Vol. 129, No. 3 Printed in U.S.A.
JOURNAL OF BACTRIOLOGY, Mar. 1977, p. 1487-1494 Copyright ©) 1977 American Society for Microbiology
Isolation and Characterization of Four Plasmids from Bacillus subtilis TERUO TANAKA,* MOTOKO KURODA, AND KENJI SAKAGUCHI Mitsubishi Kasei Institute of Life Sciences, 11, Minamiooya, Machida-shi, Tokyo, Japan
Received for publication 14 September 1976
Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 x 106. The other two strains (IAM1232, IAM1261) carried 4.9 x 106- and 5.3 x 10fi-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance or bacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.
Extrachromosomal deoxyribonucleic acid (DNA) elements have been found in sporeforming bacteria such as Bacillus pumilus (13-15), Bacillus subtilis (14), and Bacillus megaterium (3). Lovett and Bramucci (14) have found two plasmids from B. subtilis strains, one with a molecular weight of 4.7 x 106 and the other with a molecular weight of 46 x 106. Although the function of these plasmids has not been determined, the authors suggested the usefulness of these plasmids for construction of recombinant DNA molecules (14). Recently, Lovett et al. succeeded in introducing a B. pumilus plasmid into B. subtilis where the plasmid expressed its bacteriocin production function (16). Recently developed enzymatic techniques for introducing recombinant DNA molecules into Escherichia coli cells as autonomous replicons have contributed to the understanding of gene structure and function (4); at the same time, concern has been expressed over potential biohazards, despite the potential benefits of these techniques. If a genetic engineering system were established inB. subtilis, it would be a safer system, since this group of bacteria is nonpathogenic, grows aerobically, and does not inhabit the human body. Furthermore, genetic analyses can be made easily in this organism by use of transformation or transduction (F. E. Young and G. A. Wilson, Spores VI, p. 596-614, American Society for Microbiology, Washington, D.C., 1975). It has been determined that ribosomes from different bacteria show cistron specificity in the
translation of natural messenger ribonucleic acids (RNAs). Thus, E. coli ribosomes read all three cistrons of E. coli phage R17 RNA, whereas Bacillus stearothermophilus ribosomes translate only the A protein of the RNA (8). On the other hand, E. coli ribosomes cannot translate RNA from Caulobacter crescentus phage cb5 at all (12). This feature would create, in addition, a biological barrier for transmission of genetic information between E. coli and other organisms. In consideration of this cistron specificity of ribosomes in translation, we thought it would be advantageous to establish recombinant DNA systems in bacteria other than E. coli. To establish a recombinant DNA system in B. subtilis, we first started to look for plasmids among B. subtilis strains and found that four of the strains we examined harbored covalently closed circular (CCC) molecules, as revealed by CsCl-ethidium bromide (EtBr) density gradient centrifugation. The isolation and characterization of these plasmids are reported. MATERIALS AND METHODS Bacterial strains. B. subtilis strains used for the plasmid screening were obtained from the Institute for Fermentation, Osaka (IF03007, 3022, 3023, 3027, 3037, 3143, 3214, 3215, and 12250) and from the Institute of Applied Microbiology, University of Tokyo (IAM1026, 1027, 1107, 1170, 1232, 1248, 1261, 1521, 1522, and 1523). B. subtilis RM125 arg- leu- is a restriction- and modification-deficient mutant of B. subtilis YS-11 ade- arg- leu- (a derivative of B. subtilis 168) isolated by T. Uozumi et al. (unpublished data). Isolation of plasmids. Overnight cultures (100
1487
1488
TANAKA, KURODA, AND SAKAGUCHI
J. BACTERIOL.
ml) grown in enriched medium (in grams per liter: peptone, 5; yeast extract, 5; K2HPO4, 1; and glucose, 2) were harvested by centrifugation, and cells were washed twice in cold buffer consisting of 0.03 M tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 8.0), 0.05 M NaCl, and 0.005 M ethylenediaminetetraacetic acid (EDTA) (TES buffer). Cells were suspended in 3 ml of 25% sucrose in TES. A 0.5-ml portion of EDTA (0.25 M, pH 8.0) and 1.0 ml of lysozyme (5 mg/ml in TES containing 25% sucrose) were added, and the mixture was incubated for 30 min at 37°C. A 0.5-ml amount of Pronase E (5 mg/ml in TES predigested for 1.5 h at 37°C) was added, and the incubation was continued for an additional 30 min at 37°C. Lysis was brought about at room temperature by the addition of 0.5 ml of 10% sodium dodecyl sulfate in TES. NaCl (0.5 ml, 5 M) was added, and the whole mixture was left overnight at 4°C. The mixture was centrifuged for 30 min at 48,000 x g, and the supernatant fluid (approximately 4 ml) was aspirated and made up to a final volume of 6.0 ml with 50 mM EDTA (pH 8.0). A 5-g amount of solid CsCl and 0.1 ml of EtBr solution (30 mg/ml in dimethyl sulfoxide) were added, and the density was adjusted to 1.575 g/ml. The mixture was centrifuged at 38,000 rpm for 40 to 60 h at 16°C in a Hitachi RP65 rotor. The presence of CCC DNA was determined by visualization under ultraviolet illumination. When satellite bands were detected, they were collected and recentrifuged in CsCl-EtBr gradients. The purified samples were used for analyses with restriction endonucleases. For the preparation of radioactive DNA, 0.2 ml of an overnight culture in enriched medium was transferred into 10 ml of the same medium supplemented with 250 i.g of deoxyadenosine per ml and 4 ,uCi of [methyl-3H]thymidine per ml (Radiochemical Center; specific activity, 20 Ci/mmol). Incubation was continued at 37°C until the culture reached 150 Klett units (Klett-Summerson photoelectric colorimeter equipped with a no. 66 red filter). Cells were lysed as described above except that 0.12 ml of ribonuclease A (Sigma; 5 mg/ml preheated at 100°C for 10 min) was added together with lysozyme and 0.5 ml of 10% Sarkosyl NL-30 instead of sodium dodecyl sulfate was added before the Pronase treatment. The gradients were fractionated, and 0.02-ml samples from each fraction were pipetted onto filter papers (2.5 by 1.5 cm; Whatman no. 4), washed successively with 10% trichloroacetic acid and ethanol, and, finally, dried and counted in a liquid scintillation counter. Plasmid DNA was collected from the gradient, extracted with 1 volume of n-butanol three times, and dialyzed against 0.1x SSC (lx SSC contains 0.15 M NaCl plus 0.015 M sodium citrate) plus 1 mM EDTA. Sucrose density gradient centrifugation. 3H-labeled plasmid DNAs (as described above) were centrifuged in neutral 5 to 20% linear sucrose gradients in a buffer consisting of 0.03 M Tris-hydrochloride (pH 8.0), 0.005 M EDTA, and 1 M NaCl. 14C-labeled X phage DNA (obtained by heat induction of E. coli M65 lysogenized with XcI857S7[7]) was added to each sample as an internal standard for measuring S
values of the plasmids. The samples were spun at 20°C and 50,000 rpm for 100 min in a Hitachi RPS 65T rotor. Gradients were fractionated with the aid of a peristaltic pump, and portions (0.1 ml) of each fraction were applied on Whatman GF-C filters, dried, and counted in a liquid scintillation counter. Restriction enzymes. EcoRI was prepared as previously described (24). HindIII (21) was the generous gift of T. Wakabayashi of the University of Tokyo. BamNI is a restriction endonuclease obtained from B. subtilis N (20) and was kindly supplied by T. Shibata of The Physical and Chemical Institute. Digestion of the plasmid DNAs with these enzymes was for 1 h at 37°C (19, 21, 24). Electron microscopy. The procedure used for electron microscopy was essentially the same as that described by Davis et al. (5). The spreading solution contained 0.5 M ammonium acetate, 0.01% cytochrome c, and plasmid DNA, whereas hypophase contained 0.25 M ammonium acetate. The electron microscope used was the model JEOL-1OOB (JEOL Ltd., Tokyo, Japan). For the preparation of DNA samples to be examined, CCC DNA was converted to open circular DNA by incubation with 0.01 ,ug of deoxyribonuclease I per ml (Worthington Biochemicals Corp.) for 20 min at 37°C in 50 mM Tris-hydrochloride (pH 7.4) containing 10 mM MgCl2.
RESULTS
Isolation of plasmids. Among 19 B. subtilis strains tested, 4 strains, i.e., IF03022, IF03215, IAM1232, and IAM1261, were found to contain plasmids. For quantitative analysis, cells were labeled with [methyl-3H]thymidine and the whole lysates were centrifuged in CsCl-EtBr gradients. Satellite peaks were observed in these four strains (Fig. la-d). IF03022 (plasmid pLS11), IF03215 (pLS12), IAM1232 (pLS13), and IAM1261 (pLS14) contained CCC plasmid DNA to an extent of 1.8, 1.4, 2.2, and 1.8% of the chromosome DNA, respectively. Sucrose density gradient centrifugation. The 3H-labeled plasmid DNAs obtained from the CsCl-EtBr density gradients were subjected to neutral 5 to 20% sucrose density gradient centrifugation with added 14C-labeled X DNA as a marker (33.6S [2]) for determination of molecular weight. The results described in Fig. 2a-d show that the main peaks of the CCC fraction have S values of 27 (pLS11), 27 (pLS12), 25 (pLS13), and 26 (pLS14), corresponding to molecular weights of 5.4 x 106, 5.4 x 106, 4.6 x 106, and 5.0 x 106f, respectively, as calculated by the equation of Hudson et al. (10). Open circular forms (minor peaks) of the plasmids are seen in IF03022, IF03015, and IAM1232. The minimal number of copies of each plasmid can be calculated from the molecular weight and the amount of plasmid present in
VOL. 129, 1977
B. SUBTILIS PLASMIDS 10
10
a
1489
b
0 I
0
5
-
10
-f'
A
.
20
10
20
30
10
C
I
o5 x
10
20
30 FRACTION NUMBER
FIG. 1. CsCl-EtBr equilibrium centrifugation of [3H]thymidine-labeled DNA from (a) B. IF03022; (b) IF03215; (c) IAM1232; (d) IAM1261. The CsCl density decreases from left to right.
the CCC form per chromosome by assuming that the molecular weight of the B. subtilis chromosome is 2 x 109 (11). The estimated numbers of copies are presented and summarized in Table 1. Examination by electron microscopy. Each plasmid was further examined by electron miTwisted molecules were observed in plasmid samples; upon digestion with deoxyribonuclease I, they were converted to open circular forms with uniform contour lengths (Fig. 3a-d). With added R factor RSF2124 (molecular weight, 7.2 x 106) (21; unpublished data) as an internal standard, molecular weights of the croscopy.
four plasmids were determined to be 5.4 x 106 for pLS11 and pLS12, 4.9 x 106 for pLS13, and 5.3 x 106 for pLS14. The molecular weights of the plasmids obtained by electron microscopy and the sucrose density gradient centrifugation are summarized in Table 1. Digestion with restriction endonucleases. The four plasmids were digested with restric-
subtilis
tion endonucleases EcoRI, BamNI, and HindIII, followed by separation of the resultant fragments by electrophoresis in agarose gels. The digestion patterns on agarose gels are presented in Fig. 4 through 7. EcoRI cleaved pLS11 and pLS12 into single linear molecules (which could be distinguished from CCC forms by mobility on the gels [data not shown]) and pLS13 and pLS14 into four and three fragments, respectively (Fig. 4a-d). pLS11 and pLS12 contained one BamNI-susceptible site (Fig. 5a and b; the two bands in Fig. 5c and d are the relaxed and supercoiled CCC plasmid molecules), whereas neither pLS13 nor pLS14 was sensitive to this enzyme (Fig. 5c and d); in the latter two cases, bands of supercoiled (the faster-moving band) and open circular DNA are observed. Digestion of pLS11 and pLS12 with EcoRI and BamNI at the same time generated two bands, the mobilities of which were indistinguishable (Fig. 6a and b). Upon digestion with HindIII, pLS11 and pLS12 gave
1490
TANAKA, KURODA, AND SAKAGUCHI
J. BACTERIOL.
100
25 S 300
I
C
d 26 S
I
200
1
400
00I
200
10
20
30
10
20
30
40
FRACTION NUMBER
FIG. 2. Neutral sucrose density gradient centrifugation (5 to 20%0o) of [3H]thymidine-labeled plasmid DNA. Plasmid DNAs were labeled and purified as described in Materials and Methods. (a) pLS11; (b) pLS12; (c) pLS13; (d) pLS14. Arrows indicate the position of ["4C]thymidine-labeled X DNA cosedimented as a molecular weight marker. Density decreases from left to right. TABLE 1. Summary of the B. subtilis plasmids Mol wt (x 106)a
Strain
IF03022 IF03215 IAM1232 IAM1261
Plasmid
pLS11 pLS12 pLS13 pLS14
a The molecular weights centrifugation.
were
No. of cleavage sites No.
of
copies/
EM
Centrifugation
chromosome
EcoRI
BamNI
HindIII
5.4 5.4 4.9 5.3
5.4 5.4 4.6 5.0
7 5 10 7
1 1 4 3
1 1 0 0
5
determined by electron microscopy (EM) and neutral
sucrose
5 3 4
gradient
five bands with mobilities indistinguishable tomycin, to some or all of which the R-factorfrom each other (Fig. 7a and b), whereas pLS13 containing enteric bacteria frequently show reand pLS14 gave three and four bands, respec- sistance (data not shown). The ability to produce bacteriocins was extively (Fig. 7c and d). The number of cleavage sites of the plasmids is summarized in Table 1. amined by the double-layer technique of FredThese results indicate that of the four plas- ericq (6), using B. subtilis RM125 arg- leu- as mids tested pLS11 and pLS12 are indistinguish- indicator. No clear zone was observed around able and also that the four plasmids fall into the colonies of the four strains, suggesting that the presence of plasmids could not be correlated three classes. Properties of the plasmid-harboring with bacteriocin production (data not shown). strains. Plasmids have been known to specify Transformation of B. subtilis 168 with functions such as antibiotic resistance, produc- DNAs from the plasmid-harboring strains. To tion of bacteriocins, and sexual orientation of examine whether DNAs from the plasmid-conthe host cell, as well as numerous other func- taining strains show homology with that from tions summarized and reviewed by Helinski B. subtilis 168, DNA-mediated transformation and Clewell (9). In this study, the first two (1) was carried out using the restriction- and functions were tested. The plasmid-harboring modification-deficient strain RM125 as host. strains did not show resistance to any of the DNA from IF03022, IAM1232, and IAM1261 did not transform RM125 arg- leu- cells into leucommon antibiotics, such as chloramphenicol, tetracycline, ampicillin, kanamycin, and strep- cine- or arginine-independent cells (Table 2).
VOL. 129, 1977
__~~ ha~cotmewlirzgie~osn ~
B. SUBTILIS PLASMIDS
:~P;.ue¢
JOURNAL OF BACTRIOLOGY, Mar. 1977, p. 1487-1494 Copyright ©) 1977 American Society for Microbiology
Isolation and Characterization of Four Plasmids from Bacillus subtilis TERUO TANAKA,* MOTOKO KURODA, AND KENJI SAKAGUCHI Mitsubishi Kasei Institute of Life Sciences, 11, Minamiooya, Machida-shi, Tokyo, Japan
Received for publication 14 September 1976
Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 x 106. The other two strains (IAM1232, IAM1261) carried 4.9 x 106- and 5.3 x 10fi-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance or bacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.
Extrachromosomal deoxyribonucleic acid (DNA) elements have been found in sporeforming bacteria such as Bacillus pumilus (13-15), Bacillus subtilis (14), and Bacillus megaterium (3). Lovett and Bramucci (14) have found two plasmids from B. subtilis strains, one with a molecular weight of 4.7 x 106 and the other with a molecular weight of 46 x 106. Although the function of these plasmids has not been determined, the authors suggested the usefulness of these plasmids for construction of recombinant DNA molecules (14). Recently, Lovett et al. succeeded in introducing a B. pumilus plasmid into B. subtilis where the plasmid expressed its bacteriocin production function (16). Recently developed enzymatic techniques for introducing recombinant DNA molecules into Escherichia coli cells as autonomous replicons have contributed to the understanding of gene structure and function (4); at the same time, concern has been expressed over potential biohazards, despite the potential benefits of these techniques. If a genetic engineering system were established inB. subtilis, it would be a safer system, since this group of bacteria is nonpathogenic, grows aerobically, and does not inhabit the human body. Furthermore, genetic analyses can be made easily in this organism by use of transformation or transduction (F. E. Young and G. A. Wilson, Spores VI, p. 596-614, American Society for Microbiology, Washington, D.C., 1975). It has been determined that ribosomes from different bacteria show cistron specificity in the
translation of natural messenger ribonucleic acids (RNAs). Thus, E. coli ribosomes read all three cistrons of E. coli phage R17 RNA, whereas Bacillus stearothermophilus ribosomes translate only the A protein of the RNA (8). On the other hand, E. coli ribosomes cannot translate RNA from Caulobacter crescentus phage cb5 at all (12). This feature would create, in addition, a biological barrier for transmission of genetic information between E. coli and other organisms. In consideration of this cistron specificity of ribosomes in translation, we thought it would be advantageous to establish recombinant DNA systems in bacteria other than E. coli. To establish a recombinant DNA system in B. subtilis, we first started to look for plasmids among B. subtilis strains and found that four of the strains we examined harbored covalently closed circular (CCC) molecules, as revealed by CsCl-ethidium bromide (EtBr) density gradient centrifugation. The isolation and characterization of these plasmids are reported. MATERIALS AND METHODS Bacterial strains. B. subtilis strains used for the plasmid screening were obtained from the Institute for Fermentation, Osaka (IF03007, 3022, 3023, 3027, 3037, 3143, 3214, 3215, and 12250) and from the Institute of Applied Microbiology, University of Tokyo (IAM1026, 1027, 1107, 1170, 1232, 1248, 1261, 1521, 1522, and 1523). B. subtilis RM125 arg- leu- is a restriction- and modification-deficient mutant of B. subtilis YS-11 ade- arg- leu- (a derivative of B. subtilis 168) isolated by T. Uozumi et al. (unpublished data). Isolation of plasmids. Overnight cultures (100
1487
1488
TANAKA, KURODA, AND SAKAGUCHI
J. BACTERIOL.
ml) grown in enriched medium (in grams per liter: peptone, 5; yeast extract, 5; K2HPO4, 1; and glucose, 2) were harvested by centrifugation, and cells were washed twice in cold buffer consisting of 0.03 M tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 8.0), 0.05 M NaCl, and 0.005 M ethylenediaminetetraacetic acid (EDTA) (TES buffer). Cells were suspended in 3 ml of 25% sucrose in TES. A 0.5-ml portion of EDTA (0.25 M, pH 8.0) and 1.0 ml of lysozyme (5 mg/ml in TES containing 25% sucrose) were added, and the mixture was incubated for 30 min at 37°C. A 0.5-ml amount of Pronase E (5 mg/ml in TES predigested for 1.5 h at 37°C) was added, and the incubation was continued for an additional 30 min at 37°C. Lysis was brought about at room temperature by the addition of 0.5 ml of 10% sodium dodecyl sulfate in TES. NaCl (0.5 ml, 5 M) was added, and the whole mixture was left overnight at 4°C. The mixture was centrifuged for 30 min at 48,000 x g, and the supernatant fluid (approximately 4 ml) was aspirated and made up to a final volume of 6.0 ml with 50 mM EDTA (pH 8.0). A 5-g amount of solid CsCl and 0.1 ml of EtBr solution (30 mg/ml in dimethyl sulfoxide) were added, and the density was adjusted to 1.575 g/ml. The mixture was centrifuged at 38,000 rpm for 40 to 60 h at 16°C in a Hitachi RP65 rotor. The presence of CCC DNA was determined by visualization under ultraviolet illumination. When satellite bands were detected, they were collected and recentrifuged in CsCl-EtBr gradients. The purified samples were used for analyses with restriction endonucleases. For the preparation of radioactive DNA, 0.2 ml of an overnight culture in enriched medium was transferred into 10 ml of the same medium supplemented with 250 i.g of deoxyadenosine per ml and 4 ,uCi of [methyl-3H]thymidine per ml (Radiochemical Center; specific activity, 20 Ci/mmol). Incubation was continued at 37°C until the culture reached 150 Klett units (Klett-Summerson photoelectric colorimeter equipped with a no. 66 red filter). Cells were lysed as described above except that 0.12 ml of ribonuclease A (Sigma; 5 mg/ml preheated at 100°C for 10 min) was added together with lysozyme and 0.5 ml of 10% Sarkosyl NL-30 instead of sodium dodecyl sulfate was added before the Pronase treatment. The gradients were fractionated, and 0.02-ml samples from each fraction were pipetted onto filter papers (2.5 by 1.5 cm; Whatman no. 4), washed successively with 10% trichloroacetic acid and ethanol, and, finally, dried and counted in a liquid scintillation counter. Plasmid DNA was collected from the gradient, extracted with 1 volume of n-butanol three times, and dialyzed against 0.1x SSC (lx SSC contains 0.15 M NaCl plus 0.015 M sodium citrate) plus 1 mM EDTA. Sucrose density gradient centrifugation. 3H-labeled plasmid DNAs (as described above) were centrifuged in neutral 5 to 20% linear sucrose gradients in a buffer consisting of 0.03 M Tris-hydrochloride (pH 8.0), 0.005 M EDTA, and 1 M NaCl. 14C-labeled X phage DNA (obtained by heat induction of E. coli M65 lysogenized with XcI857S7[7]) was added to each sample as an internal standard for measuring S
values of the plasmids. The samples were spun at 20°C and 50,000 rpm for 100 min in a Hitachi RPS 65T rotor. Gradients were fractionated with the aid of a peristaltic pump, and portions (0.1 ml) of each fraction were applied on Whatman GF-C filters, dried, and counted in a liquid scintillation counter. Restriction enzymes. EcoRI was prepared as previously described (24). HindIII (21) was the generous gift of T. Wakabayashi of the University of Tokyo. BamNI is a restriction endonuclease obtained from B. subtilis N (20) and was kindly supplied by T. Shibata of The Physical and Chemical Institute. Digestion of the plasmid DNAs with these enzymes was for 1 h at 37°C (19, 21, 24). Electron microscopy. The procedure used for electron microscopy was essentially the same as that described by Davis et al. (5). The spreading solution contained 0.5 M ammonium acetate, 0.01% cytochrome c, and plasmid DNA, whereas hypophase contained 0.25 M ammonium acetate. The electron microscope used was the model JEOL-1OOB (JEOL Ltd., Tokyo, Japan). For the preparation of DNA samples to be examined, CCC DNA was converted to open circular DNA by incubation with 0.01 ,ug of deoxyribonuclease I per ml (Worthington Biochemicals Corp.) for 20 min at 37°C in 50 mM Tris-hydrochloride (pH 7.4) containing 10 mM MgCl2.
RESULTS
Isolation of plasmids. Among 19 B. subtilis strains tested, 4 strains, i.e., IF03022, IF03215, IAM1232, and IAM1261, were found to contain plasmids. For quantitative analysis, cells were labeled with [methyl-3H]thymidine and the whole lysates were centrifuged in CsCl-EtBr gradients. Satellite peaks were observed in these four strains (Fig. la-d). IF03022 (plasmid pLS11), IF03215 (pLS12), IAM1232 (pLS13), and IAM1261 (pLS14) contained CCC plasmid DNA to an extent of 1.8, 1.4, 2.2, and 1.8% of the chromosome DNA, respectively. Sucrose density gradient centrifugation. The 3H-labeled plasmid DNAs obtained from the CsCl-EtBr density gradients were subjected to neutral 5 to 20% sucrose density gradient centrifugation with added 14C-labeled X DNA as a marker (33.6S [2]) for determination of molecular weight. The results described in Fig. 2a-d show that the main peaks of the CCC fraction have S values of 27 (pLS11), 27 (pLS12), 25 (pLS13), and 26 (pLS14), corresponding to molecular weights of 5.4 x 106, 5.4 x 106, 4.6 x 106, and 5.0 x 106f, respectively, as calculated by the equation of Hudson et al. (10). Open circular forms (minor peaks) of the plasmids are seen in IF03022, IF03015, and IAM1232. The minimal number of copies of each plasmid can be calculated from the molecular weight and the amount of plasmid present in
VOL. 129, 1977
B. SUBTILIS PLASMIDS 10
10
a
1489
b
0 I
0
5
-
10
-f'
A
.
20
10
20
30
10
C
I
o5 x
10
20
30 FRACTION NUMBER
FIG. 1. CsCl-EtBr equilibrium centrifugation of [3H]thymidine-labeled DNA from (a) B. IF03022; (b) IF03215; (c) IAM1232; (d) IAM1261. The CsCl density decreases from left to right.
the CCC form per chromosome by assuming that the molecular weight of the B. subtilis chromosome is 2 x 109 (11). The estimated numbers of copies are presented and summarized in Table 1. Examination by electron microscopy. Each plasmid was further examined by electron miTwisted molecules were observed in plasmid samples; upon digestion with deoxyribonuclease I, they were converted to open circular forms with uniform contour lengths (Fig. 3a-d). With added R factor RSF2124 (molecular weight, 7.2 x 106) (21; unpublished data) as an internal standard, molecular weights of the croscopy.
four plasmids were determined to be 5.4 x 106 for pLS11 and pLS12, 4.9 x 106 for pLS13, and 5.3 x 106 for pLS14. The molecular weights of the plasmids obtained by electron microscopy and the sucrose density gradient centrifugation are summarized in Table 1. Digestion with restriction endonucleases. The four plasmids were digested with restric-
subtilis
tion endonucleases EcoRI, BamNI, and HindIII, followed by separation of the resultant fragments by electrophoresis in agarose gels. The digestion patterns on agarose gels are presented in Fig. 4 through 7. EcoRI cleaved pLS11 and pLS12 into single linear molecules (which could be distinguished from CCC forms by mobility on the gels [data not shown]) and pLS13 and pLS14 into four and three fragments, respectively (Fig. 4a-d). pLS11 and pLS12 contained one BamNI-susceptible site (Fig. 5a and b; the two bands in Fig. 5c and d are the relaxed and supercoiled CCC plasmid molecules), whereas neither pLS13 nor pLS14 was sensitive to this enzyme (Fig. 5c and d); in the latter two cases, bands of supercoiled (the faster-moving band) and open circular DNA are observed. Digestion of pLS11 and pLS12 with EcoRI and BamNI at the same time generated two bands, the mobilities of which were indistinguishable (Fig. 6a and b). Upon digestion with HindIII, pLS11 and pLS12 gave
1490
TANAKA, KURODA, AND SAKAGUCHI
J. BACTERIOL.
100
25 S 300
I
C
d 26 S
I
200
1
400
00I
200
10
20
30
10
20
30
40
FRACTION NUMBER
FIG. 2. Neutral sucrose density gradient centrifugation (5 to 20%0o) of [3H]thymidine-labeled plasmid DNA. Plasmid DNAs were labeled and purified as described in Materials and Methods. (a) pLS11; (b) pLS12; (c) pLS13; (d) pLS14. Arrows indicate the position of ["4C]thymidine-labeled X DNA cosedimented as a molecular weight marker. Density decreases from left to right. TABLE 1. Summary of the B. subtilis plasmids Mol wt (x 106)a
Strain
IF03022 IF03215 IAM1232 IAM1261
Plasmid
pLS11 pLS12 pLS13 pLS14
a The molecular weights centrifugation.
were
No. of cleavage sites No.
of
copies/
EM
Centrifugation
chromosome
EcoRI
BamNI
HindIII
5.4 5.4 4.9 5.3
5.4 5.4 4.6 5.0
7 5 10 7
1 1 4 3
1 1 0 0
5
determined by electron microscopy (EM) and neutral
sucrose
5 3 4
gradient
five bands with mobilities indistinguishable tomycin, to some or all of which the R-factorfrom each other (Fig. 7a and b), whereas pLS13 containing enteric bacteria frequently show reand pLS14 gave three and four bands, respec- sistance (data not shown). The ability to produce bacteriocins was extively (Fig. 7c and d). The number of cleavage sites of the plasmids is summarized in Table 1. amined by the double-layer technique of FredThese results indicate that of the four plas- ericq (6), using B. subtilis RM125 arg- leu- as mids tested pLS11 and pLS12 are indistinguish- indicator. No clear zone was observed around able and also that the four plasmids fall into the colonies of the four strains, suggesting that the presence of plasmids could not be correlated three classes. Properties of the plasmid-harboring with bacteriocin production (data not shown). strains. Plasmids have been known to specify Transformation of B. subtilis 168 with functions such as antibiotic resistance, produc- DNAs from the plasmid-harboring strains. To tion of bacteriocins, and sexual orientation of examine whether DNAs from the plasmid-conthe host cell, as well as numerous other func- taining strains show homology with that from tions summarized and reviewed by Helinski B. subtilis 168, DNA-mediated transformation and Clewell (9). In this study, the first two (1) was carried out using the restriction- and functions were tested. The plasmid-harboring modification-deficient strain RM125 as host. strains did not show resistance to any of the DNA from IF03022, IAM1232, and IAM1261 did not transform RM125 arg- leu- cells into leucommon antibiotics, such as chloramphenicol, tetracycline, ampicillin, kanamycin, and strep- cine- or arginine-independent cells (Table 2).
VOL. 129, 1977
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B. SUBTILIS PLASMIDS
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