Vol. 131, No. 1 Printed in U.S.A.
JOURNAL OF BACTZRIOLOGY, JUlY 1977, p. 374-377 Copyright C 1977 American Society for Microbiology
Transformation of Escherichia coli and Bacillus subtilis with a Hybrid Plasmid Molecule I. MAHLER* AND HARLYN 0. HALVORSON Research Center, Brandeis University, Waltham, Massachusetts 02154 Sciences Medical Basic Rosenstiel
Received for publication 25 March 1977
A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leuE. coli, converting them to leucine prototrophy, but fails to survive in strains of B. subtilis 168. In our efforts to clone and amplify gene fragments of Bacillus subtilis, we constructed hybrid deoxyribonucleic acid (DNA) molecules consisting of Escherichia coli plasmid pMB9, which carried resistance to tetracycline (tetr), and a segment of the B. subtilis genome. Covalently closed circular DNA was obtained from E. coli HB129(pMB9) by the method of Wensink et al. (15), whereas DNA from B. subtilis 168 was prepared by the method of Saito and Miura (11). The bacterial strains are listed in Table 1. The bacterial DNA (50 to 200 ,ug/ml) was incubated with restriction endonuclease EcoRI (the generous gift of P. Wensink) at 37°C for 15 to 30 min in a solution containing 12 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.6), 6 mM MgCl2, 50 mM NaCl, and 6 mM mercaptoethanol. Completeness of degradation was verified by examining the banding pattern for the disappearance of uncleaved DNA in agarose-gel electrophoresis followed by ethidium bromide staining (7). With B. subtilis JAS9 as a recipient in transformation experiments, the biological activity of the bacterial DNA was tested before and after endonuclease digestion. After enzymatic cleavage, the survival of leu+-transforming activity was reduced to 1%. A DNA segment enriched 16.6-fold for leu+-transformation activity was obtained by the method of Harris-Warrick et al. (6). Its molecular size measured against EcoRItreated phage lambda DNA (7) was estimated at 8.2 kilobases (data not shown). To prepare hybrid molecules, 0.5 ,ug of EcoRI-treated pMB9 was incubated with 1.5 ug of leu+-enriched B. subtilis DNA together with 0.4 U of T4 polynucleotide ligase (EC 6.5.11) (Biolabs, Beverly, Mass.) in a 100-,ul solution containing 30 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 8.0), 5 mM MgCl2, 0.2 mM adenosine 5'-triphosphate, 0.05 mM dithiothreitol, and 30 ,ug of bovine albumin
(fraction V; Sigma Chemical Co.) per ml. The reaction was carried out for 16 to 18 h at 140C and terminated by heating the solution at 680C for 3 min. The effectiveness of polynucleotide ligase sealing was checked by the reappearance of covalently closed circular molecules in agarose gels and the restoration of transforming activity (Table 2); the DNA thus generated was used to transformE. coli HB101. The efficiency of transformation with the treated plasmid DNAs is shown in Table 2. From the recipient strain, which is tets and leu-, double transformants (leu+ and tetr) were isolated and examined for the presence of plasmids with altered physical and biological characteristics. Two hybrid plasmids, isolated in separate experiments, were digested with EcoRI enzyme and run in a slab gel electrophoresis apparatus against EcoRI-digested phase lambda DNA (Fig. 1). The fragments of B. subtilis DNA released from two plasmids, pIM1 and pIM2, appeared very similar and were estimated at 1.5 kilobases. Since the frgaments obtained after selection in E. coli represented only 19% of the "enriched" leu+ DNA segment obtained from B. subtilis 168, a hybridization experiment was carried out with purified pIM2 DNA and 32P-labeled B. subtilis DNA. EcoRItreated pMB9 and pIM2 DNA were subjected to electrophoresis in agarose tube gels, denatured, and eluted onto a cellulose nitrate filter (13). The filter was treated as described by Denhardt (2), incubated with 32P-labeled B. subtilis DNA enriched for leu+-transforming activity, and exposed to X-ray film. Shown in Fig. 2 is a single band of radioactivity, which appears on the filter from the transferred gel of pIM2 in the region of migration of the cloned leu+ fragment. The transformation efficiency of pIM2 with both E. coli and B. subtilis as recipients is shown in Table 3. pIM2 transforms E. coli HB101 to tetracycline resistance and leucine
374
VOL. 131, 1977
NOTES
TABLE 1. Bacterial strains used in this study Bacterial
Genotype
strain
E. coli HB129(pMB9) E. coli HB1O1a
B. subtilis JAS8° B. subtilis JAS9a B. subtilis BC50c
ence
end-gal- lac- hsm+ P. Wensink hsr+ leu pro thihsm hrs recA- P. Wensink gal- pro- leuBrpsL trpC- leuB12, 16 trpC- leuC12, 16 purA- leuA- metS- 12, 16
nia-38trp+ J. Marmur a leu- defective in isopropylmalate dehydrogenase (EC 1.1.185). ° leu- defective in isopropylmalate dehydratase (EC 4.2.133). c leu- defective in 2-isopropylmalate synthetase (EC 4.1.3.12).
B. subtilis 168
TABLE 2. Transformation efficiency of plasmid DNA a Source of DNA
375
plasmid amplification (8). The 3H-labeled pIM2 was used to follow the intracellular fate of the plasmid in competent cells of E. coli and B. subtilis as described by Dubnau and Cirigliano (3). Figure 3 shows the radioactivity recovered as a function of time from the transformed and lysed cells in acid-precipitable and acid-soluble form. The sharp increase in acid-soluble counts in B. subtilis JAS9 "transformed" with 3H-labeled pIM2 DNA shortly after DNA uptake suggests that the hybrid plasmid may be subject to intracellular nuclease action. Whereas transformation of E. coli HB101 to leucine prototrophy is clearly plasmid directed, the rare leucine transformants isolated from B. subtilis appear to be the result of chromosomal integration of the B. subtilis DNA fragment.
-
_ ~~~~~~~~~I
Transformants/pg of DNA
tetp leu+ tet pMB9 5 x 105 0 7 x 103 0 pMBgb pMB9c 2.7 x 105 0 pMB9-B. subtilisc 4.1 x 104 1-_5d aE. coli HB101 was made competent by the method of Wensink et al. (15). After exposure to DNA, 5 x 108 cells were grown in LB broth (9) for 1 h and washed twice with 0.15 M NaCl-0.02 M K2HPO4. The cells (1 x 10" to 2 x 108) were then plated on LB plates containing 25 ,ug of tetracycline (Calbiochem) per ml and on minimal salt plates (1) supplemented with the required amino acids, lacking leucine, and containing 25 Mg of tetracycline per ml. b EcoRI treated. c EcoRI treated and sealed with T4 ligase (original gene selection). d Number of colonies in separate experiments.
with prototrophy with equal efficiency. The same DNA added to three leu- strains of B. subtilis produced a few transformants only with the strain having a mutation in the isopropylmalate dehydrogenase gene (16). No tetr B. subtilis colonies were found even with 25 ,ug of pIM2 DNA per ml. Although B. subtilis 168 and its derivative strains are not thought to have DNA restriction systems (4, 14), we examined the uptake and survival of pIM2 (extracted from E. coli) in B. subtilis. Radioactive pIM2 was prepared from E. coli HB1O1(pIM2) by the addition of 8 uCi of [3H]thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear) per ml at the time of
-
~
~
4
FIG. 1. Electrophoresis of DNA was carried out in 0.7% agarose (Seakem). Bromophenol blue (0.25%) in 70% sucrose was added to the samples (15 to 100 piu), and the horizontal slab gel was run at 25 mA for approximately 16 h. The gel was stained in ethidium bromide solution (1 pg/ml) for 30 min and photographed under short-wavelength ultraviolet light delivered by a Universal UV Unit (Gelman), using a yellow filter (Kodak no. 9 Wratten gelatin) and Polaroid 3,000 speed, type 107 film. (A) Lambda phage DNA digested with EcoRI (standards from top to bottom: 15.7, 13.7, 4.7, 3.7, 3.57, 3.04, 2.11 megadaltons). (B) pMB9 DNA digested with EcoRI (3.5 megadaltons). (C) pIM2 DNA digested with EcoRI. The arrow indicates the position of the B. subtilis fragment. (D) pIM1 digested with EcoRI together with EcoRI-digested lambda DNA.
376
J. BACTERIOL.
NOTES
C
E. coli (HB 101)
D
.o z
E
.Ai
0
FIG. 2. Tube gel electrophoresis of EcoRI-treated pMB9 (A) and pIM2 DNA (B). After denaturation in 0.5 M NaOH-1.5 M NaCI, the DNA was transferred to a nitrocellulose filter (13). The filter was treated as described by Denhardt (2) and hybridized with 2 x 106 cpm of 32P-labeled B. subtilis DNA having a specific activity of 2.8 x 105 cpmlpg, prepared as described by Grossman (5), using H332PO4 (carrier free, New England Nuclear). After a 16-h exposure, the filter was washed extensively (2) and exposed to
X-ray film (Kodak No Screen NS-5T). (C) pMB9. (D) pIM2 DNA. TABLE 3. Transformation efficiency of pIM2 DNAa TransfOrmants/lAg of DNA Host
tetr
eu+
8 X 104 E. coli HB101 8.5 x 104 0 0 B. subtilis JAS8 B. subtilis JAS9 80-130 0 0 B. subtilis BC50 0 a B. subtilis strains were made competent by the method of Bott and Wilson (1). Competency was checked by transforming each strain with 1 mg of DNA isolated from a wild-type revertant ofB. subtilis 168. To check for tetr transformants, 4 x 108 cells were exposed to DNA for 30 min; an equal volume of LB broth was then added to each tube, and the incubation continued for 4 h before plating on LB plates containing 10 to 25 Ag of tetracycline per ml.
Chromosomal DNA isolated from eight such colonies transformed B. subtilis JAS9 cells to leu+ with the same efficiency as DNA isolated
10
20
30 0 10 Time (Min.)
20
30
40
FIG. 3. Acid-precipitable (0) and acid-soluble (0) radioactivity recovered from transformed and lysed cells. A 1-pg amount of [3H]DNA (specific activity, 1.1 x 104 cpm/pg) was added to competent cells of E. coli HB101 and B. subtilis JAS9 at 37°C. At the times indicated, the transformed cells were diluted fivefold with cold (4°C) 0.15 M NaCI-02 M K2HPO4 buffer (pH 7.2), washed twice in the same buffer, suspended to the same volume (0.5 ml) in 0.05 M NaCI-0.05 M ethylenediaminetetraacetic acid, and lysed at 37°C by the addition of 500 pg of lysozyme (EC 2.3.1.17) (Sigma) per ml followed by 350 pg of N-lauryl sarcosine (Sigma Co.) per ml. Acid-precipitable material was collected by filtration through GFIC glass filters (Whatman) after 15 min of incubation of 0°C in a solution of 5% trichloroacetic acid and 50 pg of salmon sperm DNA (Worthington Biochemicals Corp.) per ml. Acid-soluble radioactivity was determined by collecting the nitrocellulose membrane filtrates after trichloroacetic acid precipitation. Samples were counted in Aquasol (New England Nuclear) in a Beckman scintillation spectrometer (LS-1000) .
from B. subtilis 168. The extremely low level of transformation of the cloned fragment may be a result of its low molecular weight (1.5 kilobases), which would preclude efficient integration into the chromosome (10). Construction of hybrid DNA and all operations involving DNA isolation were carried out in a P2 containment room. This research was supported by Public Health Service grant GM18904 from the National Institute of General Medical Sciel - and by National Science Foundation grant PCM 76-11693. LITERATURE CITED 1. Bott, K. F. and G. Wilson. 1967. Development of compe-
tence in the Bacillus subtilis transformation system. J. Bacteriol. 94:562-570.
VOL. 131, 1977 2. Denhardt, D. T. 1966. A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646. 3. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming DNA following uptake by competent Bacillus subtilis. HI. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J. Mol. Biol. 64:9-29. 4. Ehrlich, S. D., H. Bursztyn-Pettegrew, I. Stroynowski, and J. Lederberg. 1976. Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 73:4145-4149. 5. Gromman, L. 1967. Preparation of phosphorus-32 labeled DNA. Methods Enzymol. 12A:700-702. 6. Harris-Warrick, R. M., Y. Elkana, S. D. Ehrlich, and J. Lederberg. 1975. Electrophoretic separation of Bacillus subtilis genes. Proc. Natl. Acad. Sci. U.S.A. 72:2207-2211. 7. Helling, R. B., H. M. Goodman, and H. W. Boyer. 1974. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14:1235-1244. 8. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski. 1974. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455-3459.
NOTES
377
9. Miller, J. H. 1972. Experiments in molecular genetics, p. 432-433. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 10. Morrison, D. A., and W. R. Guild. 1972. Activity of deoxyribonucleic acid fragments of defined size in Bacillus subtilis transformation. J. Bacteriol. 112:220-223. 11. Saito, H., and K. Miura. 1963. Preparation of transforming DNA by phenol treatment. Biochim. Biophys. Acta 72:619-629. 12. Shapiro, J. A., D. H. Dean, and H. 0. Halvorson. 1974. Low-frequency specialized transduction with Bacillus subtilis bacteriophage q105. Virology 62:393-403. 13. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 14. Trautner, T. A., B. Pawlek, S. Bron, and C. Anagnostopoulos. 1974. Restriction and modification in Bacillus subtilis. Biological aspects. Mol. Gen. Genet. 131:181-191. 15. Wensink, P. C., D. J. Finnegan, J. E. Donelson, and D. S. Hogness. 1974. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell 3:315-325. 16. Young, F., and G. Wilson. 1976. Revision of the linkage map of Bacillus subtilis, p. 686-703. In Gerald D. Fasman (ed.), Handbook of biochemistry and molecular biology, vol. 2. CRC Press, Cleveland, Ohio.
JOURNAL OF BACTZRIOLOGY, JUlY 1977, p. 374-377 Copyright C 1977 American Society for Microbiology
Transformation of Escherichia coli and Bacillus subtilis with a Hybrid Plasmid Molecule I. MAHLER* AND HARLYN 0. HALVORSON Research Center, Brandeis University, Waltham, Massachusetts 02154 Sciences Medical Basic Rosenstiel
Received for publication 25 March 1977
A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leuE. coli, converting them to leucine prototrophy, but fails to survive in strains of B. subtilis 168. In our efforts to clone and amplify gene fragments of Bacillus subtilis, we constructed hybrid deoxyribonucleic acid (DNA) molecules consisting of Escherichia coli plasmid pMB9, which carried resistance to tetracycline (tetr), and a segment of the B. subtilis genome. Covalently closed circular DNA was obtained from E. coli HB129(pMB9) by the method of Wensink et al. (15), whereas DNA from B. subtilis 168 was prepared by the method of Saito and Miura (11). The bacterial strains are listed in Table 1. The bacterial DNA (50 to 200 ,ug/ml) was incubated with restriction endonuclease EcoRI (the generous gift of P. Wensink) at 37°C for 15 to 30 min in a solution containing 12 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.6), 6 mM MgCl2, 50 mM NaCl, and 6 mM mercaptoethanol. Completeness of degradation was verified by examining the banding pattern for the disappearance of uncleaved DNA in agarose-gel electrophoresis followed by ethidium bromide staining (7). With B. subtilis JAS9 as a recipient in transformation experiments, the biological activity of the bacterial DNA was tested before and after endonuclease digestion. After enzymatic cleavage, the survival of leu+-transforming activity was reduced to 1%. A DNA segment enriched 16.6-fold for leu+-transformation activity was obtained by the method of Harris-Warrick et al. (6). Its molecular size measured against EcoRItreated phage lambda DNA (7) was estimated at 8.2 kilobases (data not shown). To prepare hybrid molecules, 0.5 ,ug of EcoRI-treated pMB9 was incubated with 1.5 ug of leu+-enriched B. subtilis DNA together with 0.4 U of T4 polynucleotide ligase (EC 6.5.11) (Biolabs, Beverly, Mass.) in a 100-,ul solution containing 30 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 8.0), 5 mM MgCl2, 0.2 mM adenosine 5'-triphosphate, 0.05 mM dithiothreitol, and 30 ,ug of bovine albumin
(fraction V; Sigma Chemical Co.) per ml. The reaction was carried out for 16 to 18 h at 140C and terminated by heating the solution at 680C for 3 min. The effectiveness of polynucleotide ligase sealing was checked by the reappearance of covalently closed circular molecules in agarose gels and the restoration of transforming activity (Table 2); the DNA thus generated was used to transformE. coli HB101. The efficiency of transformation with the treated plasmid DNAs is shown in Table 2. From the recipient strain, which is tets and leu-, double transformants (leu+ and tetr) were isolated and examined for the presence of plasmids with altered physical and biological characteristics. Two hybrid plasmids, isolated in separate experiments, were digested with EcoRI enzyme and run in a slab gel electrophoresis apparatus against EcoRI-digested phase lambda DNA (Fig. 1). The fragments of B. subtilis DNA released from two plasmids, pIM1 and pIM2, appeared very similar and were estimated at 1.5 kilobases. Since the frgaments obtained after selection in E. coli represented only 19% of the "enriched" leu+ DNA segment obtained from B. subtilis 168, a hybridization experiment was carried out with purified pIM2 DNA and 32P-labeled B. subtilis DNA. EcoRItreated pMB9 and pIM2 DNA were subjected to electrophoresis in agarose tube gels, denatured, and eluted onto a cellulose nitrate filter (13). The filter was treated as described by Denhardt (2), incubated with 32P-labeled B. subtilis DNA enriched for leu+-transforming activity, and exposed to X-ray film. Shown in Fig. 2 is a single band of radioactivity, which appears on the filter from the transferred gel of pIM2 in the region of migration of the cloned leu+ fragment. The transformation efficiency of pIM2 with both E. coli and B. subtilis as recipients is shown in Table 3. pIM2 transforms E. coli HB101 to tetracycline resistance and leucine
374
VOL. 131, 1977
NOTES
TABLE 1. Bacterial strains used in this study Bacterial
Genotype
strain
E. coli HB129(pMB9) E. coli HB1O1a
B. subtilis JAS8° B. subtilis JAS9a B. subtilis BC50c
ence
end-gal- lac- hsm+ P. Wensink hsr+ leu pro thihsm hrs recA- P. Wensink gal- pro- leuBrpsL trpC- leuB12, 16 trpC- leuC12, 16 purA- leuA- metS- 12, 16
nia-38trp+ J. Marmur a leu- defective in isopropylmalate dehydrogenase (EC 1.1.185). ° leu- defective in isopropylmalate dehydratase (EC 4.2.133). c leu- defective in 2-isopropylmalate synthetase (EC 4.1.3.12).
B. subtilis 168
TABLE 2. Transformation efficiency of plasmid DNA a Source of DNA
375
plasmid amplification (8). The 3H-labeled pIM2 was used to follow the intracellular fate of the plasmid in competent cells of E. coli and B. subtilis as described by Dubnau and Cirigliano (3). Figure 3 shows the radioactivity recovered as a function of time from the transformed and lysed cells in acid-precipitable and acid-soluble form. The sharp increase in acid-soluble counts in B. subtilis JAS9 "transformed" with 3H-labeled pIM2 DNA shortly after DNA uptake suggests that the hybrid plasmid may be subject to intracellular nuclease action. Whereas transformation of E. coli HB101 to leucine prototrophy is clearly plasmid directed, the rare leucine transformants isolated from B. subtilis appear to be the result of chromosomal integration of the B. subtilis DNA fragment.
-
_ ~~~~~~~~~I
Transformants/pg of DNA
tetp leu+ tet pMB9 5 x 105 0 7 x 103 0 pMBgb pMB9c 2.7 x 105 0 pMB9-B. subtilisc 4.1 x 104 1-_5d aE. coli HB101 was made competent by the method of Wensink et al. (15). After exposure to DNA, 5 x 108 cells were grown in LB broth (9) for 1 h and washed twice with 0.15 M NaCl-0.02 M K2HPO4. The cells (1 x 10" to 2 x 108) were then plated on LB plates containing 25 ,ug of tetracycline (Calbiochem) per ml and on minimal salt plates (1) supplemented with the required amino acids, lacking leucine, and containing 25 Mg of tetracycline per ml. b EcoRI treated. c EcoRI treated and sealed with T4 ligase (original gene selection). d Number of colonies in separate experiments.
with prototrophy with equal efficiency. The same DNA added to three leu- strains of B. subtilis produced a few transformants only with the strain having a mutation in the isopropylmalate dehydrogenase gene (16). No tetr B. subtilis colonies were found even with 25 ,ug of pIM2 DNA per ml. Although B. subtilis 168 and its derivative strains are not thought to have DNA restriction systems (4, 14), we examined the uptake and survival of pIM2 (extracted from E. coli) in B. subtilis. Radioactive pIM2 was prepared from E. coli HB1O1(pIM2) by the addition of 8 uCi of [3H]thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear) per ml at the time of
-
~
~
4
FIG. 1. Electrophoresis of DNA was carried out in 0.7% agarose (Seakem). Bromophenol blue (0.25%) in 70% sucrose was added to the samples (15 to 100 piu), and the horizontal slab gel was run at 25 mA for approximately 16 h. The gel was stained in ethidium bromide solution (1 pg/ml) for 30 min and photographed under short-wavelength ultraviolet light delivered by a Universal UV Unit (Gelman), using a yellow filter (Kodak no. 9 Wratten gelatin) and Polaroid 3,000 speed, type 107 film. (A) Lambda phage DNA digested with EcoRI (standards from top to bottom: 15.7, 13.7, 4.7, 3.7, 3.57, 3.04, 2.11 megadaltons). (B) pMB9 DNA digested with EcoRI (3.5 megadaltons). (C) pIM2 DNA digested with EcoRI. The arrow indicates the position of the B. subtilis fragment. (D) pIM1 digested with EcoRI together with EcoRI-digested lambda DNA.
376
J. BACTERIOL.
NOTES
C
E. coli (HB 101)
D
.o z
E
.Ai
0
FIG. 2. Tube gel electrophoresis of EcoRI-treated pMB9 (A) and pIM2 DNA (B). After denaturation in 0.5 M NaOH-1.5 M NaCI, the DNA was transferred to a nitrocellulose filter (13). The filter was treated as described by Denhardt (2) and hybridized with 2 x 106 cpm of 32P-labeled B. subtilis DNA having a specific activity of 2.8 x 105 cpmlpg, prepared as described by Grossman (5), using H332PO4 (carrier free, New England Nuclear). After a 16-h exposure, the filter was washed extensively (2) and exposed to
X-ray film (Kodak No Screen NS-5T). (C) pMB9. (D) pIM2 DNA. TABLE 3. Transformation efficiency of pIM2 DNAa TransfOrmants/lAg of DNA Host
tetr
eu+
8 X 104 E. coli HB101 8.5 x 104 0 0 B. subtilis JAS8 B. subtilis JAS9 80-130 0 0 B. subtilis BC50 0 a B. subtilis strains were made competent by the method of Bott and Wilson (1). Competency was checked by transforming each strain with 1 mg of DNA isolated from a wild-type revertant ofB. subtilis 168. To check for tetr transformants, 4 x 108 cells were exposed to DNA for 30 min; an equal volume of LB broth was then added to each tube, and the incubation continued for 4 h before plating on LB plates containing 10 to 25 Ag of tetracycline per ml.
Chromosomal DNA isolated from eight such colonies transformed B. subtilis JAS9 cells to leu+ with the same efficiency as DNA isolated
10
20
30 0 10 Time (Min.)
20
30
40
FIG. 3. Acid-precipitable (0) and acid-soluble (0) radioactivity recovered from transformed and lysed cells. A 1-pg amount of [3H]DNA (specific activity, 1.1 x 104 cpm/pg) was added to competent cells of E. coli HB101 and B. subtilis JAS9 at 37°C. At the times indicated, the transformed cells were diluted fivefold with cold (4°C) 0.15 M NaCI-02 M K2HPO4 buffer (pH 7.2), washed twice in the same buffer, suspended to the same volume (0.5 ml) in 0.05 M NaCI-0.05 M ethylenediaminetetraacetic acid, and lysed at 37°C by the addition of 500 pg of lysozyme (EC 2.3.1.17) (Sigma) per ml followed by 350 pg of N-lauryl sarcosine (Sigma Co.) per ml. Acid-precipitable material was collected by filtration through GFIC glass filters (Whatman) after 15 min of incubation of 0°C in a solution of 5% trichloroacetic acid and 50 pg of salmon sperm DNA (Worthington Biochemicals Corp.) per ml. Acid-soluble radioactivity was determined by collecting the nitrocellulose membrane filtrates after trichloroacetic acid precipitation. Samples were counted in Aquasol (New England Nuclear) in a Beckman scintillation spectrometer (LS-1000) .
from B. subtilis 168. The extremely low level of transformation of the cloned fragment may be a result of its low molecular weight (1.5 kilobases), which would preclude efficient integration into the chromosome (10). Construction of hybrid DNA and all operations involving DNA isolation were carried out in a P2 containment room. This research was supported by Public Health Service grant GM18904 from the National Institute of General Medical Sciel - and by National Science Foundation grant PCM 76-11693. LITERATURE CITED 1. Bott, K. F. and G. Wilson. 1967. Development of compe-
tence in the Bacillus subtilis transformation system. J. Bacteriol. 94:562-570.
VOL. 131, 1977 2. Denhardt, D. T. 1966. A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646. 3. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming DNA following uptake by competent Bacillus subtilis. HI. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J. Mol. Biol. 64:9-29. 4. Ehrlich, S. D., H. Bursztyn-Pettegrew, I. Stroynowski, and J. Lederberg. 1976. Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 73:4145-4149. 5. Gromman, L. 1967. Preparation of phosphorus-32 labeled DNA. Methods Enzymol. 12A:700-702. 6. Harris-Warrick, R. M., Y. Elkana, S. D. Ehrlich, and J. Lederberg. 1975. Electrophoretic separation of Bacillus subtilis genes. Proc. Natl. Acad. Sci. U.S.A. 72:2207-2211. 7. Helling, R. B., H. M. Goodman, and H. W. Boyer. 1974. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14:1235-1244. 8. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski. 1974. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455-3459.
NOTES
377
9. Miller, J. H. 1972. Experiments in molecular genetics, p. 432-433. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 10. Morrison, D. A., and W. R. Guild. 1972. Activity of deoxyribonucleic acid fragments of defined size in Bacillus subtilis transformation. J. Bacteriol. 112:220-223. 11. Saito, H., and K. Miura. 1963. Preparation of transforming DNA by phenol treatment. Biochim. Biophys. Acta 72:619-629. 12. Shapiro, J. A., D. H. Dean, and H. 0. Halvorson. 1974. Low-frequency specialized transduction with Bacillus subtilis bacteriophage q105. Virology 62:393-403. 13. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 14. Trautner, T. A., B. Pawlek, S. Bron, and C. Anagnostopoulos. 1974. Restriction and modification in Bacillus subtilis. Biological aspects. Mol. Gen. Genet. 131:181-191. 15. Wensink, P. C., D. J. Finnegan, J. E. Donelson, and D. S. Hogness. 1974. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell 3:315-325. 16. Young, F., and G. Wilson. 1976. Revision of the linkage map of Bacillus subtilis, p. 686-703. In Gerald D. Fasman (ed.), Handbook of biochemistry and molecular biology, vol. 2. CRC Press, Cleveland, Ohio.
