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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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Published online ahead of print on 13 February 2004 as DOI 10.1099/ijs.0.02738-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces africanus CPJVR-HT is AY208912.
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Strain CPJVR-HT was isolated by C. E. Price and J. M. van Rooyen from suburban garden soil in Cape Town, South Africa in September 2001 as part of their 3rd year BSc project on actinomycetes.
The standard morphological and physiological tests recommended for characterizing Streptomyces species were carried out as described by Williams et al. (1989)
. International Streptomyces Project (ISP) media were prepared as described by Shirling & Gottlieb (1966). Antibiotic resistance was determined by incorporation of antibiotics into Bennett's medium agar plates (Atlas, 1993) at the concentrations recommended and not by using antibiotic-impregnated filter discs. Non-standard test antibiotics were tested at the following concentrations: capreomycin (20 µg ml–1), carbenicillin (100 µg ml–1), cefotaxime (100 µg ml–1), chloramphenicol (50 µg ml–1), D-cycloserine (50 µg ml–1), erythromycin (50 µg ml–1), kanamycin (10 µg ml–1), nalidixic acid (25 µg ml–1) and spectinomycin (20 µg ml–1). Antimicrobial activity was determined using 5-day-old colonies of strain CPJVR-HT grown on nutrient agar (Williams et al., 1989).
Physiological tests were carried out at 28 °C (unless otherwise indicated) and 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 a concentration 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 a concentration of 0·1 % (w/v). DL-Ornithine and 4-amino-n-butyric acid were tested as sole nitrogen sources at a concentration of 0·1 % (w/v).
Determination of the diaminopimelic acid (DAP) isomer and the whole-cell sugar pattern was 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 method of Luquin et al. (1991). The base composition of the genomic DNA of strain CPJVR-HT was determined in 0·1x SSC according to the method of Mandel & Marmur (1968).
The 16S rRNA gene of strain CPJVR-HT was amplified by PCR using universal bacterial 16S rRNA gene primers. The forward primer, F1 (5'-AGAGTTTGATCITGGCTCAG-3'; I, inosine), and reverse primer, R6 (5'-AAGGAGGTGITCCAICC-3'), were modified from primers fD1 of Weisburg et al. (1991) and p1525r of Chun & Goodfellow (1995), respectively. The 16S rRNA gene was sequenced using a MegaBACE 500 automated capillary DNA sequencing system (Molecular Dynamics) and a DYEnamic ET Dye Terminator Cycle Sequencing kit for MegaBACE; a 1488 bp sequence was determined. DNA relatedness studies were conducted in 2x SSC supplemented with 10 % (v/v) formamide at 71 °C by the identification service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) using the spectrophotometric method of De Ley et al. (1970), as modified by Escara & Hutton (1980) and Huß et al. (1983).
Strain CPJVR-HT was Gram-positive, catalase-positive and did not grow under anaerobic conditions. Light microscopy showed a branched mycelium with Spirales-type spore chains and no verticils. Scanning electron microscopy revealed Spirales-type spore chains with spiny spore sheaths. The spiny ornaments were generally straight and very long, but with a sharp curve at the tip (Fig. 1).
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Melanin was not produced on peptone/yeast extract/iron agar (ISP medium no. 6) or on tyrosine agar (ISP medium no. 7). No diffusible pigments were recorded after growth on glycerol/asparagine agar (ISP medium no. 5).
A 1488 bp 16S rRNA gene sequence was determined for strain CPJVR-HT. A BLAST search (Altschul et al., 1997) of the GenBank database using this sequence showed that it was 99 % similar to the 16S rRNA gene sequence of ‘Streptomyces steffisburgensis’ strain JCM 4833 (GenBank accession no. AB045889) over 1485 bases and 99 % similar to the 16S rRNA gene sequence of Streptomyces afghaniensis strain NRRL-ISP 5228T (GenBank accession no. AJ399483) over 1434 bases. A high degree of similarity (98–99 % over 1450–1490 bases) was also recorded to 16S rRNA gene sequences of other members of the Streptomyces cyaneus species group. A phylogenetic tree of Streptomyces 16S rRNA gene sequences was constructed using the neighbour-joining method of Saitou & Nei (1987) with CLUSTAL W (version 1.81) and MEGA (version 2.1; Kumar et al., 2001) (Fig. 2). This tree shows the close phylogenetic association of strain CPJVR-HT with certain members of the S. cyaneus species group (namely S. afghaniensis, Streptomyces caelestis and Streptomyces fumanus).
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). S. afghaniensis is one of seven allied species of S. cyaneus that produce spiny, red spores in Spirales or Retinaculiaperti spore chains (Williams et al., 1989). Strain CPJVR-HT produces spiny, blue spores and does not produce melanin. Thus, despite the high 16S rRNA gene sequence similarity between CPJVR-HT and members of the S. cyaneus species group, morphological and physiological characteristics of CPJVR-HT indicate that it is not a strain of S. cyaneus. DNA–DNA hybridization studies confirmed that strain CPJVR-HT is unique. The DNA relatedness between strain CPJVR-HT and S. afghaniensis NRRL B-5621T (=NRRL-ISP 5228T) is 46·2±0·9 % and that between strain CPJVR-HT and ‘S. steffisburgensis’ strain NRRL ISP 5547 (=JCM 4833) is 38·4±0·5 %.
Comparison of the characteristics of strain CPJVR-HT with other streptomycetes that produce blue aerial mycelia shows that it also differs from these species. These differences are discussed below.
Streptomyces amakusaensis produces smooth, blue spores in Spirales spore chains and a pH-sensitive, yellow–brown substrate mycelium. This species does not grow at 45 °C and is sensitive to penicillin G (10 IU ml–1). It is unable to grow in the presence of 7 % NaCl or 0·1 % phenol. Sucrose and (+)-L-rhamnose are not used as sole carbon sources. L-Histidine cannot be used as the sole nitrogen source.
Streptomyces glaucescens produces a red–orange substrate mycelium and red–orange diffusible pigments. Melanin is produced. Proteolytic activity is absent. It is able to grow in the presence of rifampicin (50 µg ml–1), but is sensitive to penicillin G (10 IU ml–1) and cannot grow in the presence of 7 % NaCl or 0·1 % phenol. It cannot use meso-inositol, raffinose or (+)-D-melibiose as sole carbon sources (Williams et al., 1989).
Streptomyces inusitatus produces smooth, blue–grey to grey–blue spores in Spirales spore chains and a colourless or pale-brown substrate mycelium. It cannot use (+)-L-arabinose, (–)-D-fructose, meso-inositol, D-mannitol, raffinose, (+)-L-rhamnose, salicin, sucrose or (+)-D-xylose as sole carbon source. Streptomyces ipomoeae produces smooth, blue spores in Spirales spore chains and a pale yellow or greyish-yellow substrate mycelium. Faint yellow or green diffusible pigments are produced. Streptomyces lomondensis produces warty to spiny, blue spores in Rectiflexibiles, Retinaculiaperti or Spirales spore chains. The substrate mycelium is brick-red, rust-brown or straw-coloured and a brown or pink diffusible pigment is produced. Melanin is also produced. Casein is not degraded and salicin cannot be used as the sole carbon source. Streptomyces viridochromogenes produces blue, spiny spores in Spirales spore chains and a pH-sensitive, green substrate mycelium. Green, pH-sensitive diffusible pigments are produced. This species produces melanin and is able to grow in the presence of rifampicin (50 µg ml–1) and 0·01 % sodium azide, but is sensitive to penicillin G (10 IU ml–1) and cannot grow in the presence of 7 % NaCl. It cannot use sucrose as sole carbon source or L-phenylalanine as sole nitrogen source (Williams et al., 1989).
These results support classification of strain CPJVR-HT as a representative of a novel species of Streptomyces, for which the name Streptomyces africanus sp. nov. is proposed. Additional phenetic characteristics of the strain are presented under the species description.
Description of Streptomyces africanus sp. nov.
Streptomyces africanus (af.ri.ca'nus. L. masc. adj. africanus of Africa).
Aerobic, Gram-positive, catalase-positive actinomycete that forms a blue aerial mycelium and a yellow 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 spiny spore sheaths are produced. No diffusible pigments are produced on glycerol/asparagine agar or on any other medium tested. Melanin pigment is not produced on peptone/yeast extract/iron agar or on tyrosine agar. The cell wall contains LL-DAP (cell wall type I); there are no diagnostic sugars. Excellent growth occurs on inorganic salts/starch agar (ISP medium no. 4). Very good growth occurs on yeast extract/malt extract agar (ISP medium no. 2), oatmeal agar (ISP medium no. 3) and Czapek solution agar (Atlas, 1993). Growth is good on Bennett's agar. Growth on glycerol/asparagine agar is moderate. Weak antibiotic activity is exhibited against Enterococcus faecium, but no antibiotic activity is observed against Escherichia coli ATCC 25922 or Pseudomonas aeruginosa ATCC 27853. Grows in the presence of 2-phenylethanol (0·3 %), sodium chloride (7 %, but not 10 %), carbenicillin (100 µg ml–1), cefotaxime (100 µg ml–1), D-cycloserine (50 µg ml–1), nalidixic acid (25 µg ml–1), oleandomycin (100 µg ml–1) and penicillin G (10 IU ml–1). Grows at pH 4·3 and 45 °C, but not at 4 °C or in the presence of sodium azide, capreomycin (20 µg ml–1), cephaloridine (100 µg ml–1), chloramphenicol (50 µg ml–1), erythromycin (50 µg ml–1), gentamicin (100 µg ml–1), kanamycin (10 µg ml–1), lincomycin (100 µg ml–1), neomycin (50 µg ml–1), phenol (0·1 %), rifampicin (50 µg ml–1), spectinomycin (20 µg ml–1), streptomycin (100 µg ml–1), tobramycin (50 µg ml–1) or vancomycin (50 µg ml–1). Utilizes adonitol, (+)-L-arabinose, (+)-D-cellobiose, (–)-D-fructose, (+)-D-galactose, glycerol, meso-inositol, inulin, lactose, maltose, D-mannitol, (+)-D-mannose, (+)-D-melibiose, methyl -D-glucoside, raffinose, (+)-L-rhamnose, (–)-D-ribose, salicin, sodium acetate, sodium butyrate, sodium citrate, sodium DL-malate, sodium malonate, sodium propionate, sodium pyruvate, sodium salicylate, sodium succinate, sucrose, trehalose and (+)-D-xylose as sole carbon sources, but not meso-erythritol, (+)-D-melezitose, sodium benzoate, sodium formate, sodium maleate, sodium oxalate, sodium (+)-L-tartrate, (–)-L-sorbose or xylitol. Utilizes 4-amino-n-butyric acid, DL--amino-n-butyric acid (weak growth), L-arginine, L-cysteine, L-histidine, L-hydroxyproline (weak growth), L-methionine, DL-ornithine, L-phenylalanine, potassium nitrate, L-serine, L-threonine and L-valine as sole nitrogen sources. Tests for nitrate reductase and H2S production are positive. Pectin is hydrolysed, but hippurate is not. Protease, lipase and lecithinase activities are produced on egg-yolk agar (the proteolytic reaction is weak). The organism degrades adenine, aesculin, arbutin, casein, DNA, gelatin, hypoxanthine, starch, Tween 80 and L-tyrosine, but not allantoin, cellulose, guanine, urea, xanthine or xylan.
The type strain is CPJVR-HT (=NRRL B-24143T=DSM 41829T).
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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.
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). Quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Escara, J. F. & Hutton, J. R. (1980). Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate. Biopolymers 19, 1315–1327.[CrossRef][Medline]
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis software. Arizona State University, Tempe, Arizona, USA (http://www.megasoftware.net).
Luquin, M., Ausina, V., López Calahorra, F., Belda, F., García Barceló, M., Celma, C. & Prats, G. (1991). Evaluation of practical chromatographic procedures for identification of clinical isolates of mycobacteria. J Clin Microbiol 29, 120–130.
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.
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.
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. 2452–2504. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
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.
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