Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 9, Num. 3, 1998

I. Saadoun, F. Al-Momani and A. Elbetieha

Department of Applied Biological Sciences, Jordan University of Science and Technology, Irbid-22110, Jordan

Members of the genus Streptomyces isolated from nature carry detectable covalently closed circular (CCC)-DNA. Four different extraction methods of small plasmid DNA from antibiotic-producing Streptomyces isolates and from the positive control S. lividans ATCC 35287, containing the pIJ702 plasmid, are reported. Only one procedure allowed detection of a plasmid from the positive control. No low molecular weight plasmids were observed in Streptomyces strains No. 264 and No. 404 suggesting that antibiotic production in these strains is likely to be chromosomally encoded.

Streptomycetes synthesise about two-thirds of the currently known antibiotically active substances (Crandall & Hamill, 1986). The economic importance has led to increased interest in the genetic aspects of antibiotic biosynthesis by these organisms (Fayerman, 1986; Gramajo, 1993; Hopwood et al., 1985; Jensen et al., 1993; Stutzman-Engwall et al., 1992).

Many streptomycetes carry detectable extrachromosomal elements (plasmids) and, in most cases, plasmids are present in the form of covalently closed circular (CCC)-DNA, but, occasionally, linear elements are also found (Chardon-Loiraux et al., 1986; Haruyasu et al., 1987; Hayakawa et al., 1979; Hirochika et al., 1982, 1984; Ishibashi, 1992; Keen et al., 1988; Saadoun & Blevins, 1997). So far, a number of different streptomycetes have been investigated for plasmids supposedly involved in the genetic control of the production of antibiotics (Chater et al., 1985; Kirby & Hopwood, 1977; Hayakawa et al., 1979; Hopwood, 1983). However, more detailed analyses have shown that antibiotic biosynthetic structural genes reside mostly on the chromosome (Hütter & Eckhardt, 1988).

Numerous alkaline lysis procedures for the isolation and purification of CCC-DNA have been described (Yagisawa et al., 1978; Westpheling, 1980; Omura et al., 1981; Pernodet & Guerineau, 1981; Kieser et al., 1982; Daniel & Tiraby, 1983). In this study, different plasmid extraction methods were tested to detect plasmid DNA in streptomycetes isolated from Jordanian soils and showing potential antibiotic activity.

Organisms.

The following organisms were used: 1) Streptomyces lividans ATCC 35287, containing pIJ702 plasmid (5.65 kb in size) (Katz et al., 1983), used as a positive control; 2) Escherichia coli strain X2556 (same as VA 517 according to Macrina et al., 1978), containing eight plasmids ranging in size from 1.4 megadalton (Mdal) to 35.6 Mdal, used as a source of molecular weight markers and 3) antibiotic-producing Streptomyces strains (No. 264 and No. 404) isolated from Jordanian soils.

Growth Conditions.

Streptomycetes were grown on yeast extract-malt extract (YEME) broth (Hopwood et al., 1985) (g/l: 3 yeast extract, 5 Bacto-peptone, 3 malt extract, 10 glucose, 30 sucrose, 5 glycine and 2 ml of a 2.5 M MgCl₂.6H₂O solution, the latter added after autoclaving). Streptomycetes were grown in shaken (100 rpm) broth culture at 28°C for 3 dd. E. coli was cultured on yeast extract-dextrose (YD) agar (g/l: 10 dextrose, 10 yeast extract, 5 glycerine, pH 7.5) at 37°C overnight.

Recovery of Plasmid DNA.

Lysis methods suggested by Kieser (1984), Babcock & Kendrick (1988), Okanishi & Manome (1980) and Birnboim & Doly (1979) were followed for detection of small molecular weight plasmid CCC-DNA. Gel electrophoresis was carried out with 0.7% agarose gels using a Tris-borate (0.045 M)-EDTA (0.001M) buffer (0.5 x TBE). Electrophoresis was carried out at a constant current of 35 mA. All gels were stained with 0.5 g/ml ethidium bromide solution for 30 to 60 min at room temperature followed by 30-60 min destaining in distilled water. Gels were viewed under an ultraviolet (300 nm) transilluminator Model TM-20 (Ultra Violet Products Inc., Upland, CA), then photographed using a Polaroid DS-34 Direct Screen Instant Camera (Polaroid Gel Cam, U.K.) fitted with a Tiffen 40.5 mm deep yellow 15 filter (Tiffen Manufacturing Co., Hauppauge, NY).

Following the protocols of Babcock & Kendrick (1988), Okanishi & Manome (1980) and Birnboim & Doly (1979), no small CCC plasmid DNA could be detected in either the antibiotic-producing Streptomyces strains No. 264 and No. 404 or in the positive control using conventional agarose gel electrophoresis (Fig. 1, A to C). The Birnboim & Doly (1979) procedure, used to isolate Streptomyces plasmids by several authors (Bibb et al., 1981, Doull et al., 1983) shows large amounts of chromosomal DNA which give a fluorescent streak in the gel and is therefore unsatisfactory (Omura et al., 1981). However, the extraction method suggested by Kieser (1984) shows at least one plasmid band (~ 5.4 kbp) from S. lividans ATCC 35287 (Fig. 1, D).

The Kieser method shows better recovery of small CCC plasmid DNA from S. lividans ATCC 35287 and from E. coli than the other methods. No low molecular weight plasmid DNA is observed using the different lytic techniques in either of the antibiotic-producing Streptomyces isolates. The possible presence of a low copy number plasmid cannot be excluded. However, several studies show that antibiotic production in Streptomyces sp. is encoded by either small or giant linear plasmids (Hirochika et al., 1984; Keen et al., 1988; Kinashi & Shimaji, 1987; Hershberger et al., 1989).

So far separation of large linear DNA plasmid from chromosomal DNA by conventional techniques in streptomycetes has been unsuccessful (Kinashi & Shimaji, 1987). Large linear DNAs, higher than 30 kb, have been shown to comigrate in conventional agarose gel electrophoresis and are therefore not separable.

Overall results of this study indicate that antibiotic production in the isolates is likely to be chromosomally encoded. However because of the role of giant linear plasmids in streptomycetes, which could code for antibiotic production, further study is recommended to isolate such plasmid(s) from the organisms.

This research was financed by Jordan University of Science and Technology, grant No. 12/98. Technical assistance of Ms Suha Makhloof and Mr. Khaleel Aftan are gratefully acknowledged.

Birnboim, H. C. & J. Doly (1979). A rapid alkaline extraction for screening recombinant plasmid DNA. Nuc. Acid Res., 7:1513-1523

Babcock, M. J. & K. E. Kendrick (1988). Cloning of DNA involved in sporulation of Streptomyces griseus. J. Bacteriol., 170: 2802-2808

Bibb, M. J., J. M. Ward, T. Kieser, S. N. Cohen & D. A. Hopwood (1981). Excision of chromosomal DNA sequences from Streptomyces coelicolor forms a novel family of plasmids detectable in Streptomyces lividans. Mol. Gen. Genet., 154: 230-240.

Chardon-Loiraux, I., M. Charpentier & F. Percherson (1986). Isolation and characterization of a linear plasmid from Streptomyces rimosus. FEMS Microbiol. Lett., 35: 151-155

Chater, K. F. & C. J. Bruton (1985). Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J., 4:1893-1898

Crandall, L. W. & R. L. Hamill (1986). Antibiotics produced by Streptomyces: Major structural classes. In: S. W. Queener & L. E. Day (eds.) Antibiotic-producing Streptomyces, The Bacteria. Volume IX. Academic Press, New York, pp. 355-401

Daniel, D. & G. Tiraby (1983). A survey of plasmids among natural isolates of Streptomyces. J. Antibiot., 36: 181-183

Doull, J. L., L. C. Vining & C. Stuttard (1983). A cryptic plasmid in the chloramphenicol-producing actinomycete, Streptomyces phaeochromogenes. FEMS Microbiol. Lett., 16: 349-352

Fayerman, J. T. (1986). New developments in gene cloning in antibiotic-producing microorganisms. Biotechnology, 4:786 -789

Gramajo, H. C., E. Takano & M. J. Bibb (1993). Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol. Microbiol., 7: 837-843

Haruyasu, K., M. Shimaji & A. Sakai (1987). Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes. Nature, 328: 454-456

Hayakawa, T., T. Kanaka, N. Sakaguchi, N. Otake & H. Yonchara (1979). A linear plasmid-like DNA in Streptomyces sp. producing lankacidin group antibiotics. J. Gen. Appl Microbiol., 25: 255-260

Hershberger, C. L., B. Arnold, J. Larson, P. Skatrud, P. Reynolds, P. Szoke, P. R. Rosteck, Jr., J. Swartling & D. McGilvray (1989). Role of giant linear plasmids in the biosynthesis of macrolide and polyketide antibiotics. In: C. L. Hershberger, S. W. Queener & G. Hegeman (eds.) Genetics and Molecular Biology of Industrial Microorganisms. American Society for Microbiology, Washington, D.C., pp. 147-155

Hirochika, H., K. Nakamura & K. Sakaguchi (1982). Analysis of linear plasmids isolated from Streptomyces: association of protein with the ends of the plasmid DNA. Plasmid, 7: 59-65

Hirochika, H., K. Nakamura & K. Sakaguchi (1984). A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs. EMBO J., 3: 761-766

Hopwood, D. A. (1983). Actinomycete genetics and antibiotic production. In: L. C. Vining (ed.) Biochemistry and Genetic Regulation for Commercially Important Antibiotics. Adison-Wesley, Reading, Mass., pp. 1-23

Hopwood , D. A. , M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward & H. Schrempf. (1985) Genetic Manipulation of Streptomyces. A Laboratory Manual. The John Innes Foundation, Norwich

Hütter, R. & T. Eckhardt (1988). Genetic manipulation. In: M. Goodfellow, S. T. William & M. Mordarski (eds.) Actinomycetes in Biotechnology. Academic Press, London, pp. 89-184

Ishibashi, Y. (1992). Genetic studies into musty odor production by actinomycetes. Water Sci. Tech., 25: 171-176

Jensen, S. E., D. C. Alexander, A. S. Paradkar & K. A. Aidoo (1993). Extending the b-lactam biosynthetic gene cluster in Streptomyces clavuligerus. Industrial Microorganisms: Basic and Appl. Mol. Genet., pp. 169-176

Saadoun, I. & W. T. Blevins (1997). Detection of giant linear plasmids in off-flavor compound-producing strains of Streptomyces by PFGE. Actinomycetes, 8: 58-65

The following images related to this document are available:

Photo images