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Variability of Streptomyces spectabilis 1000 N. VESSELINOVA and R. GESHEVA Institute of Microbiology, Bulgarian Academy of Sciences, 1113 - Sofia, Bulgaria
Code Number: AC91004
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Abstract. From studies carried out on the polymorphism of Streptomyces spectabilis it has been established that the strain population is heterogenous and consists of several morphological variants of essential cultural, physiological and biochemical differences. Predominant in the population is the main type, 95.67%, characterized by the specific features of the parent strain. Following in number is the yellow sporogenous variant, 4.0%. The remaining variants are rather rarely observed, 0.1%. Particularly characteristic are the colonies of the hypercoloured oligosporous variant having the highest antibiotic activity. It has been found by thin-layer chromatography that all the variants form antibiotic 1011 identical to streptovaricin except the red oligosporous variant which biosynthesizes only antibiotic 1012, new for the species S. spectabilis. Polymorphism established widens the idea of the species S.spectabilis and enhances the efficiency of selection methods. Actinomycetes are featured by marked variability and their study is of interest both for the selection of strains producing bioactive substances and for the clarification of a number of matters associated with their biology and systematics (Kuznetsov, 1987; Leblond et al., 1989; Gesheva and Gesheva, 1989).
This paper presents the results from the study of the specific biological features of variants comprising the population of Streptomyces spectabilis 1000 producing the antibiotic complex 1011 identical to streptovaricin (Ivanova et al., 1987) and antibiotic 1012 undescribed so far for the species S. spectabilis. Materials and methods
S.spectabilis, strain 1000, from the collection of the Institute of Microbiology was the subject of the present study (Vesselinova et al., 1990). A study of variability was carried out with the monospore growing colony using the Kuznetsov's method (1972). A 10-day old culture served to prepare the spore suspension. The colour of the aerial and substrate mycelia was determined using Bondartsev's colour scale (1954). Morphological, cultural and physiological properties of the variants were studied by the methods recommended by the International Streptomyces Project (ISP). Submerged cultivation was carried out on a shaker in a complex soybean medium (g/l): glucose 20.0, soy- bean meal 20.0, CaCO3 1.0, for 96-100 hrs at 28 C. Antibiotic activity was assayed by the agar diffusion method against the test culture B.subtilis ATCC 6633 and streptovaricin standard (Upjohn Co., USA). The bioautographic analysis of antibiotic extracts from mycelium and filtrates was performed according to the procedure described by us (Ivanova et al., 1987). The methods of isolation, purification and identification of antibiotic 1011 and 1012 are described in our papers (Ivanova et al., 1987; Vesselinova et al., 1988). Results It was established that in monospore inoculation of S.spectabilis 1000 on various nutrient media the population of the strain was heterogenous and consisting of different morphological variants. The population composition of strain 1000 is shown in Fig. 1. Prevaling is the main type which has the characteristic features of the parent strain, 95.67%. The other variants are comparatively rare.
The main type is characterized by protuberant colonies, regularly shaped, raised in the centre and with well formed radial folds and festoon edges. The aerial mycelium is well developed and whitish to pinky orange in colour.
The hypercoloured oligosporous colonies are distinguished by an extremely well developed substrate mycelium of carmine colour. They are highly undulated and resemble convolutions in structure. The aerial mycelium is orange and does not cover the whole colony surface. The red pigment diffuses into the nutrient medium.
The red oligosporous colonies develop a whitish or pinky orange aerial mycelium which does not extend over the whole surface. The substrate mycelium is of the characteristic carmine colour. The pigment does not diffuse into the nutrient medium. Morphological properties. Micromorphology of the aerial mycelium as observed in different variants showed that they all have long, straight sporophores of the monopodial type. On some media these form loops most frequent in the red oligosporous variant. Spores of all strains are multiple, 30 and more in a chain. They are formed in the fragmentous way and have the smooth surface of the parent strain. Granules on the spore surface which are, most probably, due to the dissolved pigment prodigiosine isolated from the mycelium of this strain, are observed in the hypercoloured oligosporous variant only (Ivanova et al., 1989).
Cultural properties. Table 1 shows the cultural properties of representatives of each morphological type: strain 63 (main), strain 36 (yellow sporogenous), strain 3 (yellow oligosporous), strain 1011 (hypercoloured red oligosporous), strain 1 (red oligosporous). As seen from the data in the Table, the variants are differentiated substantially by their cultural properties. High variability is observed in the colour of the aerial and substrate mycelia. The aerial mycelium in strains 1011 and 1 is more scarce and not covering the whole surface of the colonies or missing at all. Differences are observed also in the presence of soluble pigment which diffuses into the nutrient medium only in variant 1011.
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Medium Feature Strain 63 Strain 36
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Yeast-malt extract AM whitish to whitish
agar (ISP medium 2) pinkish-orange
SM rusty to ochre
reddish-brown
SP none none
Oatmeal agar AM whitish to whitish
(ISP medium 3) orange-pink
SM reddish-orange pale honey to ochre
SP none none
Inorganic salt- AM pale pink whitish
starch agar SM ochre-red pale honey to ochre
(ISP medium 4) SP none none
Glycerol-asparagine AM whitish whitish
agar SM whitish to pale ochre
(ISP medium 5) yellow
SP none none
Tyrosine agar AM pinkish-orange whitish
(ISP medium 7) to pink-violet
SM orange-pink to ochre
SP none none
Bennett's agar AM pinkish-orange whitish
SM blood red lemonish yellow
SP none none
Organic agar No. 2 AM pinkish-orange whitish
SM rusty pale ochre
SP none none
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Medium Feature Strain 3 Strain 1011' Strain 1
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Yeast-malt AM whitish none none
extract agar SM ochre-olive pale ochre rusty
(ISP medium 2) to rusty
SP none none none
Oatmeal agar AM pale yellow none whitish
(ISP medium 3) SM pale yellow carmin-red carmin-red
SP none none none
Inorganic salt- AM whitish to pinkish- whitish
starch agar pale lemon orange
(ISP medium 4) SM pale lemon carmin-red pinkish-
violet
SP none rusty none
Glycero]- AM whitish pinkish- none
asparagine agar orange
(ISP medium 5) SM pale yellow carmin-red pinkish-
orange to
carmin-red
SP none none none
Tyrosine agar AM whitish pinkish-orange whitish to
(ISP medium 7) pinkish-orange
SM pale yellow chestnut to rusty to
to ochre dark red chestnut
and olive
SP none dark orange none
Bennett's agar AM whitish to none none
pale gray
SM pale lemon chestnut rusty
to ochre
SP none rusty none
Organic agar AM whitish-gray none none
No. 2 SM pale yellow chestnut pale ochre
SP none brownish none
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Table 1. Cultural properties of different variants (AM:
aerial mycelium; SM: substrate mycelium; SP: soluble pigment).
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Physiological and biochemical properties. All variants studied form melanin, a characteristic taxonomic trait of the species. They liquefy gelatine and hydrolyze starch. Differences are noted in nitrate reduction, coagulation and peptonization of milk, utilization of carbon sources and biosynthesis of antibiotic substances (Table 2). All variants but strain 1 utilize the same carbon sources and synthesize antibiotics 1011 and 1012 in traces. Variant 1 uses sugars such as arabinose, sucrose and lactose and biosynthesizes antibiotic 1012 only. The results from antibiotic activity tests in different variants show that the hypercoloured variants have demonstrated the highest activity - 300 ug/ml. As known from the literature, S.spectabilis produces the antibiotic streptovaricin (British Patent 811,757), the aminoglycoside antibiotic spectinomycin (Mason et al., 1961) and other metabolites (Staley, 1990). On previous studies (Ivanova et al., 1987) we have established that S.spectabilis produces antibiotic 1011 identical to streptovaricin. The comparative thin-layer chromatographic and bioautographic tests using a streptovaricin standard kindly made available to us by Upjohn Company, U.S.A. have shown that antibiotic 1011 obtained by us represents a complex of 8 components. Essential differences were found in the composition of streptovaricin with the different variants following cultivation under identical conditions. Hypercoloured variants synthesize the whole streptovaricin complex consisting of 4 major components (A, B, C, D) and 3 minor components (E, G and J). Component F is inactive towards B.subtilis ATCC 6633. The other variants produce just some of the components. It can be assumed that all the components of the complex are present but not established because of their low antibiotic activity.
Antibiotic resistance. As illustrated in Table 3, all studied variants manifested sensitivity to aminoglycoside antibiotics, gentamicin and kanamycin, while they were resistant to the other test antibiotics. Only in variant 3 a loss in resistance to the antibiotic erythromycin was observed. Data obtained for multiple resistance to the majority of test antibiotics are in agreement with the results of Okami (1986).
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Feature Parent Variant Strains
Strain ----------------------------
1000 63 36 3 1011 1
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Melanin formation
(ISP medium 6) + + + + + +
H2S production - - - - - -
Nitrate reduction - + - - - -
Gelatine liquifaction + + + + + +
Milk coagulation - - - - - -
Milk peptonization + + + + - -
Growth on cellulose - - - - - -
Utilization of glucose,
fructose, raffinose,
xylose and inositol + + + + + +
Utilization of sucrose,
arabinose, and lactose - - - - - +
Utilization of rhammose,
Na ascorbate, and Na acetate - - - - - -
Biosynthesis of
antibiotic 1011 (ug/ml) 50 52 80 1000 300 -
Biosynthesis of
antibiotic 1012 trace trace trace trace trace trace
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Table 2. physiological and biochemical properties of the
parent strain (1000) and of the variants.
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Type Parent Variant strains
Antibiotic strain strain-----------------------------
NRRL 24 1000 63 36 3 1011 1
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Gentamicin (30ug/ml) 30 30 32 22 16 26 L5
Kanamycin (30ug/ml) 10 10 18 22 9 10 L5
Penicillin (10ug/ml) 0 0 0 0 0 0 0
Carbenicillin (10ug/ml) 0 0 0 0 0 0 0
Oxacillin (10ug/ml) 0 0 0 0 0 0 0
Bacitracin (10ug/ml) 0 0 0 0 0 0 0
Streptovaricin(500ug/ml)0 0 0 0 0 0 0
Spectinomycin(125ug/ml) 0 0 0 0 0 0 0
Tetracycline (30ug/ml) 8 8 8 9 10 15 18
Erythromycin (15ug/ml) 0 0 0 0 20 0 0
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Table 3. Antibiotic resistance of variants.
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The detailed study of morphological, cultural and physiological - biochemical properties of different variants in the population of the strain S.spectabilis 1000 has shown that it is heterogenous and the separate variants are characterized by some specific features. As a result the taxonomic characteristics of the species S.spectabilis have been extended to include also the biological properties of the separate variants within the species. Some characteristic relationships were discovered and provide for the purposeful control of the population composition and its biological productivity. Acknowledgement. The authors thank the Upjohn Company, U.S.A. for the streptovaricin standard provided. References British Patent (1959). 811,757 Bondartsev, A. (1954). Scale of Colors. The Academy of Science USSR, Moscow Gesheva, V. & R.Gesheva (1989). Natural variability of the Streptomyces hygroscopicus 111-81. Compt. Rend. Acad. Bulg. Sci., 42: 115-118. Ivanova, V., N.Vesselinova & R.Gesheva (1989). Prodigiosin-like pigments from Streptomyces sp.1011. Fifth International Conference on Chemistry and Biotechnology of Biologically Active Natural Products, 1:412-417 Ivanova, V., N.Vesselinova, R.Gesheva & M. Naidenova (1987). Comparative investigations of the components of antibiotic complex 10 11 with thin layer chromatography of variants from Streptomyces sp. 1000. Acta Microbiologica Bulg., 21:52-56 Ivanova, V., N.Vesselinova & R.Gesheva (1987). Isolation, purification and identification of antibiotic complex 1011 from Streptomyces sp. 10 11. Acta Microbiologica Bulg., 21:47-50 Kuznetsov, V.D. (1972). Studies on variation of actinomycetes producing antibiotics and other biologically active substances. Antibiotiki, 17: 666-671 Kuznetsov, V.D. (1987). Parallelism in hereditary variability and population concept of species in representatives of prokaryotes. J.G. Biologii, 48:466-476 Leblond, P.P.Demuyter, L.Moutier, M.Laakel, B.Decaris & J.-M.Simonet (1989). Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens. J.Bacteriology, 171: 419-423 Mason, D., R.Smith & A.Dietz (1961). Actinospectacin, a new antibiotic. I. Discovery and biological properties. Antibiot. Chemother., 11: 118-122 Okami, Y. (1986). Productivity of aminoglycoside antibiotics with reference to antibiotic resistance of the producer. In: Kleinkauf, H., H.Dohren, H.Dornauer & G.Nesemann (eds.). Regulation of secondary metabolite formation. Workshop Conference Hoechst, 16:333-354 Staley, A. (1990). Isolation, structure, synthesis and biosynthesis of selected bioactive microbial metabolites. Abstracts from Ph.D. dissertation, University of Illinois at Urbana-Champaign, 100-218 Vesselinova, N., R.Gesheva & V.Ivanova (1990). Streptomyces sp. 1000 producing streptovaricin complex. Folia Microbiologica (in press) Vesselinova, N., R.Gesheva and V.Ivanova (1988). Patent application form 84704, Bulgaria. Copyright 1991 C.E.T.A. The International Centre for Theoretical and Applied Ecology, Gorizia. The following images related to this document are available:Line drawing images[ac91004a.gif] |
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