Instability of the production of avermectins, sporulation, and pigmentation in Streptomyces avermitilis

JAN N o v k , JAN KOPECKY, OLGA KOFRONOVA, AND ZDENKO VANEK Institute of Microbiology, VideZiska 1083, 142 20 Praha 4, Czechoslovakia

Received February 27, 1992

Revision received September 25, 1992

Accepted October 22, 1992

NOVAK, J., KOPECKY, J., KOFRONOVA, O., and VANEK, Z. 1993. Instability of the production of avermectins, sporulation, and pigmentation in Streptomyces avermitilis. Can. J . Microbiol. 39: 265-267.

Production of avermectins, sporulation ability, and colony pigmentation were followed in Streptomyces avermitilis C-18/6 cultures during serial transfer (for eight subcultures) in four different liquid media. These phenotypes were found to be unstable and independent of each other. A procedure was established whereby depressed avermectin production could be reversed but the degree of reversion was dependent on the history of the isolate, i.e., the type of medium used. The presence of isoleucine as the sole N source caused an immediate loss of avermectin production and this could not be reversed.

Key words: avermectins, instability, pigmentation, sporulation, Streptomyces avermitilis.

NOVAK, J., KOPECKY, J., KOFRONOVA, O., et VANEK, Z. 1993. Instability of the production of avermectins, sporulation, and pigmentation in Streptomyces avermitilis. Can. J . Microbiol. 39 : 265-267.

La production d'avermectines, le pouvoir de sporulation et la pigmentation des colonies ont $tC Cvalues dans des cultures de Streptomyces avermitilis C-18/6 lors de repiquages (huit sous-cultures) dans quatre milieux de culture differents. Les phenotypes ne sont pas stables et ils sont independants l'un de I'autre. Une mithode a CtC mise au point pour inverser la production rkduite d'avermectine mais le degrC de reversion depend des conditions d'isolement, i.e., du type de milieu de culture utilid. Lorsque la seule source de N disponible est l'isoleucine, il y a une perte immediate de la production d'avermectines et ce phknomkne ne peut Ctre inversC.

Mots clgs : avermectines, instabilitb, pigmentation, sporulation, Streptomyces avermitilis. [Traduit par la redaction]

Phenotypic instability of markers, such as sporulation, pigment synthesis, secondary metabolite production, or resistance, is a common occurrence in streptomycetes (Birch et al. 1990; Cullum et al. 1988). The presence of such instability is usually described on the basis of the frequency with which mutant colonies arise from spores plated on agar media. Another approach, the study of population changes during long-term cultivation, is also possible.

Streptomyces avermitilis produces avermectins, a family I of 16-membered antiparasitic macrolides (Burg et ol. 1979). In this paper we describe the instability of three phenotypic characters, i.e., avermectin production (Avm), sporulation (Spo), and pigmentation (Pig) during growth of a serially transferred population of S. averrnitilis C18/6 for eight generations in liquid media.

Streptomyces avermitilis C-18/6, a derivative of C-18 (Cimburkova et al. 1988), was cultivated in four media (A, B, C, and D) consisting of a basal medium supplemented with four different combinations of the carbon and nitrogen sources. The basal medium contained the following (% w/v): CaC03, 0.5; NaC1, 0.2; K2HP04, 0.05; FeS04- 7H20, 0.005; ZnS04. 7H20, 0.005; MnS04 7H20, 0.005; MgS04.7H20, 0.01. The supplements were as follows (Vo w/v): medium A: glucose, 3; (NH4)2S04, 0.2; medium B: glucose, 6; (NH4)2S04, 0.2; yeast extract (Oxoid), 0.2; medium C: glucose, 3; isoleucine, 0.2; medium D: glucose, 0.4; yeast extract (Oxoid), 0.4; malt extract (Oxoid), 1 .O. The pH was adjusted in all media to 7.2. The first generation was inoculated with a 4% (v/v) inoculum

1 2 3 4 5 8 7 8

generation FIG. 1. Production of avermectins in Streptomyces avermitilis

during cultivation for eight generations in four types of liquid media: A (w), B (a), C (A), and D (0). Production of the first subculture in medium B (inoculum grown in medium A) is the ref- erence value (100070).

of culture grown in medium A for 3 days. Each generation medium on a rotary shaker (2.8 at 280C for days. Two was in containing mL of millilitres of fermentation liquid were used as inoculum for

each succeeding generation. Production of avermectins was ' ~ u t h o r to whom all correspondence should be addressed. estimated by TLC as described by Cimburkova et al. (1988).

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FIG. 2. SEM of colony surface of sporulating (A) and nonsporulating (B) strains isolated from the first and eighth generation in liquid medium B, respectively. Isolates were grown for 10 days on agar medium D at 28OC. Note the spiral sporophores in A and their absence in B. Scale bar = 5 pm.

TABLE 1. Phenotypic characters of the strains isolated from particular generations of S . avermitilis cultivated in media B and C

Culture of origin Phenotypic character

Medium Subculture AVM (%)* Strain Spo Pig AVM (%)$

Parental C-18/6 + + 100 8 1 66 YE 1 + - 94

4 8 YE 4 - - 101 8 4 YE 8 - - 44

C 1 0 IS 1 + + 9 4 0 I S 4 + / - +I-' 10 8 0 I S 8 + / - + / - 3

*Avermectin production of a particular generation in liquid media B and C, respectively, compared with the reference value.

+~vermectin production of isolated strains after subculture in liquid medium D. Complex inoculation medium (Cimburkova et al. 1988) and production medium B were used for testing. Production of the parental strain under the same conditions is the reference value (100%).

5~eterogeneous population.

Pigmentation and sporulation were examined on medium D gelled with 2.5% (w/v) agar.

The morphology of colonies grown on agar medium was observed with a TESLA BS 300 scanning electron micro- scope operating at 25 kV. Small pieces of agar medium (approx. 20 mm2) bearing streptomycete mycelium were fixed in the vapour of aqueous 2% Os04 in a vacuum desiccator. After 2 days the samples were transferred to stainless-steel sieve holders and incubated in water at 40-50°C for 30 min. They were then dehydrated in acetone

I

vapour for 2 days, mounted on aluminium sample holders with a silver conductive paint, and sputter-coated with gold, using a Polaron 11 HD sputter-coating unit.

Avermectin production declined rapidly during cultiva- tion of S. avermitilis for eight generations in media A, B, and D (Fig. 1). In media A and D production decreased, resulting in the appearance of the Avm- phenotype in later generations (third subculture and thereafter). However, in medium B, low production (approx. 6% of the original value) was detected even after eight generations. In

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NOTES 267

medium C the Avm- phenotype appeared in the first generation and the production of avermectins was not detected during the whole cultivation cycle.

Samples from the first, fourth, and eighth generations in all types of liquid media were plated on gelled medium D. Sporulation ability and pigmentation were observed as the cultures grew. Concerning these characters, relevant changes were found after cultivation in media B and C (Table I).

The Pig', Spo + phenotype was observed in the isolate from the first generation in medium C. The populations isolated from both the fourth and eighth generations were heterogeneous in these two phenotypic characters. The cul- ture from the eighth generation involved approximately 85% Pig+, Spo'; 5% Pig+, Spo-; 3% Pig-, Spof , and 7% Pig - , Spo - colonies.

The Pig - , Spo + phenotype was observed in the strain isolated from the first subculture in medium B. The Pig-, Spo - phenotype was found in the isolates from both the fourth and eighth subcultures, when the growth was homogeneous with respect to the characters followed. Examination by scanning electron microscopy of samples from the first generation in medium B for sporulation on gelled medium D showed spiral sporophores on aerial mycelium (Fig. 2A); in a similar examination of the eighth generation, sporophores were absent (Fig. 2B). The Pig', Spo + phenotype was observed in all strains isolated from media A and D (data not shown).

The strains isolated from the first, fourth, and eighth generations in all types of media were treated further in the following way: spores were used to inoculate liquid medium D and the cultures were grown without shaking for 4 days. Mycelium was then plated on gelled medium D. Avermectin production was tested in liquid medium B, using a complex inoculation medium described by Cimburkova et al. (1988). Avermectin yields were significantly higher for all isolates from medium B than for the cultures of origin (Table 1). Similar results were obtained for avermectin production when isolates from media A and D were treated in the same manner (data not shown). Strains with the

Spo - , Pig - phenotype isolated from later generations in medium B showed no reversion of these characteristics.

Different results were obtained with strains isolated from medium C (Table 1). While treatment of isolates from medium B restored the yield of avermectins almost to the original level, treatment of isolates from medium C restored production to less than 10% of that value (medium B was used for testing).

Though the instability of some phenotypic markers in streptomycetes is supposed to be dependent on the type of medium (Volff et al. 1991), the reason for the rapid loss of avermectin biosynthesis caused by growth of S. avermitilis in isoleucine-supplemented medium is unclear. Understand- ing of this regulatory phenomenon could be important in devising an effective strategy for avermectin production.

Acknowledgement The authors thank Mrs. Jarmila DvoAkova for ideas used

for reversion of depressed avermectin production and for excellent technical assistance.

Birch, A., Hausler, A., and Hiitter, R. 1990. Genome rearrange- ment and genetic instability in Streptomyces sp. J. Bacteriol. 172: 4138-4142.

Burg, R.W., Miller, B.M., Baker, E.E., Birnbaum, J., Currie, S.A., Hartman, R., Kong, Y.L., Monaghan, R.L., Olson, G., Putter, I., Tunac, J.B., Wallack, H., Stapley, E.O., Oiwa, R., and Omura, S. 1979. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob. Agents Chemother. 15: 361-367.

Cimburkova, E., Zima, J., Novkk, J., and Vanek, Z. 1988. Nitrogen regulation of avermectin biosynthesis in Streptomyces avermitilis in a chemically defined medium. J. Basic Microbiol. 28: 491-499.

Cullum, J., Flett, F., and Piendl, W. 1988. Genetic instability in streptomycetes. Microbiol. Sci. 5: 233-235.

Volff, J.N., Vandewiele, D., Sirnonet, J.M., and Decaris, B. 1991. Factors stimulating genetic instability in Streptomyces ambofaciens ATCC 23877. In International Symposium on Biol- ogy of Actinomycetes, ISBA 1991, August 11-16, 1991. Univer- sity of Wisconsin, Madison. Abstr. PI-097.

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