Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 10, Num. 1, 2000, pp. pp. 7-9

L.V. POLISHCHUK, V.V. LUKYANCHUK and B.P. MATSELYUCH

Institute of Microbiology and Virology, National Academy of Sciences, Kyiv, Ukraine

ABSTRACT. An investigation on a small collection of Streptomyces strains, freshly isolated from soil, showed the presence of plasmids in 25% of the cultures. Two plasmids were subjected to restriction enzymes.

Plasmids are found in different species of streptomycetes where they can affect various characteristics (Chater & Hopwood, 1993; Kinashi, 1992; Lin & Xue, 1992; Malina et al., 1992; Pernodet et al., 1994). Isolation and investigation of new plasmids are important both for ascertaining the effects on the hosts as well as utilising those with potential in bioengineering.

In this study the presence of plasmids in a small collection of Streptomyces strains, freshly isolated from soil, has been investigated.

Organisms. Fifty-one strains were isolated from samples of grey podzolic soil collected in the Kiev district of Ukraine.

Growth conditions. Microorganisms were grown for 2-3 dd. at 28°C as shaken cultures in medium S (Okanishi et al., 1974) plus glycine (0.1-0.5%).

DNA isolation and endonuclease screening. Plasmid DNA was isolated from the mycelium according to Kieser's (1984) method. The presence of restriction enzymes was tested according to Belavin et al. (1988).

Thirteen strains (Nos. 14 to 17, 19, 21, 22, 25 to 27, 40, 44 and 48) out of the fifty-one isolated were found to harbour plasmid DNAs.

Agarose gel electrophoresis of the plasmids showed that some were characterised by different mobility, while for a few mobility was similar. As known, different plasmids may have an identical molecular mass, as, e.g., plasmids pSAM2 and pSE101, isolated from Streptomyces ambofaciens (ATCC 23877) and Saccharopolyspora erythraea (NRRL 2338) respectively. Notwithstanding the same molecular mass (11.2 kb), restriction analysis showed that the two were different (Brown et al., 1988; Pernodet et al., 1984).

During the investigation two groups of plasmids with similar electrophoretic mobility were detected. The first (molecular mass 13.5±0.2 bb) included four plasmids, pSS14 (from Streptomyces sp., strain No. 14), pSS27, pSS40 and pSS48. Plasmids pSS19, pSS21 and pSS44 were characterised by a 60 kb mass. The biggest plasmid found was pSS15 (200 kb).

However the comparison of morphological and cultural characteristics pointed out that the cultures were different. In particular diffusable pigments were formed by strains Nos. 14, 25, 26 and 27.

Type II restriction endonuclease activity was demonstrated for strains 14 to 17, 19, 22, 25 to 27 and 48. Plasmids pSS14 and pSS27 were subjected to restriction enzymes and the results, showing the different sequence of nucleotides, are summarised in Table 1.

In conclusion the widespread (13 strains out of 51) presence of plasmids in streptomycetes, freshly isolated from soil, has been confirmed. The data (25%) are in agreement with those obtained by other investigators (Daniel & Tiraby, 1983; Hopwood et al., 1981; Okanishi et al., 1980; Omura et al., 1981; Tomyama et al., 1982).

Table 1. Restriction fragments of plasmids pSS14 and pSS27

Belavin, P.A., V.V. Dedkov & S.K. Degtyarev (1988). A simple technique for detection of restriction endonucleases in bacterial colonies. Prikladnaja Biokhimija i Mikrobiologija, 1(1):121- 124 (in Russian)

Brown, D.P., S.-J. Chiang, J.S. Tuan & L. Katz (1988) Site-specific integration in Saccharopolyspora erythrea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101. J. Bacteriol., 170(5): 2287- 2295

Daniel, D. & S. Tiraby (1983) A survey of plasmids among natural isolates of Streptomyces. J. Antibiotics, 2: 181- 183

Chater, K.F. & D.A. Hopwood (1993) Streptomyces. In: J.P. Sonenshein (ed.) Bacillus subtilis and Other Gram-Positive Bacteria. Biochemistry, Physiology and Molecular Genetics. ASM, Washington, DC, 83- 89

Hopwood, D.A., C.J. Tompson, T. Kieser, J.M. Ward & H.M. Wright (1981) Progress in the development of plasmid cloning vectors for Streptomyces., In : D. Schlesinger (ed.) Microbiology - 1981. ASM, Washington, DC, 376- 379

Kieser, T. (1984) Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid, 12: 19- 36

Kinashi, H., M. Shimaji-Murayama & T. Hanafusa (1992). Integration of SCPI, a giant linear plasmid into the Streptomyces coelicolor chromosome. Gene, 115 : 35- 41

Lin, C. & Y. Xue, (1986). Plasmid characteristics of thermophilic streptomycetes. In: S. Szabó, S. Biró & M. Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akadémiai Kiadó, Budapest, 606

Malina, H., F. Lawrence & M. Robert-Géro (1992) Cloning of sinefungin biosynthetic genes from Streptomyces incarnatus to Streptomyces lividans. Actinomycetes, 3: 25- 30

Okanishi, W.L., K. Suzuki & H. Umezawa (1974). Formation and reversion of Streptomyces protoplasts: cultural conditions and morphological study. J. gen. Microbiol., 80: 389- 400

Okanishi, M., N. Manome & H. Umezawa (1980). Isolation and characterisation of plasmid DNAs in actinomycetes. J. Antib.,. 33: 88-91

Omura, S., H. Ikeda & H. Tanaka (1981). Extraction and characterisation of plasmid from macrolide antibiotic-producing Streptomyces. J. Antib., 34 : 478- 482

Pernodet, J.-L., J.-M. Simonet & M. Guerineau (1984). Plasmid in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2. Mol. Gen. Genetics, 184: P35- 41

Rhodes, P.M., I.S. Hunter, E.J. Friend & M. Warren (1984). Recombinant DNA method for the oxytetracycline producer Streptomyces rimosus. Biochem. Soc. Trans., 12: 586- 587

Toyama, H., E. Hayashi, C. Nojiri, K. Katsumata, A. Miyata & Y. Yanada (1982). Isolation and characterization of small plasmids from Streptomyces. J. Antib., 35: 369- 373.

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