Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 10, Num. 1, 2000, pp. pp. 13-15

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

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

ABSTRACT. Production of restriction enzymes is widespread among soil streptomycetes. More than 15% of fresh soil isolates showed this ability. Of eight strains showing enzyme activity, seven formed isoschizomers of AsuII. Enzymes with such specificity were not previously found amongst streptomycetes.

Enzymes of restriction-modification (RM} systems are present in large amounts in streptomycetes and some strains are strong producers of endonucleases (e.g., SacI, SacII, SalGI}. Streptomycetes also produce isoschizomers of EcoRI, Pst1 and others. Studies on these enzymes are valuable for understanding regulation and functioning of RM systems, actual production of the enzymes themselves and for pharmaceutical and other biotechnological applications (Rodicio & Chater, 1988}. The aim of the present study was to investigate the amount of restrictases among fresh soil isolates.

Fifty-one streptomycetes, randomly chosen from a collection of strains isolated from podzolic soils in the Kiev district of Ukraine, were investigated. The strains were grown as shaken cultures on medium S (Okanishi et al., 1974) for 2- 3 dd at 28°C.

The presence of restriction enzymes was tested according to Belavin et al. (1988). Site-specificity was determined by comparison with known DNA electrophoretic fragments from the literature and computer data. Phage l and T7 as well as plasmid pBR322 and pUC19 DNAs were used as reference.

Of the fifty-one strains randomly chosen streptomycetes, eight (over 15%) show site-specific activity (Table 1). The percentage is in agreement with the results of other investigators (Kessler et al., 1985}.

In addition 42 strains showed non-specific endonuclease activity. It is possible that some of these also possess site-specific, but masked endonucleases. Therefore the 15% should be taken as an underestimate.

Seven restriction enzymes cut l DNA at seven sites, T7 at six sites, but did not cleave pUC19 and pBR322 DNA. Ssp27 endonuclease shows one site at each of l, T7 and pBR322, but none at pUC19.

Table 1. Enzyme production by the different Streptomyces strains (?: the site specificity of the restrictase has not yet been ascertained)

By comparing recognition sites at the level of phage and plasmid DNAs with available data it is possible to ascertain that the seven strains produce isoschizomers of AsuII. The site-specificity of Ssp27 is under investigation.

Morphological and physiological characteristics of AsuII producers show that strains 42 and 43 are very similar, in addition they were isolated from the same sample.

The other isolates show morphological and physiological differences in the colour of aerial mycelium, soluble pigments and presence of plasmids. Anyway other authors have demonstrated the production of restrictase isoschizomers by strains randomly picked from culture collections and belonging to different actinomycete genera (Baltz, 1986; Kessler et al., 1986; Peczynska-Czoch & Mordarski, 1988; Roberts & Macelis, 1992).

In conclusion the results of the investigation show that six strains out of 51 are able to produce isoschizomers of AsuII, a feat not previously recorded for streptomycetes.

Work on the site-specificity of Streptomyces sp. 27 restrictase (Ssp27) is in progress.

Baltz, R. H. (1986}. Mutation in Streptomyces. In: S.W. Queener & L.E. Day (Eds.) The Bacteria. Vol. IX. Academic Press, New York, pp. 61- 94

Belavin, P.A., V.V. Dedkov & S.K. Degtyarev (1988). A simple technique for the detection of restriction endonucleases in bacterial colonies. Priklad. Biokhim. Mikrobiol., 1: 121-124 (in Russian)

Kessler C., T.S. Neumaier & W. Wolf (1985). Recognition sequences of restriction endonucleases and methylases - a review. Gene, 33 : 1-120

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

Peczynska-Czoch, W. & M Mordarski (1988}. Actinomycete enzymes In: M. Goodfellow, S.T. Williams & M. Mordarski (eds.) Actinomycetes in Biotechnology. Academic Press, London, pp. 220 - 283

Roberts, R.J. & D. Macelis (1992}. Restriction enzymes and their isoschizomers. Nucleic Acids Research, 20: 2167- 2168

Rodicio, M.R. & K.F. Chater (1988}. The Sal1(SalG1} restriction - modification system of Streptomyces albus. Gene, 74: 39-42.

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