JOURNAL
OF
BACTERIOLOGY, Feb. 1991, p. 1523-1529
Vol. 173, No. 4
0021-9193/91/041523-07$02.00/0 Copyright C 1991, American Society for Microbiology
Physical Characterization of SCP1, a Giant Linear Plasmid from Streptomyces coelicolor HARUYASU KINASHIt* AND MIYUKI SHIMAJI-MURAYAMA Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan Received 4 September 1990/Accepted 7 December 1990
SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with k exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein. The application of pulsed-field gel electrophoresis (PFGE) (6, 37) to Streptomyces DNA enabled the detection of giant linear plasmids of 90 to 590 kb from antibiotic-producing strains such as Streptomyces lasaliensis, S. violaceoruber, S. fradiae, S. parvulus, S. venezuelae, and S. rochei (23). This method also revealed that SCP1, a plasmid in S. coelicolor which had been sought for a long time as a genetic determinant of methylenomycin biosynthesis (25, 26), was a giant linear plasmid (24). Southern blot analysis with pIJ601 (1), a plasmid containing the methylenomycin resistance gene, confirmed that SCP1 carries the methylenomycin biosynthetic gene cluster (7). Extensive genetic studies by Hopwood and his collaborators revealed that SCP1 is present in various states in S. coelicolor strains (for reviews, see reference 8), namely as an autonomous replicating plasmid, as an autonomous replicating plasmid containing a chromosomal fragment (SCP1'cysB), and integrated into the chromosome (NF strains). We confirmed these various states of SCP1 by PFGE analysis in a preliminary investigation (22) and further demonstrated that S. violaceoruber JCM4979 contains a series of plasmids (410 to 590 kb) with a size difference of about 30 kb (22). These plasmids were suggested to be formed by integration of a circular sex plasmid, SCP2 (4, 27), into SCP1 and by subsequent amplification and deletion. S. violaceoruber JCM4979 was derived from Erikson's S. coelicolor A3(2) strain (11), with which Hopwood started the genetic study (16). Therefore, S. violaceoruber JCM4979 and S. coelicolor 1147, a wild-type culture of S. coelicolor A3(2) at the John Innes Institute, are apparently identical. We thus believed at first that strain 1147 had the same series of plasmids as had strain JCM4979. This was not found to be the case, however; strain 1147 has only SCP1 and not the plasmid ladder, which is also a characteristic of strain M138 (24). To study the details of the structures of various states of SCP1 in S. coelicolor strains and to elucidate the mechanism of its dynamic changes, we undertook the physical charac*
Corresponding author.
t Present address: Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashihiroshima 724,
Japan.
1523
terization of SCP1 as the first goal. In this paper, we report physical maps of SCP1 which revealed the presence of unusually long terminal inverted repeats (TIRs) of about 80
kb on both ends. The IS466 insertion sequence was also found at the end of the right TIR (TIR-R). Exonuclease digestion revealed that a protein is bound to the 5' ends of SCP1. MATERIALS AND METHODS
Bacterial strains and plasmids. The strains and plasmids used in this study are described in Table 1. S. coelicolor 1147 and M138 and plasmid pIJ601 were provided by D. A. Hopwood, John Innes Institute. S. coelicolor JCM4979 was obtained from the Japan Collection of Microorganisms, Wako, Saitama, Japan. Escherichia coli JM109 and plasmid pUC18 were used for the cloning of SCP1 fragments. pSCP201, pSCP211, and pSCP221 are pUC18 derivatives containing the terminus of SCP1 and the endpoint of TIR-R or the left TIR (TIR-L), respectively. Details of the cloning, restriction maps, and DNA sequences of these plasmids will be described elsewhere. pMT644, a plasmid containing the 0.8-kb Sall fragment of IS466, was provided by C. Conzelmann, University of Manchester. Media and buffers. YEME medium was prepared as described by Hopwood et al. (18). P3 and PWP buffers were as described by Shirahama et al. (36). TSE buffer was composed of 30 mM Tris hydrochloride (pH 8.0), 50 mM NaCl, and 5 mM EDTA. TBE buffer was as described by Sambrook et al. (35). Actinase E (Kaken Pharmaceuticals, Tokyo, Japan) used as pronase was dissolved in TSE buffer (5 mg/ml) and incubated at 37°C for 2 h before being used to digest contaminated nucleases. A Exonuclease and exonuclease III were purchased from New England BioLabs, Beverly, Mass., and Boehringer, Mannheim, Federal Republic of Germany, respectively. Low-melting-point (LMP) agarose (type VII) was purchased from Sigma, St. Louis, Mo. Preparation of DNA inserts for PFGE. S. coelicolor M138 was grown for about 3 days at 30°C in YEME medium containing 0.5% glycine to the early stationary phase and washed twice with 10.3% sucrose. About 5 g of wet mycelium was suspended in 40 ml of P3 buffer and treated
1524
J. BACTERIOL.
KINASHI AND SHIMAJI-MURAYAMA TABLE 1. Bacterial strains and plasmids
Strain or plasmid
Strains S. coelicolor 1147
S. coelicolor M138 S. violaceoruber JCM4979 Plasmids pU601 pMT644
pSCP201 pSCP211
pSCP221
Source or reference
Description or genotype
John Innes Institute wild-type culture of S. coelicolor A3(2), SCP1+
Erikson's S. coelicolor A3(2) strain (11)
SCP2+ SCP1+ SCP2- argAl proAl cysD18 Japan Collection of Microorganisms culture, SCP1+ SCP2+
Strain 1147 of Bibb and Hopwood (4) Erikson's S. coelicolor A3(2) strain (11)
pBR322 derivative containing the mmr gene pBR322 derivative containing the 0.8-kb SalI fragment of IS466 pUC18 derivative containing the terminal 4.1-kb Spel fragment of SCP1 pUC18 derivative containing the end BamHI fragment of TIR-R pUC18 derivative containing the end BamHI fragment of TIR-L
with 2 mg of lysozyme per ml at 300C for 10 to 30 min with gentle shaking. Protoplasts were filtered through glass wool, centrifuged at 2,700 x g for 10 min, and resuspended in 2 ml of PWP buffer. Since the addition of EDTA to protoplasts in solution caused their quick lysis, a previously described procedure (24) was modified as follows. The protoplast suspension was transferred to a Falcon 3002 plate to which 4 ml of molten 1.5% LMP agarose in TSE buffer was added and mixed gently. After the agarose was solidified at 4°C, it was overlaid with 2 ml of 0.5 M EDTA (pH 8.0) and kept at 4°C for 2 h. One milliliter of pronase solution was added to the EDTA layer and incubated at 37°C for 2 h, and 1 ml of 10% sodium dodecyl sulfate (SDS) was added and incubated at 37°C overnight. The liquid layer was replaced by 0.5 M EDTA, and the plate was stored at 4°C. DNA inserts (1 by 3 by 10 mm) were cut out of the agarose gel and used for PFGE analysis. Agarose gel eectrho resis. Conventional agarose gel electrophoresis was carried out with 0.7% agarose in lx TBE buffer at 100 V. Orthogonal-field alternation gel electrophoresis was conducted as described before (24). Contourclamped homogeneous electric fields (CHEF) gel electrophoresis was conducted overnight with 1% agarose in 0.5x TBE buffer at 180 V in the hexagonal-array apparatus of Chu et al. (9). Pulse times for CHEF analysis were from 18 to 30 s, depending on the length of DNA to be separated. SDSCHEF analysis was carried out by adding 0.2% SDS to both the buffer and the agarose gel. We found that the addition of SDS required a prolonged pulse time for CHEF analysis; therefore, we used pulse times of 24 s for CHEF analysis and 48 s for SDS-CHEF analysis in the experiments shown in Fig. 5. Restriction endonuclease ditin of SCP1 in agarose gels. The DNA inserts were subjected to CHEF analysis in LMP agarose. The SCP1 band was excised from the gel, cut into pieces, washed twice for 2 h each time in TE buffer (pH 8.0) (35), and stored in TE buffer at 4°C. Before use, the gel pieces were washed twice in the digestion buffer for 2 h each time and treated overight with gentle shaking with a restriction endonuclease in digestion buffer supplemented with 100 ,ug of bovine serum albumin per ml. Non-pronase-treated SCP1 was obtained by omitting the pronase treatment of the DNA inserts. After separation by SDS-CHEF gel electrophoresis in LMP agarose, the untreated SCP1 band was cut out of the gel and washed in TE buffer to remove SDS before restriction endonuclease digestion. Exonucleae digestion of SCP1. A mixture of SCP1 and the
1 20 This work
This work This work
SmaI fragments of pSCP201 (used as internal markers) was digested with A exonuclease or exonuclease III at 37°C for 5 or 15 min in 67 mM glycine (pH 9.4)-3 mM MgCl2-3 mM 2-mercaptoethanol or 66 mM Tris hydrochloride (pH 7.6)0.66 mM MgCl2-1 mM 2-mercaptoethanol, respectively. After digestion, each reaction mixture was extracted twice with phenol, precipitated with 70% ethanol, and digested with SpeI. Samples before and after the SpeI digestion were analyzed by conventional agarose gel electrophoresis. Southern blot analysis. The DNA fragments separated by CHEF gel electrophoresis or conventional agarose gel electrophoresis were transferred to a Nytran N membrane filter (Schleicher & Schuell, Dassel, Federal Republic of Germany) as described by Hopwood et al. (18). DNA probes were prepared by electroelution of large (>20-kb) DNA fragments from the agarose gel and phenol extraction. Small (
OF
BACTERIOLOGY, Feb. 1991, p. 1523-1529
Vol. 173, No. 4
0021-9193/91/041523-07$02.00/0 Copyright C 1991, American Society for Microbiology
Physical Characterization of SCP1, a Giant Linear Plasmid from Streptomyces coelicolor HARUYASU KINASHIt* AND MIYUKI SHIMAJI-MURAYAMA Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan Received 4 September 1990/Accepted 7 December 1990
SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with k exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein. The application of pulsed-field gel electrophoresis (PFGE) (6, 37) to Streptomyces DNA enabled the detection of giant linear plasmids of 90 to 590 kb from antibiotic-producing strains such as Streptomyces lasaliensis, S. violaceoruber, S. fradiae, S. parvulus, S. venezuelae, and S. rochei (23). This method also revealed that SCP1, a plasmid in S. coelicolor which had been sought for a long time as a genetic determinant of methylenomycin biosynthesis (25, 26), was a giant linear plasmid (24). Southern blot analysis with pIJ601 (1), a plasmid containing the methylenomycin resistance gene, confirmed that SCP1 carries the methylenomycin biosynthetic gene cluster (7). Extensive genetic studies by Hopwood and his collaborators revealed that SCP1 is present in various states in S. coelicolor strains (for reviews, see reference 8), namely as an autonomous replicating plasmid, as an autonomous replicating plasmid containing a chromosomal fragment (SCP1'cysB), and integrated into the chromosome (NF strains). We confirmed these various states of SCP1 by PFGE analysis in a preliminary investigation (22) and further demonstrated that S. violaceoruber JCM4979 contains a series of plasmids (410 to 590 kb) with a size difference of about 30 kb (22). These plasmids were suggested to be formed by integration of a circular sex plasmid, SCP2 (4, 27), into SCP1 and by subsequent amplification and deletion. S. violaceoruber JCM4979 was derived from Erikson's S. coelicolor A3(2) strain (11), with which Hopwood started the genetic study (16). Therefore, S. violaceoruber JCM4979 and S. coelicolor 1147, a wild-type culture of S. coelicolor A3(2) at the John Innes Institute, are apparently identical. We thus believed at first that strain 1147 had the same series of plasmids as had strain JCM4979. This was not found to be the case, however; strain 1147 has only SCP1 and not the plasmid ladder, which is also a characteristic of strain M138 (24). To study the details of the structures of various states of SCP1 in S. coelicolor strains and to elucidate the mechanism of its dynamic changes, we undertook the physical charac*
Corresponding author.
t Present address: Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashihiroshima 724,
Japan.
1523
terization of SCP1 as the first goal. In this paper, we report physical maps of SCP1 which revealed the presence of unusually long terminal inverted repeats (TIRs) of about 80
kb on both ends. The IS466 insertion sequence was also found at the end of the right TIR (TIR-R). Exonuclease digestion revealed that a protein is bound to the 5' ends of SCP1. MATERIALS AND METHODS
Bacterial strains and plasmids. The strains and plasmids used in this study are described in Table 1. S. coelicolor 1147 and M138 and plasmid pIJ601 were provided by D. A. Hopwood, John Innes Institute. S. coelicolor JCM4979 was obtained from the Japan Collection of Microorganisms, Wako, Saitama, Japan. Escherichia coli JM109 and plasmid pUC18 were used for the cloning of SCP1 fragments. pSCP201, pSCP211, and pSCP221 are pUC18 derivatives containing the terminus of SCP1 and the endpoint of TIR-R or the left TIR (TIR-L), respectively. Details of the cloning, restriction maps, and DNA sequences of these plasmids will be described elsewhere. pMT644, a plasmid containing the 0.8-kb Sall fragment of IS466, was provided by C. Conzelmann, University of Manchester. Media and buffers. YEME medium was prepared as described by Hopwood et al. (18). P3 and PWP buffers were as described by Shirahama et al. (36). TSE buffer was composed of 30 mM Tris hydrochloride (pH 8.0), 50 mM NaCl, and 5 mM EDTA. TBE buffer was as described by Sambrook et al. (35). Actinase E (Kaken Pharmaceuticals, Tokyo, Japan) used as pronase was dissolved in TSE buffer (5 mg/ml) and incubated at 37°C for 2 h before being used to digest contaminated nucleases. A Exonuclease and exonuclease III were purchased from New England BioLabs, Beverly, Mass., and Boehringer, Mannheim, Federal Republic of Germany, respectively. Low-melting-point (LMP) agarose (type VII) was purchased from Sigma, St. Louis, Mo. Preparation of DNA inserts for PFGE. S. coelicolor M138 was grown for about 3 days at 30°C in YEME medium containing 0.5% glycine to the early stationary phase and washed twice with 10.3% sucrose. About 5 g of wet mycelium was suspended in 40 ml of P3 buffer and treated
1524
J. BACTERIOL.
KINASHI AND SHIMAJI-MURAYAMA TABLE 1. Bacterial strains and plasmids
Strain or plasmid
Strains S. coelicolor 1147
S. coelicolor M138 S. violaceoruber JCM4979 Plasmids pU601 pMT644
pSCP201 pSCP211
pSCP221
Source or reference
Description or genotype
John Innes Institute wild-type culture of S. coelicolor A3(2), SCP1+
Erikson's S. coelicolor A3(2) strain (11)
SCP2+ SCP1+ SCP2- argAl proAl cysD18 Japan Collection of Microorganisms culture, SCP1+ SCP2+
Strain 1147 of Bibb and Hopwood (4) Erikson's S. coelicolor A3(2) strain (11)
pBR322 derivative containing the mmr gene pBR322 derivative containing the 0.8-kb SalI fragment of IS466 pUC18 derivative containing the terminal 4.1-kb Spel fragment of SCP1 pUC18 derivative containing the end BamHI fragment of TIR-R pUC18 derivative containing the end BamHI fragment of TIR-L
with 2 mg of lysozyme per ml at 300C for 10 to 30 min with gentle shaking. Protoplasts were filtered through glass wool, centrifuged at 2,700 x g for 10 min, and resuspended in 2 ml of PWP buffer. Since the addition of EDTA to protoplasts in solution caused their quick lysis, a previously described procedure (24) was modified as follows. The protoplast suspension was transferred to a Falcon 3002 plate to which 4 ml of molten 1.5% LMP agarose in TSE buffer was added and mixed gently. After the agarose was solidified at 4°C, it was overlaid with 2 ml of 0.5 M EDTA (pH 8.0) and kept at 4°C for 2 h. One milliliter of pronase solution was added to the EDTA layer and incubated at 37°C for 2 h, and 1 ml of 10% sodium dodecyl sulfate (SDS) was added and incubated at 37°C overnight. The liquid layer was replaced by 0.5 M EDTA, and the plate was stored at 4°C. DNA inserts (1 by 3 by 10 mm) were cut out of the agarose gel and used for PFGE analysis. Agarose gel eectrho resis. Conventional agarose gel electrophoresis was carried out with 0.7% agarose in lx TBE buffer at 100 V. Orthogonal-field alternation gel electrophoresis was conducted as described before (24). Contourclamped homogeneous electric fields (CHEF) gel electrophoresis was conducted overnight with 1% agarose in 0.5x TBE buffer at 180 V in the hexagonal-array apparatus of Chu et al. (9). Pulse times for CHEF analysis were from 18 to 30 s, depending on the length of DNA to be separated. SDSCHEF analysis was carried out by adding 0.2% SDS to both the buffer and the agarose gel. We found that the addition of SDS required a prolonged pulse time for CHEF analysis; therefore, we used pulse times of 24 s for CHEF analysis and 48 s for SDS-CHEF analysis in the experiments shown in Fig. 5. Restriction endonuclease ditin of SCP1 in agarose gels. The DNA inserts were subjected to CHEF analysis in LMP agarose. The SCP1 band was excised from the gel, cut into pieces, washed twice for 2 h each time in TE buffer (pH 8.0) (35), and stored in TE buffer at 4°C. Before use, the gel pieces were washed twice in the digestion buffer for 2 h each time and treated overight with gentle shaking with a restriction endonuclease in digestion buffer supplemented with 100 ,ug of bovine serum albumin per ml. Non-pronase-treated SCP1 was obtained by omitting the pronase treatment of the DNA inserts. After separation by SDS-CHEF gel electrophoresis in LMP agarose, the untreated SCP1 band was cut out of the gel and washed in TE buffer to remove SDS before restriction endonuclease digestion. Exonucleae digestion of SCP1. A mixture of SCP1 and the
1 20 This work
This work This work
SmaI fragments of pSCP201 (used as internal markers) was digested with A exonuclease or exonuclease III at 37°C for 5 or 15 min in 67 mM glycine (pH 9.4)-3 mM MgCl2-3 mM 2-mercaptoethanol or 66 mM Tris hydrochloride (pH 7.6)0.66 mM MgCl2-1 mM 2-mercaptoethanol, respectively. After digestion, each reaction mixture was extracted twice with phenol, precipitated with 70% ethanol, and digested with SpeI. Samples before and after the SpeI digestion were analyzed by conventional agarose gel electrophoresis. Southern blot analysis. The DNA fragments separated by CHEF gel electrophoresis or conventional agarose gel electrophoresis were transferred to a Nytran N membrane filter (Schleicher & Schuell, Dassel, Federal Republic of Germany) as described by Hopwood et al. (18). DNA probes were prepared by electroelution of large (>20-kb) DNA fragments from the agarose gel and phenol extraction. Small (
