore mass, reddish- or yellowish-brown substrate mycelium and orange or red, pH-sensitive diffusible pigments. Melanin pigment is not produced (Williams et al., 1989).
These results support the classification of strain PK-BlueT as a novel species of Streptomyces, for which we propose the name Streptomyces speibonae sp. nov. Additional data from the phenotypic characterization of the strain are presented below
Description of Streptomyces speibonae sp. nov.
Streptomyces speibonae (spei.bo'nae. L. n. spes -ei hope; L. adj. bonus good; N.L. masc. adj. speibonae of good hope, to indicate Cape Town, the Cape of Good Hope, South Africa, the geographical location from which the type strain was isolated).
Aerobic, Gram-positive, catalase-positive actinomycete that forms a grey aerial mycelium and a blue substrate mycelium. The colour of the substrate mycelium is not pH sensitive. Verticils are not present. The mycelium does not fragment. Spirales-type spore chains with hairy spore sheaths are produced. No diffusible pigments are produced on glycerol-asparagine agar (ISP medium no. 5) or on any other medium. Melanin pigment is produced on both peptone-yeast extract-iron agar (ISP medium no. 6) and tyrosine agar (ISP medium no. 7). Although growth on inorganic salts-starch agar (ISP medium no. 4) is initially slow, very good growth with profuse sporulation is observed on this medium after 14 days. Very good growth occurs on yeast extract-malt extract agar (ISP medium no. 2). Good growth is observed on oatmeal agar (ISP medium no. 3) and moderate growth on Czapek solution agar (Atlas, 1993). Growth on glycerol-asparagine agar is poor. The substrate mycelium is blue on yeast extract-malt extract agar and oatmeal agar, but light grey on the Czapek and glycerol-asparagine media. The cell wall contains LL-DAP (cell wall type I). The whole-cell sugar pattern contains no diagnostic sugars. No antibiosis is exhibited against Enterococcus faecium (clinical isolate), Escherichia coli ATCC 25922 or Pseudomonas aeruginosa ATCC 27853. Grows in the presence of (µg ml-1 unless stated otherwise): cefotaxime (100), cephaloridine (100), D-cycloserine (50), lincomycin (100), oleandomycin (100), penicillin G (10 IU ml-1), phenol (0·1 %), 2-phenylethanol (0·1 %), sodium chloride (10 %) and vancomycin (50) and at 45 °C but not at 4 °C, pH 4·3 or in the presence of sodium azide (0·01 %), capreomycin (20), gentamicin (100), kanamycin (10), neomycin (50), rifampicin (50), streptomycin (100), tobramycin (50) or viomycin (8). Uses DL--amino-n-butyric acid, 4-amino-n-butyric acid, L-arginine, DL-citrulline, L-cysteine, L-histidine, L-methionine, DL-ornithine, potassium nitrate, L-serine, L-threonine and L-valine as sole nitrogen sources, but not L-hydroxyproline or L-phenylalanine. Uses L(+)-arabinose, D(+)-cellobiose, D(-)-fructose, D(+)-galactose, glycerol, meso-inositol, lactose, maltose, D-mannitol, D(+)-mannose, L(+)-rhamnose, D(-)-ribose, sodium acetate, sodium butyrate, sodium DL-malate, sodium malonate, sodium propionate, sodium pyruvate, sodium succinate, sucrose (weak growth), trehalose and D(+)-xylose as sole carbon sources but not adonitol, meso-erythritol, inulin, D(+)-melezitose, D(+)-melibiose, methyl -D-glucoside, raffinose, salicin, sodium benzoate, sodium citrate, sodium formate, sodium maleate, sodium oxalate, sodium salicylate, sodium L(+)-tartrate, L(-)-sorbose or xylitol. Tests for nitrate reductase and the production of H2S are positive, but pectin is not hydrolysed. Lipase and lecithinase are produced on egg-yolk agar, but protease activity is not seen on this medium after the recommended 2 days of incubation (there is weak activity after 6 days). Degrades casein, DNA, gelatin, guanine, hypoxanthine, starch (weakly), Tween 80, L-tyrosine, xanthine and xylan but not adenine, allantoin or urea.
The DNA G+C content of the type strain, strain PK-BlueT (=DSM 41797T=ATCC BAA-411T), is 73·4 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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]
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.
Elander, R. P. (1987). Microbial screening, selection and strain improvement. In Basic Biotechnology, pp. 217–251. Edited by J. Bu'Lock and B. Kristiansen. London: Academic Press.
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
Kataoka, M., Ueda, K., Kudo, T., Seki, T. & Yoshida, T. (1997). Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces. FEMS Microbiol Lett 151, 249–255.[CrossRef][Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis software. Arizona State University, Tempe, AZ, USA. http://www.megasoftware.net
Mandel, M. & Marmur J. (1968). Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol 12, 195–206.[CrossRef]
Pernodet, J.-L., Boccard, F., Alegre, M.-T., Gagnat, J. & Guérineau, M. (1989). Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens. Gene 79, 33–46.[CrossRef][Medline]
Prauser, H. (1989). Genus Nocardioides Prauser 1976, 61AL. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2371–2375. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
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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