Vol. 15, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, Jan. 1975, p. 16-21 Copyright 0 1975 American Society for Microbiology
Bacteriophage Transformation of PBS2 in Bacillus subtilis M. B. HERRINGTON AND I. TAKAHASHI* Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Received for publication 29 August 1974
Transformation of temperature-sensitive mutants of bacteriophage PBS2 for Bacillus subtilis was demonstrated. The number of transformants was linearly related to the concentration of DNA within a range of 0.01 to 1 Ag/ml. No transformants were obtained when the DNA was pretreated with DNase. PBS2 DNA sheared to approximately 1% of the total chromosome length was centrifuged in Cs2SO4-Hg gradients to fractionate the DNA according to the base composition. Transformation experiments carried out with the fractionated DNA indicated the possibility of determining the base composition of different regions of the phage chromosome. The distribution of sheared DNA from Bacillus subtilis (18) and transducing phage PBS1 (H. Yamagishi, unpublished data) in CS2SO4Hg density gradients is quite broad, reflecting a segmental distribution of nucleotides in the DNA. To determine the biological activity of the phage DNA fractionated by the above technique, phage transformation similar to that observed with SP82 for B. subtilis (1) or with T4 for Escherichia coli (15) has been developed. This technique measures genetic recombination between intracellular phage chromosome and fragments of DNA taken up by B. subtilis cells. This paper reports the optimal condi tions for transformation of PBS2, a clear-plaque derivative of PBS1, and preliminary results obtained with the fractionated-phage DNA. Results obtained in the present study suggest the possibility of determining the base composition of different regions of PBS2 chromosome. This technique would also enable us to concentrate DNA fragments carrying a specific marker.
numbers representing the order of isolation. Thus, mutants induced with FUdR are prefixed TFU; with hydroxylamine, THA; with nitrosoguanidine, TNG; and with 2-aminopurine, TAP. Media. Adsorption medium (17) was used for phage dilutions. Penassay broth (Difco) was used to prepare broth cultures. The basic growth medium (BGM) and the competence medium (CM) were modified from those described by Mahler (4). The BGM was a minimal medium (9) supplemented with tryptophan (50 jig/ ml), yeast extract (Difco; 1 mg/ml), Casamino Acids (Difco; 200 ,g/ml), tyrosine (50 pg/ml), and phenylalanine (50 ,g/ml). Immediately before use arginine (1 mg/ml) was added. The CM was a minimal medium (9) supplemented with tryptophan (5 Ag/ml), yeast extract (Difco; 1 mg/ml), Casamino Acids (Difco; 100 ,ggml), tyrosine (25 ,g/ml), histidine (25 ,g/ml), and shikimic acid (25 ug/ml). Immediately before use, 0.25 ml of 0.125 M CaCl2, 0.25 ml of 0.1 M MgCl2, and 0.1 ml of 0.05 M spermine (Aldrich Chemical Co.) were added to 10 ml of CM. Phage techniques. Lysates were prepared as described by Takahashi (11) except that lysates of ts mutants were incubated at 30 C. Plaque assays were made as described previously (11). Phage transformation. Competent cells of SB202 were infected with a ts mutant at a multiplicity of 1 to 2, at which time the phage DNA was added. The transformation mixture was incubated at 30 C with shaking for 100 min at which time lysis was complete. The lysate was then diluted and plated. The plates were incubated at the nonpermissive temperature (45 C) for 4 to 8 h to determine the number of wild-type transformants present in the lysate. Controls were routinely made to determine the number of wild-type revertants present in lysates in the absence of transforming DNA and to determine plaque-forming activities in transformation mixtures where mutant phages were omitted. Induction of competence. Cells of SB202 were made competent (method I) as described by Takahashi (13), except that the second incubation was for 60
MATERIALS AND METHODS Bacterial strains and phage. Strain SB19E (strr, er/) (12) of B. subtilis was used as host for PBS2.
Strain SB202 (aro-2, trp-2, hisB2, tyr-1) (5) was used as host in phage transformation.
A single plaque isolate of a clear-plaque mutant was obtained from a lysate of PBS1 (10). This mutant was called PBS2 (11) because it was isolated at the same time as PBS1 and was considered to be distinct from PBS1. Temperature-sensitive (ts) mutants were isolated from lysates of PBS2 treated with 5-fluoro-
deoxyuridine (FUdR), hydroxylamine, N-methyl-N'nitro-N-nitrosoguanidine (nitrosoguanidine) and 2aminopurine (2). Temperature-sensitive mutants are identified by the prefix T followed by two letters referring to the mutagen employed and two or three 16
VOL. 15, 1975
17
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
min. Cells were also made competent by the following technique (method II), which is a modification of the method of Mahler (4). Cells of SB202 were grown in BGM, and the growth was followed by measuring the turbidity of the culture with a Klett-Summerson colorimeter with a no. 59 filter. At the end of the exponential phase, the cells were diluted 10-fold in CM and incubated further for 90 min at 37 C. Competent cells prepared by method II gave higher levels of transformation than did competent cells prepared by method I. Preparation of DNA. Phage lysates were centrifuged at 3,000 x g for 15 min to remove bacterial debris. Phage particles were collected from the supernatant by centrifugation at 40,000 x g for 1 h. The phage particles were suspended in a small volume of 0.15 M NaCl and 0.1 M phosphate buffer (pH 7.0) and then treated with lysozyme (100 pg/ml), DNase (10 ug/ml), and RNase (10 ug/ml) at 37 C for 30 min. The phage was subjected to two more cycles of differential centrifugation and was finally suspended in 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate). The purified phage in 1 x SSC was shaken for 10 min with an equal volume of cold phenol saturated with 1 x SSC. This was centrifuged at 3,000 x g for 10 min, and the aqueous layer was extensively dialyzed against 1 x SSC to remove phenol. The DNA was precipitated by adding 2 volumes of 95% ethanol and stored in 75% ethanol for 3 days prior to use. The DNA was dissolved in sterile 0.1 x SSC, and then the SSC concentration was adjusted to 1 x SSC by the addition of 20 x SSC. Solutions of DNA were stored at -10 C. The concentration of DNA was estimated by measuring the absorbance at 260 nm. An optical density of 20 at 260 nm was taken as a DNA concentration of 1 mg/ml. Fractionation of sheared DNA. Solutions of DNA in 1 x SSC (140 ug/ml) were sheared by stirring at 33,000 rpm for 20 min in a VirTiis homogenizer to obtain DNA fragments of about 1.5 x 10" daltons (18). The sheared DNA was then dialyzed extensively against 0.1 M Na2SO4 to remove chloride ions. Gradients of Cs,SO4-Hg were prepared as described by Yamagishi and Takahashi (18). The ingredients were added directly to a 12-ml nitrocellulose centrifuge tube in the following order: Cs2SO4, 2.52 g; DNA (in 0.1 M Na,SO4), 2.5 ml; 0.1 M sodium borate solution, 0.125 ml; HgCI2 (100 Ag/ml), volume determined for individual experiment; 0.1 M Na2SO4-0.005 M sodium borate, to a final weight of 6.04 g. The amount of HgCl2 used depended on the R, value (mole of Hg/mole of nucleotide) used and was calculated from the following formula: the amount of HgCl, in micrograms equals the amount of DNA in micrograms x 0.7882 (a constant factor) x R, value. For transformation experiments, 220 jig of DNA was centrifuged at an R, value of 0.08. Gradients were overlaid with mineral oil and centrifuged for 48 h at 4 C and 36,000 rpm in a 5OTi rotor in a Beckman L2-65 ultracentrifuge. After centrifugation, fractions were collected from the bottom of the tube. The fractions were collected in 1 ml of 1.0 M NaCl. The amount of DNA in the fractions was estimated by measuring the absorbance
at 260 nm in a Unicam spectrophotometer fitted with 0.2-ml cuvettes (1-cm path length). Fractions used for transformation were pooled as shown in Fig. 4 and dialyzed against 1 x SSC. The dialyzed samples were stored at -10 C. Treatment of DNA by enzymes. A solution of DNA (100 Mg/ml) in 0.02 M Tris buffer-0.1 M NaCl-0.01 M MgSo4 (pH 7.5) was treated with DNase at 1 jg/ml at 37 C for 30 min. Treatment of DNA (50 ;g/ml) with trypsin (50 Mg/ml) was done in 0.05 M Tris buffer (pH 8.0) plus 0.02 M CaCl2 at 37 C for 60 min, and with Pronase (200 Ag/ml) in 0.01 M potassium phosphate buffer (pH 7.0) at 37 C for 45 min. A solution of DNA (5 jg/ml) was treated with RNase at 10 ;g/ml in 0.1 M acetate buffer (pH 7.0) at 37 C for 30 min.
RESULTS Transformation of PBS2. Figure 1 shows that ts mutant TAP02 could be transformed to the wild type with DNA extracted from wildtype phage. Although the experiments were carried out under different conditions, the number of transformants obtained was always linearly related to the concentration of DNA up to about 1 jig/ml. Competence, or the ability to take up DNA,
E 107
~
/ ~~~~~~~~~
z 4 0~~~~~~~~~~ U-.0
o-J
0
DNA CONCENTRATION
(gg/mi)
FIG. 1. Effect of DNA concentration on PBS2 transformation. Competent cells were infected with TAP02 and treated with different concentrations of wild-type DNA. Symbols: 0, unsheared DNA, cells made competent by method II; A, unsheared DNA, cells made competent by method I; 0, sheared DNA, cells made competent by method II.
18
HERRINGTON AND TAKAHASHI
can be induced in B. subtilis in a variety of ways. Competent cells in the present study were prepared by two methods. Method I was found to be much less effective than method II and, in fact, no transformants were obtained with sheared DNA when method I was used (Fig. 1). As expected, sheared DNA gave a considerably reduced level of transformation (Fig. 1). This has also been observed with sheared DNA in bacterial transformation (6). The following experiments were carried out to determine the optimal conditions for PBS2 transformation. In the first experiment, competent cells were mixed with DNA, and the mutant phage was added at different times (Table 1). Wild-type transformants were assayed after the cells lysed. The amount of transformation decreased rapidly in the first 10 min, indicating that the DNA might be degraded during the incubation period. A DNase which specifically degrades the uracil-containing PBS2 DNA has been isolated from uninfected B. subtilis cells (F. Tomita and I. Takahashi, manuscript in preparation). This DNase may be responsible for the inactivation of PBS2 DNA in vivo, thereby causing the reduction in the amount of transformation. In the second experiment, DNA was added to competent cells at different times after infection. Transformation could be observed for at least 40 min, but the amount of transformation decreased considerably after 30 min (Table 1). These experiments indicate that transformation is most efficient when DNA and mutant phage are added to competent cells at the same time. The effects of various enzymes and serum albumin on transformation of PBS2 are summarized in Table 2. As expected, DNase greatly
J. VIROL.
reduced the transforming activity of the DNA. Treatment of the DNA with high concentrations of other enzymes showed a slight reduction in the transforming activity. This slight reduction was probably due to an inhibitory effect of protein, since the addition of bovine serum albumin directly to the transformation mixture reduced the amount of transformation. Alternatively, this inactivation might be due to contaminating nucleases. The time course of the transformation process was determined by assaying, at different times, the number of infective centers present in a mixture for transformation of TAP02. Figure 2 shows the total number of infective centers (assayed at 30 C) and the number of wild-type infective centers (assayed at 45 C) at different times after infection. The total infective centers TABLE 2. Effect of various treatments on transforming DNA Transformation (% of untreated control)a
Treatment
DNase ............................ Trypsin ............................. Pronase ............................ RNase .............................. Bovine serum albumin ......... ......
0.7 100 75 91 78
aTransformation was performed as follows: competent cells (method H) were infected with TAP02, and treated DNA was added to give a final concentration of 1 ,g/ml for DNase-treated sample and 0.5 Ag/ml for all other samples. Bovine serum albumin (0.5 ,g/ml) was added directly to the transformation mixture.
TABLE 1. Dependence of transformation on the time of phage and DNA additiona i
uAJ 4
Time of infection or DNA addition (min)
Wild-type transformants/ml DNA added
atOmin
TAP02 added atOmin
4
zI
0
9
4c
0.
0 aJ
0 5 10 15 20 25 30 35 40
2.2 x 106 2.5 x 105
4.0 x 105 1.5 1.3 8.1 1.5
x 105 x 10 x 104 x 100
2.2 1.8 1.5 1.8 1.1
x 106 x 106
x 106 x 106 x 106
1.1 x 106
TIME (min)
FIG. 2. Single-step growth curve in transformation. Competent cells (method II) were infected with TAP02 and 2 sg of wild-type DNA per ml was added. a Cells were made competent by method II. The Samples were taken at different times and were concentration of DNA was 2 tsg/ml, and the multiplic- plated at 30 and 45 C. Symbols: 0, wild-type PFU/ ity of infection was 1. ml; 0, total PFU/ml. 4.3 x 105 3.6 x 105
VOL. 15, 1975
19
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
followed a typical single-step growth curve with a latent period of 63 min, a rise period of 22 min, and a burst size of 7.7. The appearance of wild-type infective centers was more complex. Wild-type infective centers appeared rapidly during the first 15 min, and then appeared at a lower rate throughout the remainder of the latent period and the rise period. The time required for the uptake of DNA by competent cells was determined by adding DNase to the transformation mixture at different times and assaying for transformants after the cells lysed. When the cells were in contact with DNA for less than 10 min, only a few transformants were obtained (Fig. 3). As the time of contact with DNA increased, the amount of transformation increased sharply until 40 min. There was a slight decrease in the number of transformants thereafter. These results indicate that DNA must be present at least for 10 min to cause transformation, and the uptake of DNA may be complete after 40
DNA were centrifuged in a Cs,SO-Hg density gradient (Fig. 4A). Results of transformation performed with the fractionated DNA were expressed as the percentage of the total number of transformants in the gradient (Fig. 4B and Table 4). Transforming activities for various mutants were distributed differently in the gradient, indicating hetTABLE 3. Frequency of transformants of different mutantsa
Transformants/ml Transformants/ml with unsheared with sheared
Mutant
DNA
TAP02 TFU121 TNG04 TFU34 TFU91
4.1 x 8.8 x 6.0 x 1.7 x 7.6 x
106 101 104 106 105
DNA
3.7 4.4 1.4 1.2
X 104 x 104
x 104 x 104
1.1 X 104
aCells were made competent by method II, and DNA (0.1 Ag/ml) from wild-type phage was used.
min.
The frequencies of transformants observed with various phage mutants are listed in Table 3. These mutants gave frequencies of transformants varying from 4.1 x 10/ml to 6.0 x 104/ml with unsheared DNA and from 4.4 x 104/ml to 1.1 x 104/ml with sheared DNA (Table 3). Transformation by fractionated DNA. To obtain DNA fractionated with respect to nucleotide composition, small fragments of PBS2
Ec
0
N
A
(0 0.300
~~1a 57
C 0.200 z
4
M
0
0.100
co) ui
a 4 (A
C,)
LAJ
z
a
o
IL z
0 UL
0
0
z
4 Iz
4 at.
a-
LL
U1)
z 0
4c
TIME (min)
FIG. 3. Kinetics of transformation by PBS2 DNA. Competent cells (method II) were infected with TAP02 at a multiplicity of infection of 1 to 2; wild-type PBS2 DNA was added at a concentration of 2 ug/ml. The mixtures were incubated at 30 C with shaking. At different times 10 sg of pancreatic DNase per ml was added.
10 20 30 40 FIG. 4. Transformation of ts mutants by fractionated DNA. The histogram shows the distribution of DNA in the gradient. Fractions were pooled and numbered as indicated by the horizontal lines, and their transforming activity was measured after dialysis. Transformation was measured by incubating 0.05 ml of mutant phage (1.0 x 109 PFU/ml), 0.05 ml of fractionated DNA, and 0.4 ml of competent cells (method II) for 100 min and then assaying for wildtype transformants. Symbols: 0, TFUI03; 0, TFU91; A, TAP11.
J. VIROL.
HERRINGTON AND TAKAHASHI
20
TABLE 4. Transformation of ts mutants by fractionated DNAa
Pooled fraction number
THA01
TFU11
TFU34
TFU12
TAP02
TFU73
TFU171
TFU102
1 2 3 4 5 6 7 8
1.9 4.8 7.6 11.0 16.0 23.0 23.0 12.0
0.8 4.9 3.2 15.0 26.0 17.0 21.0 11.0
0 2.7 9.7 17.0 39.0 20.0 6.2 5.3
1.0 0 5.8 11.0 54.0 20.0 9.1 0
0.6 27.0 39.0 14.0 4.3 9.3 0.9 4.6
0 4.3 0.5 6.5 4.2 4.6 46.0 34.0
0.9 13.0 1.8 62.0 14.0 9.0 0 0
2.7 2.2 3.8 30.0 42.0 13.0 6.3 1.2
a The method used to determine transforming activity is described in the footnote to Fig. 4. The pooled fraction numbers refer to those in Fig. 4A. The results for each mutant are expressed as the percentage of the total number of transformants obtained with fractionated DNA.
base composition of various genes in PBS2 chromosome. The transforming activity for some mutants (e.g., TFU73 and TFU102 in Table 4) was concentrated in a few fractions, whereas the activity for other mutants showed a rather broad distribution. erogeneous
The transforming activity for TFU91 was present in 5 of the 8 fractions tested (Fig. 4A), whereas the activity for TFU34 was present in only 3 of the 8 fractions (Table 4). It is possible that the broadness in the distribution of transforming activity in the gradient is an indication of heterogeneous base composition of the region of the chromosome involved. If a mutation is located in a region of the chromosome composed of two segments of quite different nucleotide composition, then, since breakage by shear is not absolutely specific (7), fragments of DNA containing this mutation will be distributed more widely in the gradient than fragments carrying mutations from a homogeneous region. Therefore, the broadness in the distribution of transforming activity may be used to estimate the degree of heterogeneity in the base composition around a particular marker.
DISCUSSION The foregoing results indicate that ts mutants of PBS2 can be transformed to the wild type in competent B. subtilis cells and that this transformation is mediated by free DNA molecules. Transfection, which has been observed with a number of other B. subtilis phages, has not been successful with PBS2 under our conditions. The absence of transfection by PBS2 DNA might be due to the large size (1.9 x 108 daltons) of the DNA (3). Centrifugation in gradients of Cs2SO,-Hg separates DNA molecules according to their ACKNOWLEDGMENTS nucleotide composition (8) and can be used to This work was supported by grant A1996 from the National determine whether the nucleotide composition Research Council of Canada. of a DNA molecule is uniform throughout the We would like to thank H. Yamagishi and C. T. Chow foi molecule or if the molecule is made up of several assistance with the gradients. segments of different nucleotide composition. LITERATURE CITED The DNA of bacteriophage lambda (8) is comD. 1. M. 1964. Infectivity of DNA isolated from Green, posed of several large segments of different subtilis Bacillus bacteriophage, SP82. J. Mol. Biol. DNA the In contrast, nucleotide composition. 10:438-451. from E. coli shows little or no intramolecular 2. Herrington, M. B., and I. Takahashi. 1973. Mutagenesis of bacteriophage PBS 2. Mutat. Res. 20:275-278. heterogeneity when large fragments of the moleB. I., H. Yamagishi, and I. Takahashi. 1967. cule are examined, but does show heterogeneity 3. Hunter, Molecular weight of bacteriophage PBS 1 deoxyribonu. when smaller fragments (1.5 x 106 daltons) are cleic acid. J. Virol. 1:841-842. examined (16). A similar situation has been 4. Mahler, I. 1968. Procedures for Bacillus subtilis transformation, p. 846-850. In S. P. Colowick and N. 0. found for PBS1 and PBS2 DNA. According to Kaplan (ed.), Methods in enzymology, vol. XII, part B. H. Yamagishi (personal communication), the Academic Press Inc., New York. base composition of small fragments of PBS1 5. Nester, E. W., M. Schafer, and J. Lederberg. 1963. Gene DNA fractionated by centrifugation in the gralinkage in DNA transfer: a cluster of genes concerned with aromatic biosynthesis in Bacillus subtilis. Genetdients ranged from 25 to 35% guanine plus ics 48:529-551. cytosine. The average base composition of PBS1 6. Rosenberg, B. H., F. M. Sirotnak, and L. F. Cavalieri. DNA determined by chemical analysis is 28% 1959. On the size of genetic determinants in Pneumococcus and the nature of the variables involved guanine plus cytosine (14).
VOL. 15, 1975
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8. 9. 10. 11.
12.
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
in transtormation. Proc. Nat. Acad. Sci. U.S.A. 45:144-156. Skalka, A. 1971. A method for the breakage of DNA and resolution of the fragments, p. 341-350. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. XXI, part D. Academic Press Inc., New York. Skalka, A., E. Burgi, and A. D. Hershey. 1968. Segmental distribution of nucleotides in the DNA of bacteriophage lambda. J. Mol. Biol. 34:1-16. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 4:1072-1078. Takahashi, I. 1961. Genetic transduction in Bacillus subtilis. Biochem. Biophys. Res. Commun. 5:171-175. Takahashi, I. 1963. Transducing phages for Bacillus subtilis. J. Gen. Microbiol. 31:211-217. Takahashi, I. 1965. Transduction of sporogenesis in Bacillus subtilis. J. Bacteriol. 89:294-298
21
13. Takahashi, I. 1965. Localization of spore markers on the chromosome of Bacillus subtilis. J. Bacteriol. 89:1065-1067. 14. Takahashi, I., and J. Marmur. 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature (London) 197:794-795. 15. Van de Pol, J. H., G. Veldhuisen, and J. A. Cohen. 1961. Phage transformation: a new criterium for the biological activity of bacteriophage DNA. Biochim. Biophys. Acta 48:417-418. 16. Yamagishi, H. 1970. Nucleotide distribution in the DNA of Escherichia coli. J. Mol. Biol. 49:603-608. 17. Yamagishi, H., and I. Takahashi. 1968. Transducing particles of PBS 1. Virology 36:639. 18. Yamagishi, H., and I. Takahashi. 1971. Heterogeneity in nucleotide composition of Bacillus subtilis DNA. J. Mol. Biol. 57:369-371.
JOURNAL OF VIROLOGY, Jan. 1975, p. 16-21 Copyright 0 1975 American Society for Microbiology
Bacteriophage Transformation of PBS2 in Bacillus subtilis M. B. HERRINGTON AND I. TAKAHASHI* Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Received for publication 29 August 1974
Transformation of temperature-sensitive mutants of bacteriophage PBS2 for Bacillus subtilis was demonstrated. The number of transformants was linearly related to the concentration of DNA within a range of 0.01 to 1 Ag/ml. No transformants were obtained when the DNA was pretreated with DNase. PBS2 DNA sheared to approximately 1% of the total chromosome length was centrifuged in Cs2SO4-Hg gradients to fractionate the DNA according to the base composition. Transformation experiments carried out with the fractionated DNA indicated the possibility of determining the base composition of different regions of the phage chromosome. The distribution of sheared DNA from Bacillus subtilis (18) and transducing phage PBS1 (H. Yamagishi, unpublished data) in CS2SO4Hg density gradients is quite broad, reflecting a segmental distribution of nucleotides in the DNA. To determine the biological activity of the phage DNA fractionated by the above technique, phage transformation similar to that observed with SP82 for B. subtilis (1) or with T4 for Escherichia coli (15) has been developed. This technique measures genetic recombination between intracellular phage chromosome and fragments of DNA taken up by B. subtilis cells. This paper reports the optimal condi tions for transformation of PBS2, a clear-plaque derivative of PBS1, and preliminary results obtained with the fractionated-phage DNA. Results obtained in the present study suggest the possibility of determining the base composition of different regions of PBS2 chromosome. This technique would also enable us to concentrate DNA fragments carrying a specific marker.
numbers representing the order of isolation. Thus, mutants induced with FUdR are prefixed TFU; with hydroxylamine, THA; with nitrosoguanidine, TNG; and with 2-aminopurine, TAP. Media. Adsorption medium (17) was used for phage dilutions. Penassay broth (Difco) was used to prepare broth cultures. The basic growth medium (BGM) and the competence medium (CM) were modified from those described by Mahler (4). The BGM was a minimal medium (9) supplemented with tryptophan (50 jig/ ml), yeast extract (Difco; 1 mg/ml), Casamino Acids (Difco; 200 ,g/ml), tyrosine (50 pg/ml), and phenylalanine (50 ,g/ml). Immediately before use arginine (1 mg/ml) was added. The CM was a minimal medium (9) supplemented with tryptophan (5 Ag/ml), yeast extract (Difco; 1 mg/ml), Casamino Acids (Difco; 100 ,ggml), tyrosine (25 ,g/ml), histidine (25 ,g/ml), and shikimic acid (25 ug/ml). Immediately before use, 0.25 ml of 0.125 M CaCl2, 0.25 ml of 0.1 M MgCl2, and 0.1 ml of 0.05 M spermine (Aldrich Chemical Co.) were added to 10 ml of CM. Phage techniques. Lysates were prepared as described by Takahashi (11) except that lysates of ts mutants were incubated at 30 C. Plaque assays were made as described previously (11). Phage transformation. Competent cells of SB202 were infected with a ts mutant at a multiplicity of 1 to 2, at which time the phage DNA was added. The transformation mixture was incubated at 30 C with shaking for 100 min at which time lysis was complete. The lysate was then diluted and plated. The plates were incubated at the nonpermissive temperature (45 C) for 4 to 8 h to determine the number of wild-type transformants present in the lysate. Controls were routinely made to determine the number of wild-type revertants present in lysates in the absence of transforming DNA and to determine plaque-forming activities in transformation mixtures where mutant phages were omitted. Induction of competence. Cells of SB202 were made competent (method I) as described by Takahashi (13), except that the second incubation was for 60
MATERIALS AND METHODS Bacterial strains and phage. Strain SB19E (strr, er/) (12) of B. subtilis was used as host for PBS2.
Strain SB202 (aro-2, trp-2, hisB2, tyr-1) (5) was used as host in phage transformation.
A single plaque isolate of a clear-plaque mutant was obtained from a lysate of PBS1 (10). This mutant was called PBS2 (11) because it was isolated at the same time as PBS1 and was considered to be distinct from PBS1. Temperature-sensitive (ts) mutants were isolated from lysates of PBS2 treated with 5-fluoro-
deoxyuridine (FUdR), hydroxylamine, N-methyl-N'nitro-N-nitrosoguanidine (nitrosoguanidine) and 2aminopurine (2). Temperature-sensitive mutants are identified by the prefix T followed by two letters referring to the mutagen employed and two or three 16
VOL. 15, 1975
17
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
min. Cells were also made competent by the following technique (method II), which is a modification of the method of Mahler (4). Cells of SB202 were grown in BGM, and the growth was followed by measuring the turbidity of the culture with a Klett-Summerson colorimeter with a no. 59 filter. At the end of the exponential phase, the cells were diluted 10-fold in CM and incubated further for 90 min at 37 C. Competent cells prepared by method II gave higher levels of transformation than did competent cells prepared by method I. Preparation of DNA. Phage lysates were centrifuged at 3,000 x g for 15 min to remove bacterial debris. Phage particles were collected from the supernatant by centrifugation at 40,000 x g for 1 h. The phage particles were suspended in a small volume of 0.15 M NaCl and 0.1 M phosphate buffer (pH 7.0) and then treated with lysozyme (100 pg/ml), DNase (10 ug/ml), and RNase (10 ug/ml) at 37 C for 30 min. The phage was subjected to two more cycles of differential centrifugation and was finally suspended in 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate). The purified phage in 1 x SSC was shaken for 10 min with an equal volume of cold phenol saturated with 1 x SSC. This was centrifuged at 3,000 x g for 10 min, and the aqueous layer was extensively dialyzed against 1 x SSC to remove phenol. The DNA was precipitated by adding 2 volumes of 95% ethanol and stored in 75% ethanol for 3 days prior to use. The DNA was dissolved in sterile 0.1 x SSC, and then the SSC concentration was adjusted to 1 x SSC by the addition of 20 x SSC. Solutions of DNA were stored at -10 C. The concentration of DNA was estimated by measuring the absorbance at 260 nm. An optical density of 20 at 260 nm was taken as a DNA concentration of 1 mg/ml. Fractionation of sheared DNA. Solutions of DNA in 1 x SSC (140 ug/ml) were sheared by stirring at 33,000 rpm for 20 min in a VirTiis homogenizer to obtain DNA fragments of about 1.5 x 10" daltons (18). The sheared DNA was then dialyzed extensively against 0.1 M Na2SO4 to remove chloride ions. Gradients of Cs,SO4-Hg were prepared as described by Yamagishi and Takahashi (18). The ingredients were added directly to a 12-ml nitrocellulose centrifuge tube in the following order: Cs2SO4, 2.52 g; DNA (in 0.1 M Na,SO4), 2.5 ml; 0.1 M sodium borate solution, 0.125 ml; HgCI2 (100 Ag/ml), volume determined for individual experiment; 0.1 M Na2SO4-0.005 M sodium borate, to a final weight of 6.04 g. The amount of HgCl2 used depended on the R, value (mole of Hg/mole of nucleotide) used and was calculated from the following formula: the amount of HgCl, in micrograms equals the amount of DNA in micrograms x 0.7882 (a constant factor) x R, value. For transformation experiments, 220 jig of DNA was centrifuged at an R, value of 0.08. Gradients were overlaid with mineral oil and centrifuged for 48 h at 4 C and 36,000 rpm in a 5OTi rotor in a Beckman L2-65 ultracentrifuge. After centrifugation, fractions were collected from the bottom of the tube. The fractions were collected in 1 ml of 1.0 M NaCl. The amount of DNA in the fractions was estimated by measuring the absorbance
at 260 nm in a Unicam spectrophotometer fitted with 0.2-ml cuvettes (1-cm path length). Fractions used for transformation were pooled as shown in Fig. 4 and dialyzed against 1 x SSC. The dialyzed samples were stored at -10 C. Treatment of DNA by enzymes. A solution of DNA (100 Mg/ml) in 0.02 M Tris buffer-0.1 M NaCl-0.01 M MgSo4 (pH 7.5) was treated with DNase at 1 jg/ml at 37 C for 30 min. Treatment of DNA (50 ;g/ml) with trypsin (50 Mg/ml) was done in 0.05 M Tris buffer (pH 8.0) plus 0.02 M CaCl2 at 37 C for 60 min, and with Pronase (200 Ag/ml) in 0.01 M potassium phosphate buffer (pH 7.0) at 37 C for 45 min. A solution of DNA (5 jg/ml) was treated with RNase at 10 ;g/ml in 0.1 M acetate buffer (pH 7.0) at 37 C for 30 min.
RESULTS Transformation of PBS2. Figure 1 shows that ts mutant TAP02 could be transformed to the wild type with DNA extracted from wildtype phage. Although the experiments were carried out under different conditions, the number of transformants obtained was always linearly related to the concentration of DNA up to about 1 jig/ml. Competence, or the ability to take up DNA,
E 107
~
/ ~~~~~~~~~
z 4 0~~~~~~~~~~ U-.0
o-J
0
DNA CONCENTRATION
(gg/mi)
FIG. 1. Effect of DNA concentration on PBS2 transformation. Competent cells were infected with TAP02 and treated with different concentrations of wild-type DNA. Symbols: 0, unsheared DNA, cells made competent by method II; A, unsheared DNA, cells made competent by method I; 0, sheared DNA, cells made competent by method II.
18
HERRINGTON AND TAKAHASHI
can be induced in B. subtilis in a variety of ways. Competent cells in the present study were prepared by two methods. Method I was found to be much less effective than method II and, in fact, no transformants were obtained with sheared DNA when method I was used (Fig. 1). As expected, sheared DNA gave a considerably reduced level of transformation (Fig. 1). This has also been observed with sheared DNA in bacterial transformation (6). The following experiments were carried out to determine the optimal conditions for PBS2 transformation. In the first experiment, competent cells were mixed with DNA, and the mutant phage was added at different times (Table 1). Wild-type transformants were assayed after the cells lysed. The amount of transformation decreased rapidly in the first 10 min, indicating that the DNA might be degraded during the incubation period. A DNase which specifically degrades the uracil-containing PBS2 DNA has been isolated from uninfected B. subtilis cells (F. Tomita and I. Takahashi, manuscript in preparation). This DNase may be responsible for the inactivation of PBS2 DNA in vivo, thereby causing the reduction in the amount of transformation. In the second experiment, DNA was added to competent cells at different times after infection. Transformation could be observed for at least 40 min, but the amount of transformation decreased considerably after 30 min (Table 1). These experiments indicate that transformation is most efficient when DNA and mutant phage are added to competent cells at the same time. The effects of various enzymes and serum albumin on transformation of PBS2 are summarized in Table 2. As expected, DNase greatly
J. VIROL.
reduced the transforming activity of the DNA. Treatment of the DNA with high concentrations of other enzymes showed a slight reduction in the transforming activity. This slight reduction was probably due to an inhibitory effect of protein, since the addition of bovine serum albumin directly to the transformation mixture reduced the amount of transformation. Alternatively, this inactivation might be due to contaminating nucleases. The time course of the transformation process was determined by assaying, at different times, the number of infective centers present in a mixture for transformation of TAP02. Figure 2 shows the total number of infective centers (assayed at 30 C) and the number of wild-type infective centers (assayed at 45 C) at different times after infection. The total infective centers TABLE 2. Effect of various treatments on transforming DNA Transformation (% of untreated control)a
Treatment
DNase ............................ Trypsin ............................. Pronase ............................ RNase .............................. Bovine serum albumin ......... ......
0.7 100 75 91 78
aTransformation was performed as follows: competent cells (method H) were infected with TAP02, and treated DNA was added to give a final concentration of 1 ,g/ml for DNase-treated sample and 0.5 Ag/ml for all other samples. Bovine serum albumin (0.5 ,g/ml) was added directly to the transformation mixture.
TABLE 1. Dependence of transformation on the time of phage and DNA additiona i
uAJ 4
Time of infection or DNA addition (min)
Wild-type transformants/ml DNA added
atOmin
TAP02 added atOmin
4
zI
0
9
4c
0.
0 aJ
0 5 10 15 20 25 30 35 40
2.2 x 106 2.5 x 105
4.0 x 105 1.5 1.3 8.1 1.5
x 105 x 10 x 104 x 100
2.2 1.8 1.5 1.8 1.1
x 106 x 106
x 106 x 106 x 106
1.1 x 106
TIME (min)
FIG. 2. Single-step growth curve in transformation. Competent cells (method II) were infected with TAP02 and 2 sg of wild-type DNA per ml was added. a Cells were made competent by method II. The Samples were taken at different times and were concentration of DNA was 2 tsg/ml, and the multiplic- plated at 30 and 45 C. Symbols: 0, wild-type PFU/ ity of infection was 1. ml; 0, total PFU/ml. 4.3 x 105 3.6 x 105
VOL. 15, 1975
19
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
followed a typical single-step growth curve with a latent period of 63 min, a rise period of 22 min, and a burst size of 7.7. The appearance of wild-type infective centers was more complex. Wild-type infective centers appeared rapidly during the first 15 min, and then appeared at a lower rate throughout the remainder of the latent period and the rise period. The time required for the uptake of DNA by competent cells was determined by adding DNase to the transformation mixture at different times and assaying for transformants after the cells lysed. When the cells were in contact with DNA for less than 10 min, only a few transformants were obtained (Fig. 3). As the time of contact with DNA increased, the amount of transformation increased sharply until 40 min. There was a slight decrease in the number of transformants thereafter. These results indicate that DNA must be present at least for 10 min to cause transformation, and the uptake of DNA may be complete after 40
DNA were centrifuged in a Cs,SO-Hg density gradient (Fig. 4A). Results of transformation performed with the fractionated DNA were expressed as the percentage of the total number of transformants in the gradient (Fig. 4B and Table 4). Transforming activities for various mutants were distributed differently in the gradient, indicating hetTABLE 3. Frequency of transformants of different mutantsa
Transformants/ml Transformants/ml with unsheared with sheared
Mutant
DNA
TAP02 TFU121 TNG04 TFU34 TFU91
4.1 x 8.8 x 6.0 x 1.7 x 7.6 x
106 101 104 106 105
DNA
3.7 4.4 1.4 1.2
X 104 x 104
x 104 x 104
1.1 X 104
aCells were made competent by method II, and DNA (0.1 Ag/ml) from wild-type phage was used.
min.
The frequencies of transformants observed with various phage mutants are listed in Table 3. These mutants gave frequencies of transformants varying from 4.1 x 10/ml to 6.0 x 104/ml with unsheared DNA and from 4.4 x 104/ml to 1.1 x 104/ml with sheared DNA (Table 3). Transformation by fractionated DNA. To obtain DNA fractionated with respect to nucleotide composition, small fragments of PBS2
Ec
0
N
A
(0 0.300
~~1a 57
C 0.200 z
4
M
0
0.100
co) ui
a 4 (A
C,)
LAJ
z
a
o
IL z
0 UL
0
0
z
4 Iz
4 at.
a-
LL
U1)
z 0
4c
TIME (min)
FIG. 3. Kinetics of transformation by PBS2 DNA. Competent cells (method II) were infected with TAP02 at a multiplicity of infection of 1 to 2; wild-type PBS2 DNA was added at a concentration of 2 ug/ml. The mixtures were incubated at 30 C with shaking. At different times 10 sg of pancreatic DNase per ml was added.
10 20 30 40 FIG. 4. Transformation of ts mutants by fractionated DNA. The histogram shows the distribution of DNA in the gradient. Fractions were pooled and numbered as indicated by the horizontal lines, and their transforming activity was measured after dialysis. Transformation was measured by incubating 0.05 ml of mutant phage (1.0 x 109 PFU/ml), 0.05 ml of fractionated DNA, and 0.4 ml of competent cells (method II) for 100 min and then assaying for wildtype transformants. Symbols: 0, TFUI03; 0, TFU91; A, TAP11.
J. VIROL.
HERRINGTON AND TAKAHASHI
20
TABLE 4. Transformation of ts mutants by fractionated DNAa
Pooled fraction number
THA01
TFU11
TFU34
TFU12
TAP02
TFU73
TFU171
TFU102
1 2 3 4 5 6 7 8
1.9 4.8 7.6 11.0 16.0 23.0 23.0 12.0
0.8 4.9 3.2 15.0 26.0 17.0 21.0 11.0
0 2.7 9.7 17.0 39.0 20.0 6.2 5.3
1.0 0 5.8 11.0 54.0 20.0 9.1 0
0.6 27.0 39.0 14.0 4.3 9.3 0.9 4.6
0 4.3 0.5 6.5 4.2 4.6 46.0 34.0
0.9 13.0 1.8 62.0 14.0 9.0 0 0
2.7 2.2 3.8 30.0 42.0 13.0 6.3 1.2
a The method used to determine transforming activity is described in the footnote to Fig. 4. The pooled fraction numbers refer to those in Fig. 4A. The results for each mutant are expressed as the percentage of the total number of transformants obtained with fractionated DNA.
base composition of various genes in PBS2 chromosome. The transforming activity for some mutants (e.g., TFU73 and TFU102 in Table 4) was concentrated in a few fractions, whereas the activity for other mutants showed a rather broad distribution. erogeneous
The transforming activity for TFU91 was present in 5 of the 8 fractions tested (Fig. 4A), whereas the activity for TFU34 was present in only 3 of the 8 fractions (Table 4). It is possible that the broadness in the distribution of transforming activity in the gradient is an indication of heterogeneous base composition of the region of the chromosome involved. If a mutation is located in a region of the chromosome composed of two segments of quite different nucleotide composition, then, since breakage by shear is not absolutely specific (7), fragments of DNA containing this mutation will be distributed more widely in the gradient than fragments carrying mutations from a homogeneous region. Therefore, the broadness in the distribution of transforming activity may be used to estimate the degree of heterogeneity in the base composition around a particular marker.
DISCUSSION The foregoing results indicate that ts mutants of PBS2 can be transformed to the wild type in competent B. subtilis cells and that this transformation is mediated by free DNA molecules. Transfection, which has been observed with a number of other B. subtilis phages, has not been successful with PBS2 under our conditions. The absence of transfection by PBS2 DNA might be due to the large size (1.9 x 108 daltons) of the DNA (3). Centrifugation in gradients of Cs2SO,-Hg separates DNA molecules according to their ACKNOWLEDGMENTS nucleotide composition (8) and can be used to This work was supported by grant A1996 from the National determine whether the nucleotide composition Research Council of Canada. of a DNA molecule is uniform throughout the We would like to thank H. Yamagishi and C. T. Chow foi molecule or if the molecule is made up of several assistance with the gradients. segments of different nucleotide composition. LITERATURE CITED The DNA of bacteriophage lambda (8) is comD. 1. M. 1964. Infectivity of DNA isolated from Green, posed of several large segments of different subtilis Bacillus bacteriophage, SP82. J. Mol. Biol. DNA the In contrast, nucleotide composition. 10:438-451. from E. coli shows little or no intramolecular 2. Herrington, M. B., and I. Takahashi. 1973. Mutagenesis of bacteriophage PBS 2. Mutat. Res. 20:275-278. heterogeneity when large fragments of the moleB. I., H. Yamagishi, and I. Takahashi. 1967. cule are examined, but does show heterogeneity 3. Hunter, Molecular weight of bacteriophage PBS 1 deoxyribonu. when smaller fragments (1.5 x 106 daltons) are cleic acid. J. Virol. 1:841-842. examined (16). A similar situation has been 4. Mahler, I. 1968. Procedures for Bacillus subtilis transformation, p. 846-850. In S. P. Colowick and N. 0. found for PBS1 and PBS2 DNA. According to Kaplan (ed.), Methods in enzymology, vol. XII, part B. H. Yamagishi (personal communication), the Academic Press Inc., New York. base composition of small fragments of PBS1 5. Nester, E. W., M. Schafer, and J. Lederberg. 1963. Gene DNA fractionated by centrifugation in the gralinkage in DNA transfer: a cluster of genes concerned with aromatic biosynthesis in Bacillus subtilis. Genetdients ranged from 25 to 35% guanine plus ics 48:529-551. cytosine. The average base composition of PBS1 6. Rosenberg, B. H., F. M. Sirotnak, and L. F. Cavalieri. DNA determined by chemical analysis is 28% 1959. On the size of genetic determinants in Pneumococcus and the nature of the variables involved guanine plus cytosine (14).
VOL. 15, 1975
7.
8. 9. 10. 11.
12.
BACTERIOPHAGE TRANSFORMATION IN B. SUBTILIS
in transtormation. Proc. Nat. Acad. Sci. U.S.A. 45:144-156. Skalka, A. 1971. A method for the breakage of DNA and resolution of the fragments, p. 341-350. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. XXI, part D. Academic Press Inc., New York. Skalka, A., E. Burgi, and A. D. Hershey. 1968. Segmental distribution of nucleotides in the DNA of bacteriophage lambda. J. Mol. Biol. 34:1-16. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 4:1072-1078. Takahashi, I. 1961. Genetic transduction in Bacillus subtilis. Biochem. Biophys. Res. Commun. 5:171-175. Takahashi, I. 1963. Transducing phages for Bacillus subtilis. J. Gen. Microbiol. 31:211-217. Takahashi, I. 1965. Transduction of sporogenesis in Bacillus subtilis. J. Bacteriol. 89:294-298
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13. Takahashi, I. 1965. Localization of spore markers on the chromosome of Bacillus subtilis. J. Bacteriol. 89:1065-1067. 14. Takahashi, I., and J. Marmur. 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature (London) 197:794-795. 15. Van de Pol, J. H., G. Veldhuisen, and J. A. Cohen. 1961. Phage transformation: a new criterium for the biological activity of bacteriophage DNA. Biochim. Biophys. Acta 48:417-418. 16. Yamagishi, H. 1970. Nucleotide distribution in the DNA of Escherichia coli. J. Mol. Biol. 49:603-608. 17. Yamagishi, H., and I. Takahashi. 1968. Transducing particles of PBS 1. Virology 36:639. 18. Yamagishi, H., and I. Takahashi. 1971. Heterogeneity in nucleotide composition of Bacillus subtilis DNA. J. Mol. Biol. 57:369-371.
