Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 2, Num. 1, 1998, pp. 30-39

Ljubomira Yocheva^1* and Stefka Antonova-Nikolova²

¹National Bank for Industrial Microorganisms and Cell Cultures, 1113 Sofia, P.O. Box 239, Bulgaria;
²The Sofia University, Biological Faculty, Department of Microbiology, 8 "Dragan Tsankov" st., 1421 Sofia, Bulgaria

Code Number: cc98006

Summary

The natural variability of strain Streptomyces galbus (F) subsp. achromogenes 695 was examined. The changes of three characteristics: morphology of the colonies, formation of diffusible pigment and antibiotic activity, were studied on 21 agar media.

It was established that the polymorphism of the strain was the greatest on Gauze's No1 medium. Its population divided into four morphological types of colonies and one subtype of type I, which differed in the development stage of the aerial mycelium and in the colour intensity of the aerial and vegetative mycelium and of the soluble pigment.

A positive correlation between the antibiotic activity and the biosynthesis of diffusible pigment was observed.

The variants of the basic (I) morphological type had the highest antibiotic activity. Strain 695-3, which activity was with 20% higher than the one of the initial culture, was chosen for further selection.

Introduction

It was necessary to know the composition of the microbial population and the stage and character of its variability in order to maintain a high biosynthetic activity of the microorganisms - producers of biologically active substances.

The present paper is a part of a complex investigation of the biology of strain Streptomyces galbus (F.) subsp. achromogenes 695 - a producer of actinomycin antibiotic complex [10]. The aim is to study the natural polymorphism of the strain and the possible correlation between the macromorphology of the variants and their antibiotic activity.

Materials and Methods

The object of the investigation was strain Streptomyces galbus (F.) subsp. achromogenes 695 [11], a producer of an antibiotic complex with antitumor activity [10]. The strain was placed at our disposal by the Department of Microbiology at the Sofia University "St. Kliment Ohridski" and preserved by lyophilization in the National Bank for Industrial Microorganisms and Cell Cultures.

Its morphological and cultural properties and natural variability were investigated on nutrient media and after a method offered by Kusnetsov [5].

The parental strain and its morphological variants were cultivated at temperature 28°C, at a different duration, as single colonies or a thick sowing on solid media. For the final scheme of the composition of the strain's population a five-times straining of the medium was done and the highest number of morphologically changed colonies was obtained. The isolation of morphological variants after a natural spread was performed according to the method of the total manual choice [1]. A spore suspension of a 14-day culture with spore density 10⁹ spores/ml was used as an inoculum in all of the investigations.

The cultural colouring and the colour of the soluble pigment were determined according to Bondartsev colour scale [2].

The antibiotic activity of the initial strain and its variants of the different morphological types was determined according to the diffusive method on agar plate [3] compared to the test-microorganisms UF₂ and Sacrina lutea. The results were given: 1./ in mm of diameter sterile zone and recalculated in percentage compared to the control; 2./ in (mg/ml according to Dmitrieva's table towards standard actinomycin D[3].

They were worked up according to Plohinski's variational-statistic analysis [7]. The following indices were calculated: mean arithmetical (M); standard error of the mean arithmetical meaning (m); sample standard deviation (s) and coefficient of variation (CV).

Results and Discussion

Choice of medium for studying the spontaneous variability of strain 695

The growth, the sporulation time and the antibiotic activity of the studied strain were followed on 21 nutrient media (Table 1a, 1b). A noticeable growth was observed even on the third day and was heavy for the whole period of examination. The only exception was the Raistrik's medium on which the strain did not grow probably because of the high sugar concentration in the molasses, being the single carbon source in the medium. The strain 695, depending on the medium, formed white to whitish, pale sandy or grey-pink aerial mycelium.

The colour of the diffusible pigment on the different media was different and varied from straw-yellow through orange-yellow to pale reddish-brown. The colour intensity of the pigment grew with the cultivation time.

The spore formation, on most of the media, began on the third day. On media CPI according to Krasilnikov, glucose-asparagine agar, Waksman's organic medium and Chapek's medium with glucose and maize extract, the strain sporulized scantily and the culture lysed as early as the seventh day. Chapek's medium with glucose, Gauze's medium No 1 and potato agar were the most suitable for the growth and spore-formation of strain 695.

Strain 695 showed the highest biological activity when cultivated on Gauze's medium No 1, Waksman's synthetic medium and potato agar. Despite of the moderate growth on starch agar and starch - ammonia agar, there was no antibiotic activity against the examined test - microorganisms (Table 1a, 1b).

Most frequently strain 695 dissociated in two, three or four types of colonies. Its morphological variability was the greatest on Chapek's medium with glucose - 7 types of colonies and on Gauze's medium No 1 - 6 types. This fact made these media suitable for studying the natural variability of the strain. Its population was homogeneous on potato and starch agar (Table 2).

Population composition of strain 695 on the media with the highest number of morphological variants. Scheme of the population splitting

Usually when spreading on solid nutrient media is performed, unequal microconditions for the development of the population colonies are established. As a result a part of the spores germinated later than the basic mass. These colonies could differ considerably in their morphological and other properties. These differences had a modification character and they could not be considered as hereditary consolidated morphological variants.

In order to differentiate the real variants from the modifications, a five-times straining at an interval of 10-14 days was carried out of all the isolated morphological variants of the initial strain, on the media stimulating the highest morphological variability of strain 695.

Only 4 of the primarily established 7 morphological types of colonies, differing mainly in the development of the aerial mycelium, remained relatively homogeneous on Chapek's medium with glucose (Fig. 1). The remaining variants could be considered as modifications, because their colonies dissociated in colonies of I, Ia, II and III types. Based on the results about the connections between the separate types of colonies on this medium, a scheme for the population splitting of the strain 695 was made up.

At natural cultivation on mineral Gauze's medium No 1 the population of strain 695 dissociated in 6 types of colonies and one subtype of type one (Fig. 2). After a five-times straining of the separate variants and their spreading on the same medium, the culture again dissociated in 4 type of colonies and one subtype of type I.

Gauze's medium No 1 was chosen for further selection because the strain had a higher antibiotic activity on it. A potato agar, on which the strain 695 remained morphologically homogeneous and saved its biosynthetic abilities, was suitable for its maintenance.

Characteristic of the morphological variants, isolated by natural cultivation of strain 695 on mineral Gauze's medium No 1

The population of strain 695 is a dynamic community, in which the separate morphological variant types are in a definite connection (Fig. 3).

On the medium mentioned above, the population included 4 morphological types of colonies and one subtype with the following characteristics (Fig. 4):

First type (dominating): round, flat colonies with a peak in the centre and undulating edges, with a diameter of 3-5 mm. The aerial mycelium covering the whole colony was white to greyish-pink. The vegetative mycelium was goldish to pale lemon-yellow; pale coppery soluble pigment. Subtype Ia: its colonies were flat with a diameter of 3-5 mm, without aerial mycelium in the centre.

Second type: round colonies with a diameter of 4-5 mm, with slightly undulating edges, flat, with a peak in the centre, scarce aerial mycelium and only in the centre, white. The vegetative mycelium was pale lemon-yellow. Pale coppery-yellow pigment.

Third type: round, slightly protruding colonies with a diameter of 4 mm, slightly undulating edges, a crater in the centre, with a small umbilicus. The aerial mycelium was granular, grey-pink to grey-violet and it covered tightly the whole colony. The vegetative mycelium was lemon-yellow, and the soluble pigment - pale coppery.

Fourth type: round, domed colonies with wrinkled surface and undulating edges, scarce aerial mycelium and only in centre, white. The vegetative mycelium and the soluble pigment were pale lemon-yellow.

By some streptomycetes strains the quantity of the first morphological variant varied 98% to 75% from the whole amount of colonies [4, 6, 9]. Strain 695 characterised relatively small number of colonies from the basic morphological type (47.58%) of the population and relatively high content of colonies from type II (20.93%) and type III (26.35%).

Natural variability of strain 695 according to its antibiotic formation capability. A correlation between variant's morphology and the quantity of the synthesised antibiotic

The results of the investigation of the natural variability of strain 695 according to its antibiotic activity showed that the colonies from the basic morphological type of the population had the highest antibiotic activity, which was 102.68% when compared to the activity of the initial strain (Table 3). The lowest was the antibiotic activity of the variants from type IV - mean 80.25% towards the parental strain's activity. The highest coefficient of variation - 12.80% was a characteristic for this morphological type. Nevertheless there was no variant of this morphological type which antibiotic activity was higher than that of the control. The mean antibiotic activity of the colonies from types II and III was respectively 94.4% and 89.75% from the activity of the initial strain. A higher variability according characteristic antibiotic formation, had the colonies from type II. Their limits of variation were 53.75% - 110% in respect to the control activity.

The distribution of the variants according to the quantity of the produced antibiotic 695, shown in percents towards the control, was a normal (Gauss) one and it was characterised with a modal class in the variation line (Fig. 5). The modal class in morphological type I consisted of variants with antibiotic activity 100-120%, for types II and III - these were the variants with activity 80-100% towards the control. The quantity of the variants with antibiotic activity 60-80% and 80-100% towards the control was equal for type IV. This untypical distribution could be explained with the comparatively small number of isolated colonies from this type (Table 3).

For conditional limits, between which the whole distribution of the variants according to characteristic antibiotic activity was situated, M ± 2s was accepted, because of the possibility this presumption to be true was 0,95000, i.e. high enough. As "plus" variants M + 2s were registered, as "minus'' M - 2s . All the isolated colonies, despite of their morphological type, produced antibiotic substance. Every morphological type was characterised with a determined mean antibiotic activity. In one and the same type, a considerable variation in the level of the investigated characteristic was observed. This variation was revealed even when considerably small number of colonies was investigated. In our studies, "plus'' variants were established only at the natural cultivation of type I colonies (Table 3). This determined type I as the most suitable for the choice of high productive variants.

Despite of the level of the aerial mycelium development, all the colonial types formed soluble pale lemon-yellow to orange pigment. The results show that the colour intensity of the pigment correlates to the antibiotic activity, e.g. a correlation between macromorphology and the biosynthetic activity of the colony is a characteristic of the strain. The production of yellow-orange pigment is an exclusively important criterion. Such a correlation is established for another producers of actinomycins [4, 6] and for Actinomyces coeruleorubidus - producer of the antitumor antibiotic rubomicyn [8].

Several variants from the natural cultivation of strain 695, belonging to type I, were isolated during the investigations. Variant 695-3, producing 168 (mg/ml antibiotic, which was 120% towards the control, was chosen for further investigation.

References

1. Alihanjan, S., 1968. Selection of industrial microorganisms, Moskva: Nauka,391 (in Russian).
2. Bondartsev, A., 1954. Colour scale. Moskva: Acad. Sciences USSR, 27 (in Russian).
3. Egorov, N., 1965. Microorganisms-antagonists and biological methods for evaluation of antibiotic activity . Moskva: High scool, 200 (in Russian).
4. Filippova, S., V. Kusnetsov, 1974. Antibiotics, 19(2), 104-107 (in Russian).
5. Kusnetsov, V., 1972. Antibiotics, 17(7), 666-671 (in Russian).
6. Kusnetsov, V., S. Filippova, 1974. Antibiotics, 19(2), 201-208 (in Russian).
7. Plohinskii, N., 1970. Biomethry, Moskva: High scool, 364.
8. Saburova, T., O. Lapchinskaya, E. Kruglyak, 1974. Antibiotics, 19(6), 509-513 (in Russian).
9. Sokolova, Z., K. Vinogradova. A. Polin, 1987. Antibiot. Med. Biothecnol., 32(1), 15-19 (in Russian).
10. Yocheva, L., 1996. Doctoral thesis, Sofia (in Bulgarian).
11. Yocheva, L., L. Kominkov, St. Antonova, 1998. Compt. Rend. Acad. Bulg. Sci., (in press).

Copyright 1998 - National Bank for Industrial Microorganisms and Cell Cultures - Bulgaria

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