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IDENTIFICATION OF THE STREPTOTHRICIN PRODUCER STRAIN SC4 ISOLATED IN TAIWAN
R. Y. WU AND M. H. CHEN Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan, 11529, Republic of China
ABSTRACT. A previously isolated streptomycete, designated strain SC4 and able to produce a new streptothricin type antibiotic, was compared with Streptomyces lavendulae type strain ATCC 8664, producer of streptothricin. The two strains are quite similar in morphology, cell wall composition and spore morphology but can be differentiated by some cultural and physiological characteristics. The high (87.1%) DNA homology suggests the identity of strain SC4 with S.lavendulae.
Streptomyces strain SC4, isolated from soil (Wu, 1984), produces SC4-X, a new member of the streptothricin type antibiotics (Wu et al., 1983; Wu, 1985), active against Gram-positive and -negative bacteria and fungi. Streptothricin is produced by Streptomyces lavendulae ATCC 8664 (Waksman and Woodruff, 1942) and in this paper results of a comparison of morphological, cultural, physiological, genomic and antimicrobial characteristics of the two organisms are reported. MATERIALS and METHODS Bacterial strains and culture conditions. Strain SC4 was isolated from a soil sample collected at Tapehu, Taiwan (Wu, 1984). Streptomyces lavendulae subsp. lavendulae (Waksman and Curtis) Waksman and Henrici, strain ATCC 8664, was purchased from the American Type Culture Collection. Except when otherwise specified, both strains were cultured at 28 C on tryptone-yeast extract-glucose (TYG) agar medium (10g glucose, 3 g yeast extract, 5 g peptone, 1g KH2PO4, 1g K2HPO4, 20g agar, 1,000 ml distilled water). Cultural and morphological characterization. Cultural characteristics of the strains were determined after 7, 14 and 21dd incubation on Czapek-Dox agar, nutrient agar, Sabouraud agar and ISP media (Shirling and Gottlieb, 1966). Morphology was examined by light and scanning electron microscopy (Zeiss DSM model 950).
Physiological tests. Utilization of carbohydrates was investigated on a basal carbon nutrient medium (Pridham and Gottlieb, 1948; Waksman, 1967). Methods and media used for physiological tests were as described by Luedemann and Brodsky (1964), Waksman (1967), Luedemann (1971) and Neyra et al. (1977). All cultures were incubated at 28 C for 10dd, except for the gelatine liquefaction (15 C for 21dd).
Cell chemistry. Determination of the cell-wall composition, including diaminopimelic acid isomers and sugars, was carried out according to Becker et al. (1965), Pine and Boone (1967), Boone and Pine (1968), Lechevalier and Lechevalier (1970, 1980) and Kawamoto et al. (1981).
DNA-DNA homology study. The DNA-DNA relatedness was determined according to Ezaki et al. (1989). RESULTS and DISCUSSION Cultural characteristics. Cultural characteristics are shown in Table 1.
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STRAIN
Medium Character SC4 ATCC
8664
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Czapek-Dox agar Growth* Moderate, 2mm Poor, 0.5mm
Reverse color Colourless Light brown
Aerial mycelium Light yellow Light yellow
Diffusible pigment None None
Nutrient agar Growth Moderate, 2mm Moderate, 2mm
Reverse color Light brown Light brown
Aerial mycelium White White
Diffusible pigment Light brown Light brown
Sabouraud agar Growth Moderate, 2mm Very poor, 1mm
Reverse color Colorless Colorless
Aerial mycelium White Light yellow
Diffusible pigment None None
ISP medium 2 Growth Good, 2-3mm Abundant, 3-5mm
Reverse color Colorless Brown
Aerial mycelium Pale yellow White
Diffusible pigment None Black
ISP medium 3 Growth Good, 3-5mm Abundant, 5mm
Reverse color Brown Colorless
Aerial mycelium White White
Diffusible pigment Light brown None
ISP medium 4 Growth Moderate, 3-5mm Abundant, 3mm
Reverse color Colorless Brown
Aerial mycelium White White
Diffusible pigment None None
ISP medium 5 Growth Poor, 1mm Abundant, 3mm
Reverse color Colorless Colorless
Aerial mycelium White White
Diffusible pigment None Light brown
ISP medium 6 Growth Moderate, 1-2mm Moderate,2mm
Reverse color Brown Light brown
Aerial mycelium White Yellow
Diffusible pigment Brown to black None
ISP medium 7 Growth Moderate, 2-3mm Abundant,3-5mm
Reverse color Pink Black
Aerial mycelium White White to pink
Diffusible pigment Pink Black
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Table 1. Cultural characteristics of strain SC4 and
S.lavendulae ATCC 8664 on various media after 3-weeks
incubation at 28 C (* Measurements refer to colony
diameter).
Strain SC4 grows well on most organic and synthetic media, with the exception of glycerol asparagine agar (ISP medium 5). Colonies are typically convex with indented edges and covered with white aerial mycelium and spores. Diffused melanoid pigments may be observed on ISP media 6 and 7.
S.lavendulae shows growth characteristics similar to those of strain SC4. It can be differentiated from strain SC4 by its poor growth on synthetic media, such as Czapek-Dox agar and Sabouraud agar, and by its pigmentation on ISP media 2 and 5 but not on ISP media 3 and 6.
Morphological characteristics. Strain SC4 proliferates on TYG and nutrient agar showing velvety aerial mycelium and powdery spores. Aerial filaments are long, branched and flexuous. Terminal aerial hyphae develop into short conidiophores (0.2 x 1.5um) bearing (Fig. 1A) straight, curved or hooked spore chains (5 to 7 spores).
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Strain
Character SC4 ATCC 8664
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Spore surface Smooth Smooth
Spore shape Globose to elliptical Oblong to
rectangular
Spore mass color Grey to pink Pink to violet
Spore chain Flexibiles
Recti-flexibiles
Spore size 1.0 x 1.0 or
1.2 x 1.8um 0.6 x 0.8um
Average number
of spores 5 to 7 10 to 20
Conidiophore 0.5 x 1.5um 0.5 x 1.0um
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Table 2. Morphological characteristics of strain SC4
and Streptomyces lavendulae ATCC 8664.
Spores are globular (1.0m in diameter) or oval to elliptical (1.2 x 1.8um) in shape and show a smooth surface. S.lavendulae forms lumpy colonies on organic media. Both aerial and substrate mycelia are well branched and do not fragment. Monopodially branched aerial hyphae bear long, straight spore chains (10 to 20 or more spores). Spores are smooth surfaced, rectangular or oblong (0.6 x 0.8um) in shape (Fig. 1B). Morphological characteristics are summarized in Table 2. Strain SC4 appears morphologically similar to S.lavendulae, but could be distinguished by the number of spores (Tresner et al., 1961).
Physiological characteristics. Physiological properties are compared in Table 3. Both strains show similar reactions with differences only in the level of activity. For example, gelatine liquefaction, milk peptonization and starch hydrolysis are stronger in strain SC4, whereas S.lavendulae reduces nitrate better and forms more melanin and H2S. As for antimicrobial activity (Table 4) both antibiotic SC4-X (Wu et al., 1983) and streptothricin show antibacterial and antifungal activity. Streptothricin appears more potent than antibiotic SC4-X.
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Strain
Physiological property SC4 ATCC 8664
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----
Gelatine liquefaction ++ +
Milk coagulation + -
Milk peptonization +
+/-
Starch hydrolysis ++ +
Nitrate reduction + ++
Melanin* + ++
NaCl tolerance 2% 4%
Growth temperature 10-40 C
10-40 C
Optimum temperature 28 C
28-30 C
H2S production ++ +
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Table 3. Physiological properties of strain SC4 and
Streptomyces lavendulae ATCC 8664 (+: positive;
-: negative; * on peptone-yeast extract agar). No
strain decomposes cellulose or liquefies serum.
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TEST ORGANISM Minimal inhibitory concentration
(g/ml)
SC4-X streptothricin
Staphylococcus aureus,
strain 209 P 10 20
Sarcina lutea,
strain ATCC 9341 10 20
Candida albicans,
strain YU 1200 20 100
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Table 4. Antibiotic activity of SC4-X and
streptothricin.
Carbohydrate utilization. The utilization of carbohydrates is summarized in Table 5. Both strains utilize D-glucose, salicin and D-fructose and none of them D-mannitol, D-xylose, cellulose, i-inositol and L-rhamnose. Assimilation of D-galactose, raffinose, L-arabinose and sucrose is different in the two strains.
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Strain
Carbohydrate SC4 ATCC 8664
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Control - -
D-Galactose - +
Raffinose - +
L-Arabinose + -
Sucrose + -
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Table 5. Carbohydrate utilization by strain SC4 and
S.lavendulae ATCC 8664 (+: utilized; -: not utilized).
Both strains utilize D-glucose, D-fructose and salicin; none
of them D-mannitol, D-xylose, cellulose, i-inositol and
L-rhamnose.Chemotaxonomy. Analysis of whole cell hydrolysates show the presence of LL-diaminopimelic acid and no diagnostic sugars in both strains, suggesting a type I cell wall, typical of streptomycetes.
DNA homology. As determined by fluorometric DNA-DNA hybridization, DNA similarity between strain SC4 and S.lavendulae is equal to 87.1%. This suggests a significant genomic relatedness between the two strains. A number of producers of streptothricin group antibiotics have been reported in the past (Table 6). Strain SC4 appears to be the first such strain isolated in Taiwan. It produces streptothricin type antibiotics, namely SC4-X, which have a streptothricin structure with modification on the aminosugar moiety (Wu et al., 1983). The antimicrobial spectra (Table 4) suggest an efficacy of strain SC4 equivalent to that of S.lavendulae.
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Antibiotic Producer Reference
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Streptothricin S.lavendulae Waksman and Woodruff, 1942;
Waksman, 1943
Streptidine S.lavendulae Peck et al.,1946
Streptolin Streptomyces sp., strain S-11 Larson et al.,1953
Roseothricin S.roseochromogenus Nakanishi et al., 1954
LL-AC 541
(= E-749-C, Citromycin, BY-81)
S.hygroscopicus
S.hygroscopicus
Streptomyces sp., strain IN-1483
S.olivoreticuli Borders et al., 1967
Shoji et al., 1968
Taniyama et al., 1972
Furumai et al., 1968
Deforminino-LL-AC 541 S.hygroscopicusBorders et al., 1967;
Zbinovsky et al.,
1968; Borders et al.,
1970
Yazumycin S.lavendulae Akasak et al.,1968
Sclerothricin S.sclerogranulatus Kono et al., 1969;
Shimazu et al., 1969
Fucothricin A, B S.fradiae Thirumalachar et al., 1971
SF-701 (= LL-BL 136) S.griseochromogenus
Streptomyces sp. Tsuruoka et al., 1968
Borders et al., 1970
LL-AB 664 (= BD-12) S.candidus
S.luteocolor Borders et al., 1970
Furumai et al., 1968
Deformimino-LL-AB 664 S.griseus Sawada et al., 1977
Streptothricin R4H S.lavendulae,
strain R4 Sawada et al., 1974
Streptothricin S15-1 S.purpeofuscus Kawamura et al., 1976; Brown
et al., 1977
Glycinothricin S.griseus Sawada et al., 1977
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Table 6. Streptothricin group antibiotics and producing
strains.Results from the above comparisons indicate that strain SC4 is a streptomycete closely related to S.lavendulae in cell wall composition and morphology. Although they could be differentiated by few distinct cultural and physiological characteristics, the high degree of DNA-DNA homology suggests the identity of strain SC4 with S.lavendulae. Acknowledgements. This study was supported by the Academia Sinica and the National Science Council, R.O.C. (NSC 80-0211-B-001-37). The authors would like to thank Dr. A.Seino and Miss M.Tseng for their valuable discussion and encouragement. REFERENCES Akasak, K., H.Abe, A.Seino & S.Shiratol (1968). Yazumycin, a new antibiotic produced by Streptomyces lavendulae. J.Antib., 21: 98-105 Becker, B., M.P.Lechevalier & H.A.Lechevalier (1965). Chemical composition of cell-wall preparations from strains of various form-genera of aerobic actinomycetes. Appl.Microbiol., 13: 236-243
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