Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 1, Num. 3, 1990, pp. 70-82

DNA Restriction Endonuclease Analysis Of Some Streptomyces Species

J.ZAKRZEWSKA-CZERWINSKA, K. KUCZEK, A. GASZEWSKA-MASTALARZ and M.MORDARSKI

Institute of Immunology and Experimental Therapy, Wroclaw, Poland.

Code Number: AC90012
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Abstract.

Relationships between some Streptomyces species were determined by restriction endonuclease analysis (REA). DNA isolated from twelve Streptomyces strains was analysed using SalI endonuclease. The results indicate that REA patterns can be used both to distinguish between species of Streptomyces and to recognize variation within species.

Restriction endonuclease analysis (REA) of genomic DNA has been used to classify bacterial strains within the genus Campylobacter (De Lisle et al., 1987), Clostridium (Devli et al., 1987), Escherichia (Marshall et al., 1985), Legionella (van Ketel 1988), Mycobacterium (Collins and De Lisle, 1985), Neisseria (Bjorvatan et al., 1984), Staphylococcus (Renaud et al., 1988) and Streptococcus (Skjold et al., 1987). The technique has also been adopted to distinguish between Streptomyces coelicolor, S. noursei, S.parvullus, S.peucetius and S. scabies (Crameri et al., 1983) and between S.glaucescens strains (Hintermann et al., 1981).

In the present study, the REA technique has been used to determine the relationships between representatives of nine Streptomyces species some of which were included in an extensive numerical taxonomic study of the genus Streptomyces (Williams et al., 1983).

Materials And Methods

Bacterial strains and growth conditions. The test strains (Table 1) were grown at 30 C in yeast extract-malt extract (YEME + 34% sucrose) broth (Chater et al., 1982).

Restriction endonuclease analysis. High molecular weight, genomic DNA was prepared as described by Hopwood et al. (1985). DNA samples (2 ug) were digested with the following restriction enzymes: AvaI, BamHI, BglII, ClaI, HindIII, KpnI, PstI, PvuII, SalI, TaqI and XhoI (Boehringer, Mannheim, FRG). The restriction fragments were separated by overnight electrophoresis in a 1.0% agarose gel at 30 V in TAE buffer (Maniatis et al., 1982) then stained with ethidium bromide and photographed under UV light.

Results And Discussion

Streptomyces lividans, TK 21, was screened against eleven restriction endonucleases. The SalI digest was chosen for further analysis as it gave the best-resolved bands.

The SalI digest patterns of DNA from the twelve Streptomyces strains are shown in Figure 1. Differences in DNA profiles of the test strains were observed in the 4.0 kb to 23 kb range. The intense bands of the S.coriofaciens, ISP 5485, DNA digest (Fig. 1 D) differentiate this strain from the closely related S.coelicolor, ISP 5233. These strains were assigned to cluster 1A on the basis of numerical taxonomic-data (Williams et al., 1983) and have been shown to have a high DNA-DNA homology (>90%; Mordarski et al., 1986). The differences in the DNA profiles of these strains are due to the presence of repeated DNA in the S.coriofaciens, ISP 5485, genome (Kuczek and Mordarski, 1986).

Table 1. Test strains.
(ATCC, American Type Culture Collection, Rockville, Md., USA; IAW, Institute of Antibiotics, Warsaw, Poland; IMET, Akademie der Wissenschaften der DDR, Zentralinstitut fur Mikrobiologie und Experimentelle Therapie, Jena, GDR.; ISP, International Streptomyces Project; PCM, Polish Collection of Microorganisms, Wroclaw, Poland. * Cluster defined in the numerical classification of Williams et al. (1983). ** Saccharopolyspora erythraea, ISP 5517, was previously classified as Streptomyces erythraeus (Labeda, 1987).

Strains                   No. of    Source 
                          Cluster* 
-------------------------------------------------
Saccharopolyspora 
erythraea,** 
ISP 5517 (ATCC 11635)       1C       M.Goodfellow 
Streptomyces 
albus, 
ISP 5313 (ATCC 3004)        16       M.Goodfellow 
Streptomyces 
coelicolor, 
(S. violaceoruber 
sensu Kutzner & Waksman)    --       H.Prauser 
IMET 40271 
Streptomyces 
coelicolor, 
ISP 5233 (ATCC 23899)       1A       M.Goodfellow 
Streptomyces 
coriofaciens, 
ISP 5485 (ATCC 14155)       1A       M.Goodfellow 
Streptomyces 
fradiae, 
PCM2330 (ATCC 10745)        68       S.Rowinski 
Streptomyces 
griseus, 
ISP 5236 (ATCC 23345)       1B       M.Goodfellow 
Streptomyces 
griseus, 
W 3478 (ATCC 3478)          --       K.Trojanowska 
Streptomyces 
lividans, TK 21             --       D.Hopwood 
Streptomyces 
rimosus, 
IAW 116 (ATCC 10970)        42       J.Wolf 
Streptomyces 
vinaceus, 
IAW 126 (ATCC 11861)        --       J.Wolf 
Streptomyces 
viridogenes, 
IAW 129 (ATCC 3372)          3       J.Wolf

Figure 1. DNA fragment patterns of Streptomyces strains after digestion with SalI. Lanes: A, S. albus, ISP 5313; B, S. coelicolor (S.violaceoruber sensu Kutzner and Waksman), IMET 40271; C, S.coelicolor, ISP 5233; D, S.coriofaciens, ISP 5485; E, Saccharopolyspora erythraea, ISP 5517; F, S.fradiae, PCM 2330; G, S.griseus, ISP 5236; H, S.griseus, W 3478; I, S.lividans, TK 21; J, S.rimosus, IAW 116; K, S. vinaceus, IAW 126; L, S. viridogenes, IAW 129.

Two other closely related strains, namely S. coelicolor (S.violaceoruber sensu Kutzner & Waksman), IMET 40271, and S. lividans, TK 21 (Cullum et al., 1986), gave similar restriction fragment patterns (Fig. 1 B, I). The major difference observed between these patterns is due to the presence of a high copy plasmid in S.coelicolor, IMET 40271 (3.75 kb; J.Zakrzewska-Czerwinska, unpublished results).

The two S.griseus strains gave nearly identical REA profiles (Fig. 1 G, H). However, the DNA digest of S.griseus, ISP 5236 (ATCC 23345), had a band (indicated by arrow) that was not visible in the pattern for S.griseus, W 3478 (ATCC 3478). The remaining strains, which represented Streptomyces species assigned to separated clusters in the numerical taxonomic study (Table 1; Williams et al., 1983), showed different REA patterns (Fig. 1A, E, F, J, K, L). DNA from S.albus, ISP 5313, was completely resistant to digestion by SalI (Fig. 1A), possibly due to the methylation of the cytosine residue in the sequences recognized by SalI. Sequence-specific methylation has been found in S. albus G which produces SalI (Kessler et al., 1985).

It is evident from the present study that REA patterns can be used both to distinguish between species of Streptomyces and to recognize variation within species. The ability of REA analysis to differentiate between closely related strains has also been noted for other Streptomyces species (Hintermann et al., 1981) as well as for other bacterial genera (Marshall et al., 1985; Collins et al., 1985; Bjorvatan et al., 1984).

The most comprehensive numerical phenetic survey (Williams et al., 1983) has been based on comparison of morphological and biochemical characters, many of them known as genetically unstable. Therefore, Schrempf et al. (1988) have postulated that REA analyses could be of great use in distinguishing different Streptomyces species.

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Copyright 1990 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia