A LINEAR PLASMID-LIKE DNA IN STREPTOMYCES SP. PRODUCING LANKACIDIN

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1 J. Gen. App!. Microbiol., 25, (1979) A LINEAR PLASMID-LIKE DNA IN STREPTOMYCES SP. PRODUCING LANKACIDIN GROUP ANTIBIOTICS TAKAKI HAYAKAWA, TERUO TANAKA,* KENJI SAKAGUCHI,* NOBORU OTAKE, AND HIROSHI YONEHARA Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan *Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan (Received April 11, 1979) A linear plasmid-like DNA was isolated by Agarose gel electrophoresis from a lysate of Streptomyces sp AN4 which produces lankacidin group antibiotics. The DNA (psla2) with a molecular weight of 11.2 x 106 was cleaved into five and three fragments, respectively, with XmaI and BamNI on the definite sites from the end, but not digested by EcoRI and HindIII. Upon treatment of the strain with ethidium bromide, variants were obtained which have lost the ability to produce the antibiotics. These variants were found to have lost psla2. These results suggest that the linear plasmid-like DNA is involved in the production of lankacidin group antibiotics. Since the first observation by OKANISHI et al. (1) that plasmids may be involved in the production of kasugamycin and aureothricin, lines of evidence have accumulated on plasmid involvement in antibiotic production (2, 3). These observations are based on genetic analysis (4, 5) or on the loss of antibiotic production by host cells upon treatment with curing agents (1, 6). In several antibiotic-producing Streptomyces species (3, 7, 8), extrachromosomal DNA elements have been found as covalently closed circular (ccc) DNA. We have extended these studies by screening 34 antibiotic-producing strains of Streptomyces for plasmid, in order to obtain further insight into possible involvement of plasmids in antibiotic production. During these processes, we found a linear plasmid-like DNA which may be involved in the production of lankacidin group antibiotics. Results obtained are described in this paper. 255

2 256 HAYAKAWA, TANAKA, SAKAGUCHI,, OTAKE, and YONEHARA VOL. 25 MATERIALS AND :METHODS Bacterial strains. Streptomyces sp AN4 which produces lankacidin group antibiotics was obtained from the Institute of Applied Microbiology, University of Tokyo. Xanthomonas oryzae used for assaying the activity of lankacidin group antibiotics was kindly supplied by Dr. M. Uramoto of the Institute of Physical and Chemical Research. Isolation of a linear plasmid like DNA. Mycelia grown in 100 ml of Y-P medium (in grams pre liter: glucose, 3; polypeptone, 3; and yeast extract, 4; ph 7.0) were harvested and processed by the same procedure as for the isolation of Bacillus subtilis plasmid (9). Two volumes of cold (-20 ) ethanol was added to the lysate to precipitate the DNA, and the mixture was kept overnight at -20. The precipitate was collected by centrifugation, suspended in 10 ml of TES buffer containing 30 mm Tris-HCI, ph 8.0, 50 mm NaCI, 5 mm EDTA and 0.4 % sarkosyl, 0.4 ml of ribonuclease A (Sigma; 5 mg/ml preheated for 10 min at 100 ) was added, and the mixture was incubated for 30 min at 37. For preparation of plasmid DNA, disc gel electrophoresis was used. The DNA sample obtained was subjected to electrophoresis in 0.7 % Agarose (10). The areas of gels containing plasmid DNA were sliced and the plasmid DNA was purified by hydroxyapatite column chromatography. The gel slices were incubated with 1 volume of 5 M sodium perchlorate until they melted at 65. The gel solution was applied to the same volume of hydroxyapatite column which had been washed successively with 5 volumes of 0.5 M sodium phosphate (ph 6.9) and 10 volumes of 0.01 M sodium phosphate (ph 6.9). The column was washed with 10 volumes of 0.01 M sodium phosphate (ph 6.9), and then the plasmid DNA was eluted with 4 volumes of 0.4 M sodium phosphate (ph 6.9). The eluate was dialysed against 0.1 M NaCI, 0.01 M Tris buffer (ph 7.9), and M EDTA. The plasmid DNA was recovered by ethanol precipitation and dissolved in 0.1 x SSC (0.15 M NaC1, M sodium acetate) plus 1 mm EDTA. Electron microscopy. The procedure used for electron microscopy has been described previously (9). The electron microscope was the model JEOL-100B (Japan Electron Optics Laboratory, Tokyo). Digestion of DNA with restriction endonucleases. DNA was digested with endonuclease EcoRI, HindIII, BamNI, or XmaI for 1 hr at 37. The resultant DNA fragments were subjected to 0.7 % Agarose gel electrophoresis. Treatment with ethidium bromide and assay for antibiotic production. Streptomyces sp AN4 was grown in Y-P medium supplemented with ethidium bromide (1.56 µg/ml). Mycelia were cut with Polytron (Kinematica) and plated on lankacidin-producing plates described by URAMOTO et al. (11), in which yeast extract was substituted for the corn steep liquor. Ability of clones to produce the antibiotics was examined by the method of AKAZAWA et al. (4). The test strain for the bioassay was Xanthomonas oryzae.

3 1979 Linear Plasmid-like DNA in Streptomyces Producing Lankacidin 257 RESULT Isolation of plasmid When a lysate prepared from Streptomyces sp AN4 by the method using lysozyme, pronase and sarkosyl (9) was subjected to CsCI-ethidium bromide centrifugation, we failed to detect ccc-dna. However, an extrachromosomal DNA band was observed in the agarose gel when a DNA sample was subjected to gel electrophoresis, as shown in Fig. la. The upper band corresponds to the chromosomal DNA since it gave diffuse DNA bands upon treatment with SmaI (data not shown). The DNA in the lower band (psla2) was isolated from the gels (Fig. lb) and used for further study. Properties of psla2 plasmid More than 100 molecules of the psla2 plasmid-like DNA were examined by electron microscopy and all of them were found to be linear. The molecular weight was determined to be 11.2 x 106 by the use of RSF 1010 plasmid molecule as internal standard (5.5 x 106) (Fig. 2). Restriction endonucleases XmaI and BamNI cleaved psla2 into five and three discrete fragments, respectively (Fig. 3b, c). The molecular weight of psla2 was determined to be 11.3 x 106 by agarose gel electrophoresis (Fig. 3a). Sum of the molecular weights of the DNA fragments was 10.2 x 106 and 10.3 x 106 for XmaI and BamNI digestion, respectively. psla2 was not cleaved by EcoRI and HindIII. Fig. 1. Agarose (0.7%) gel electrophoresis of the psla2 plasmid DNA. (a) The upper band corresponds to chromosomal DNA and the lower to psla2; (b) psla2 plasmid DNA purified by hydroxyapatite column chromatography. Electrophoresis was from top to bottom. Fig. 2. Electron micrograph of plasmid DNA molecules. (a) Linear plasmid DNA of psla2; (b) circular plasmid DNA of RSF1010 as internal standard (5.5 x 106).

4 258 HAYAKAWA, TANAKA, SAKAGUCHI, OTAKE, and YONEHARA VOL. 25 Fig. 3. Cleavage of plasmid DNA. Digests were fractionated on Agarose gel electrophoresis. (a) Undigested psla2; (b) psla2 digested with XmaI; (c) psla2 digested with BamNI; (d) ADNA digested with Hind III. Fig. 4. (b) antibiotic nonproducing Agarose gel electrophoresis of psla2 plasmid DNA from: (a) parent strain; variant. Elimination of the ability to produce antibiotics by ethidium bromide treatment To test whether any plasmid is involved in the antibiotic synthesis in this strain, the mycelia were treated with ethidium bromide (1.56 µg/ml) as described in the Methods. Six among 135 colonies tested (4.4%) were found to have lost the ability ro produce antibiotics effective against Xanthomonas oryzae. The six variants were further purified by two cycles of the Polytron treatment and subsequent growth on Y-P plate. In the first cycle, 9.5 % to 38 % of the progeny colonies from the six variants lacked the ability to produce the antibiotics. One of the nonproducing variants from each group was subjected to the second cycle purification and it was found that every single colony was a nonproducer. This indicates that we obtained a homogeneous population of cells by the two cycles of purification. One of the clones obtained after the second cycle was used for further study.

5 1979 Linear Plasmid-like DNA in Streptomyces Producing Lankacidin 259. Properties of the antibiotic nonproducing variant The variant grew less well than the parent strain. This explains why the nonproducing colonies appeared at a low rate in the first cycle of purification described above. The DNA preparation from the variant was subjected to agarose gel electrophoresis and it was found that the variant lacked psla2 (Fig. 4). This observation was also true for other variants, suggesting that psla2 is involved in the antibiotic production. The variant also lost resistance against the antibiotics produced by the parent strain. For example, minimum inhibitory concentration (MIC) of lankacidin C against the variant was 3.13 µg/ml, whereas MIC against the parent strain was more than 100 µg/ml. DISCUSSION This paper describes the isolation and characterization of a linear plasmidlike DNA (psla2) from Streptomyces sp AN4 which produces lankacidin group antibiotics, and antibiotic nonproducing variants. The argument that psla2 is an extrachromosomal element and thus possesses the plasmid nature is substantiated from the following criteria. First, digestion of psla2 with restriction endonucleases (XmaI and BamNI) gave rise to discrete DNA segments (Fig. 3) and the sum of their molecular weights were in agreement with the molecular weight of psla2. These results rule out the possibility that psla2 was made by random cleavage of the chromosomal DNA, since the chromosomal DNA gave diffuse DNA bands upon treatment with these enzymes. Second, psla2 was lost by the treatment of ethidium bromide which is known to cure extrachromosomal DNA (12). Recently, OKANisHI and UMEZAWA (8) have reported that a plasmid possibly involved in the production of kasugamycin contains a protein(s) and that upon treatment withh pronase most of the plasmid molecules are converted to a linear form. This result and the results reported in this paper suggest that linear plasmids exist in nature. The question whether psla2 is the circular form in vivo remains to be determined. We are grateful to Dr. M. Okanishi for his valuable advices and discussions. We are also grateful to Dr. M. Uramoto for supplying Xanthomonas oryzae, lankacidin C, and for helpful advices and discussions. REFERENCES 1) 2) 3) 4) 5) M. OKANISHI, T. OHTA, and H. UMEZAWA, J. Antibiot., 23, 45 (1970). D. A. HopwooD, Annu. Rev. Microbiol., 32, 373 (1978). M. YAGISAWA, T-S. R. HUANG, and J. E. DAvIs, J. Antibiot., 31, 809 (1978). H. AKAZAWA, M. OKANISHI, and H. UMEZAWA, J. Gen. Microbiol., 90, 336 (1975). R. KIRBY and D. A. HopwooD, J. Gen. Microbiol., 98, 239 (1977).

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