JOURNAL OF VIROLOGY, Mar. 1976, p. 718-726 Copyright 0 1976 American Society for Microbiology
Vol. 17, No. 3 Printed in U.S.A.
Recombinational-Type Transfer of Viral DNA During Bacteriophage 2C Replication in Bacillus subtilis PHILIPPE HOET, GEORGES FRASELLE, AND CARLO COCITO* Department of Microbiology and Genetics, International Institute of Cell Pathology, University of Louvain Medical School, B Brussels 1200, Belgium
Received for publication July 1975
The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 101 daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil or [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegetative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 106 daltons.
The problem of conservation of viral genomes during the replication cycle and of their transfer to offspring virions is closely related to: (i) the mechanism of viral nucleic acid duplication; (ii) the host- and virus-dependent systems of genetic recombination; and (iii) the process of assembly of viral components into mature particles. Transfer of viral DNA has been extensively investigated in the T-even bacteriophages of Escherichia coli. Early work showing partial conservation of parental DNA components (39) has been subsequently confirmed (27), whereas
lently joined end-by-end, and their accumulation is prevented by protein synthesis inhibitors
(35).
In the present work, the problem of transfer of viral DNA has been investigated in the phage 2C-Bacillus subtilis system. The latter differs from T4-infected E. coli in that formation of cell components, which is halted by infection with T4, continues after the attachment of phage 2C to the host cell (6, 38). Although the genome of phage 2C is a double-stranded DNA molecule similar in size to T4 DNA, it contains hydroxymethyluracil in the place of thymine (33, 46). In addition, 2C DNA has two strands of different buoyant densities that are transcribed to different extents during the replication cycle (18). Because of the peculiar features and functions of 2C DNA, it was thought that a study of conservation and transfer of 2C DNA would furnish data on the regulatory systems which catalyze DNA duplication and recombination in virus-infected gram-positive organisms.
breakdown of input viral genomes and reutilization of the degradation products were excluded (9). It was then shown that T. DNA, whereas replicating in a semiconservative fashion, undergoes a fragmentary transfer (23, 30). Pieces of parental phage DNA, relatively homogeneous in size and comprising 5 to 10% of the genome, are inserted into newly formed DNA molecules by the process of genetic recombination (5, 25, 26). The latter is also responsible for MATERIALS AND METHODS the production of very large replicative intermediates at late stages of the replication cycle (8). Growth of bacteria and bacteriophage. Virus 2C These strips, 4 to 5 phage equivalent units long, was multiplied in the 168/2 strain of B. subtilis (leuapparently are formed by DNA molecules cova- tryp-). The composition of synthetic YS medium for 718
VOL. 17, 1976
growth of this microorganism (7), as well as the techniques for concentration and titration of virus 2C (6), has been previously described. Labeling of parental phage. 32P-labeled phage were obtained by infecting bacteria (multiplicity of infection of 5) in low-phosphate YS medium (7) containing 0.25 mCi of [32Pjorthosphosphate and 20 ,gg of [31P]orthophosphate for 2.5 x 107 cells per ml. The multiplication cycle was continued to complete lysis. Virions with [3H Juracil-labeled DNA were produced by lytic infection of B. subtilis in YS medium containing 0.1 mCi of [3H]uracil and 2.5 x 107 cells/ml. "Heavy" labeled parental particles were prepared by infecting overnight cultures of B. subtilis 168/2 in YS medium containing 0.1 mCi of [3H]uracil, 5 x 107 cells/ml and with D20 (99.75%) replacing H2O. Infected cells were shaken at 37 C until lysis was complete. Centrifugal analysis of phage particles and viral DNA. Phage particles, concentrated by ultracentrifugation (15,000 rpm, 50 min, 4 C) were purified by equilibrium banding in CsCl (p at 23 C = 1.52 g/cm3, 33,000 rpm, SW50.1 Spinco rotor, 4 C, 16 h). For density gradient analysis of DNA, CsCl was added to DNA solutions in SSC buffer (0.15 M NaCl plus 0.015 M sodium citrate) (p at 23 C = 1.75 g/cm3), and centrifugation was performed either in an angular rotor (R65, Spinco) or in a swing-out rotor (SW 50.1, Spinco) (33,000 rpm, 19 C, 65 h). Apparent buoyant densities in CsCl were 1.742 g/cm3 for native DNA, 1.762 g/cm3 for the heavy (H) strand, and 1.752 g/cm 3 for the light (L) strand. Sonic treatment and shearing of viral DNA. Sonic treatment of viral DNA (10 Mg/ml in SSC) was accomplished in a Raytheon ultrasonic vibrator at 10,000 cycles/min for 2 min at 15 C. Controlled shear degradation of 2C DNA was achieved by stirring DNA solutions for 15 min at 4 C, at different speeds, with a stainless-steel blade (5 cm in diameter) driven by a Cole-Parmer stirrer coupled with a solid-state speed controller. Concentration of DNA in 0.1 M NaCl-0.003 M EDTA-0.05 M Tris buffer (pH 7.4) was 1 ug/ml (3). Molecular weight measurements of sheared DNA. For sedimentation velocity evaluation of molecular weight, 0.2 ml of sheared DNA solution was layered on top of a 4-ml linear 5 to 20% sucrose gradient in 1.0 M NaCl-0.02 M Tris-hydrochloride buffer (pH 7.4), and samples were centrifuged in a SW50.1 rotor (Spinco) for 120 min at 35,000 rpm at 19 C. Fractions were collected on Whatmann 3MM filter papers, and radioactivity was measured by liquid scintillation spectrometry. Molecular weights were calculated by the formula of Burgi and Hershey (3): D2/Dj = (M2Ml) 035, where D is the distance from the meniscus and M is the molecular weight. Intact 2C DNA used as marker has a molecular weight of 1.08 x
108.
Denaturation and separation of DNA strands. Phages, suspended in SSC containing 0.01 M EDTA, were extracted with water-saturated redistilled phenol and 1% sodium dodecyl sulfate at 18 C. DNA (2 optical density at 260 nm units/ml) was mixed with a solution of poly (G) (50 Mg) pretreated at 95 C for 2 min. After incubation at 95 C for 6 min, the mixture
PHAGE 2C REPLICATION IN B. SUBTILIS
719
was cooled rapidly in ice, CsCl was added to a density of 1.76 g/cm3, and the mixture was centrifuged at 33,000 rpm for 70 h at 19 C in a Spinco SW 50.1 rotor. Fractions collected were analyzed by radioactivity or spectrophotometric (absorbance at 260 nm) measurements. After binding of poly (G), the density of the H strand was shifted to 1.810 g/cm3, and that of the L strand was shifted to 1.762. Hydrolysis of viral DNA and determination of nucleotide composition. Enzymatic hydrolysis of 2C DNA was accomplished as follows. To a solution of radioactive DNA (2 optical density at 260 nm units/ ml) exhaustively dialyzed against H20 at 4 C, 50,umol of Tris-hydrochloride (pH 7.4), 10 mol of MgCl2, and 100 Mg of DNase were added, and the mixture was incubated for 1 h at 37 C. The pH was shifted to 9 with NaOH, snake venom phosphodiesterase (20 Mg) was added, and digestion was pursued for 2 h at 37 C. After heating (95 C, 2 min) and centrifugation, the supernatant containing free nucleotides was applied to polyethyleneimine-cellulose plates for thin-layer chromatography. Development was achieved with two solvent systems, the first containing 1 N acetic acid and the second containing 3 M LiCl plus 1 N acetic acid (1:9, vol/vol). In control experiments, column chromatography (Dowex 1 x 8, 100 to 200 mesh) was used. Stepwise elution of nucleotides was accomplished with 0.01 M HCOOH (dCMP), 0.1 M HCOOH (dAMP), 0.1 M HCOOH plus 0.05 M HCOONH4 (dHMUMP), and 0.1 M HCOOH plus 0.2 M HCOONH4 (dGMP) (11). Additional controls were done by use of a microanalytical procedure of lowvoltage electrophoresis on cellulose acetate strips (10). Fractionation was obtained with citrate buffer (pH 3.15) and 150-min runs at 300 V and 20 C. Spots of nucleotides were cut and eluted in 0.05 N KOH for 3 h at 37 C, and radioactivity was measured by scintillation spectrometry. Radioisotopes and chemicals. [32P]orthophosphate (20 mCi/mg) and [6-3HJuracil (28 Ci/mmol) were obtained from IRE (Mol, Belgium). Deuterium oxide (99.75% purity), CsCl (analytical grade), and snake venom phosphodiesterase I were products from Merck (Darmstadt, West Germany). Poly (G), 5-hydroxymethyluracil, and DNase C (1 x crystallized) were obtained from Sigma Chemical Co. (St. Louis, Mo.). L broth, Casamino Acids (acid hydrolysate of casein), and yeast extract were purchased from Difco Laboratories (Detroit, Mich.). EDTA, trichloroacetic acid, and sodium dodecyl sulfate (SDS) were products from BDH (Poole, England). SSC buffer was at pH 7. Polyethyleneimine-impregnated cellulose plates were obtained from Macherey-Nagel (Duren, West Germany). Radioactivity measurements. For evaluation of 32p_ and 3H-labeled DNA, bovine serum albumin (40 Mg) and ice-cold 0.5 M trichloroacetic acid were added, and samples were filtered through 0.45-Mm micropore membranes (Schleicher and Schull, Dassel, West Germany). Precipitates were washed with ice-cold 0.5 M trichloroacetic acid, dried, and counted in a liquid scintillation spectrometer. Alternatively, ultracentrifugal fractions collected on 3MM filters (Whatman, Springfield Mill, England) were dried and counted in glass vials. Toluene containing 4 Mg of
720
HOET, FRASELLE, AND COCITO
J. VIROL.
Omnifluor [98% 2,5-diphenyloxazole and 2% 1,4-bis(5-phenyloxazol)-2-benzene] per ml was used as scintillation fluid.
RESULTS (i) Analysis of native DNA in parental and progeny phage particles. Data from the literature indicate that some viral nucleic acids are transferred to progeny virions and others are not. To find out whether 2C DNA is conserved during the replication cycle and incorporated into offspring phage, virions carrying 3H-labeled DNA were prepared by growing infected B. subtilis in the presence of [8H]uracil. Since 2C DNA contains hydroxymethyluracil, labeled uracil is a specific marker for viral DNA (8). One aliquot of purified labeled virions was extracted with phenol, and the remaining part was used to infect exponential cells in the presence of unlabeled uracil. After termination of the multiplication cycle, viral particles were purified, and DNA was extracted. Solutions of parental and progeny viral DNA, after the addition of 32P-labeled 2C DNA as reference, were centrifuged at equilibrium in CsCl gradients. Figure 1A and B show overlapping peaks of 3H and 32P tracings, thus indicating conservation and transfer of parental DNA to progeny
virions. In these experiments, about 40% of the radioactivity of the adsorbed parental phages was present as acid-precipitable counts in DNase-treated lysates and 20 to 30% in purified progeny phages. These values are comparable to the ones reported for T-even phages (9). It can still be argued, however, that an unusual base such as hydroxymethyluracil, hypothetically derived from DNA degradation, would be preferentially incorporated into newly made viral chromosomes. To rule out such a possibility, 32P-labeled phage was used to infect unlabeled B. subtilis in high- [31P]phosphate medium, and progeny viral DNA was analyzed. Identical results were obtained (Fig. 1C). (ii) Analysis of denatured viral DNA. The presence in the 2C genome of two strands of different buoyant densities offers the possibility of comparing the efficiency of transfer of single strands. This was done by using 3H-labeled purified phage particles to infect unlabeled cells. Virions liberated by lysis of host cells were harvested and purified. DNA extracted from parental and progeny phage was denatured in the presence of poly (G) and submitted to equilibrium centrifugation. Figure 2 shows the two strands of 2C DNA
B
A
C \ 1.4050 c
\1.4040 X)
32P 3H x
2 1.4030 z)
~~3H32P
32P 2-
2-10
E
4-8 5.~
5-
~
~
~
~
-
I,
~~~~~ U-
3
4~~~2 FRACT IONS FIG. 1. Conservation of parental label and its transfer to progeny. DNA extracted from purified parental and progeny virus particles was centrifuged in CsCI gradient after the addition of reference phage DNA. (A) Parental DNA labeled with [3Hjuracil and reference DNA labeled with [32Pjphosphate; (B) progeny DNA labeled with [3HJuracil and reference DNA labeled with [(2Plphosphate; (C) progeny DNA labeled with [32Plphosphate. Symbols: 0, 3H;-0, 32P.
VOL. 17, 1976
~~~~PHAGE 2C
REPLICATION IN B. SUB TILIS
721
B
A
C~~~~~~~~~~~~~~~~~~~~~~~~~~C,
¶~ ~ ~ 4~ ~ ~ ~Q ~1 75
z -
0~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I E
0
FRACTIONS
FIG. 2. Efficiency of transfer of 2C DNA strands. Unlabeled bacteria
were
infected
with
Hjuracil-labeled
phage, and offspring particles were purified. DNA extracted from parental and progeny virions was denatured in the presence of poly (G) and centrifuged in CsCl gradients. (A) Parental DNA strands (B) progeny DNA strands.
widely separated after CsCl centrifugation. Integration of radioactivity within the two peaks gave similar values for parental and progeny
TABLE 1. Nucleotide composition of the two strands of parental and progeny phage DNAa
DNA. This indicates that the two strands of 2C are transferred with equal efficiency to offspring phage particles. (iii) Nucleotide composition of the two DNA strands of parental and offspring phage. Although data in Fig. 1 and 2 point to
DNA
Composition of the DNA digest (%)
GenSource
era-
tion
C
HMU
A
G
G+ C
L strand
1 2
19.9 20.2
27.1 26.7
31.6 33.3
21.2 19.7
41.1 39.9
H strand
1 2
23.3 21.8
32.0 32.2
27.8 28.9
16.8 17.1
40.1 38.9
Native DNA
1
19.8
30.7
31.2
18.1
38.0
the conservation and transfer of 2C DNA at the
they cannot hypothetical degradation of parental chromosomes and use of the building blocks for progeny DNA formation. This possibility was checked by nucleotide analysis of parental and level of both molecules and strands, rule out
progeny
a
DNA strands. For this purpose
32P-
phage was used to infect unlabeled high-phosphate medium. DNA extracted from purified virions of the first and DNA second generations was denatured. strands, separated in CsCl gradients, were hydrolyzed to nucleotides, which were then analyzed either by thin-layer or column chromatography or by electrophoresis. labeled
bacteria in
Data in Table 1 indicate very similar nucleo-
tide composition for the H strands of parental and progeny virions. The
same
held true for the
L strands. versus dispersive transAlthough data presented above with a transfer without degrada-
(iv) Conservative fer of 2C DNA. are
tion
consistent of 2C
between
DNA,
the two
we
were
unable
to
decide
models of conservative
fragmentary transfer. This problem
was
and
investi-
a Progeny (second generation) virions were produced by infection of unlabeled cells in unlabeled medium with 32P-labeled parental (first generation)
phage. 'C, Deoxycytidylic acid; HMU, hydroxymethyldeoxyuridylic acid; A, deoxyadenylic acid; G, deoxyguanylic acid. Data are average values of four experiments of the same kind made with three separation techniques.
gated by double labeling experiments. Heavy labeled virions were obtained by multiplication of 2C in B. subtilis in medium containing (3H uracil and with D,0 replacing water. Purified phages were used to infect cells in H20 medium containing unlabeled uracil. Native DNA extracted from parental and progeny virions was analyzed in CsCl density gradients after the addition of H20 2C DNA as density marker.
722
J. VIROL.
HOET, FRASELLE, AND COCITO
Figure 3 shows the banding of the first (Fig. broad heterogeneous peaks of DNA molecules of 3A) and second (Fig. 3B) generation virions: the densities ranging from that of completely light latter type formed a large band at a density to that of the hybrid species. A considerable slightly higher than that of light phage. portion of 2C DNA overlapped the light 82P The [ lH]uracil tracings in Fig. 4B show very band of newly formed phage DNA, but most of A
REFRACTIV
(120)
\~ ~
B
INDEX
3850 3840
3830 3820 3
3H 32p
A32p E.0
5-2
0.5
C4 w
z
0Q25 4110 I
2
4~~~~~~~c
FRACTIONS
FIG. 3. Buoyant density of progeny phage derived from heavy and radioactive parental virions. Purified phage particles which had incorporated [3Hluracil and D20 (see Materials and Methods) were used to infect bacteria in H20 medium containing 100 ug of uracil/ml. Parental (A) and progeny (B) virions were centrifuged in CsCI (p at 23 C = 1.52 g/cm2) after the addition of a small number of 32P-labeled light phage and of an excess of unlabeled light phage (B only) as density markers. Symbols: ---, optical density at 260 nm; *, 3H; 0, 92p.
B
A
1.4060 1.4050 1.4040
t s t . Ix 3H32i
3H 32p
E
.43 '-
0
U. .
4~ ~ ~ ~ ~ ~
Il
w~~~~~~~~z
a-.
Vol. 17, No. 3 Printed in U.S.A.
Recombinational-Type Transfer of Viral DNA During Bacteriophage 2C Replication in Bacillus subtilis PHILIPPE HOET, GEORGES FRASELLE, AND CARLO COCITO* Department of Microbiology and Genetics, International Institute of Cell Pathology, University of Louvain Medical School, B Brussels 1200, Belgium
Received for publication July 1975
The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 101 daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil or [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegetative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 106 daltons.
The problem of conservation of viral genomes during the replication cycle and of their transfer to offspring virions is closely related to: (i) the mechanism of viral nucleic acid duplication; (ii) the host- and virus-dependent systems of genetic recombination; and (iii) the process of assembly of viral components into mature particles. Transfer of viral DNA has been extensively investigated in the T-even bacteriophages of Escherichia coli. Early work showing partial conservation of parental DNA components (39) has been subsequently confirmed (27), whereas
lently joined end-by-end, and their accumulation is prevented by protein synthesis inhibitors
(35).
In the present work, the problem of transfer of viral DNA has been investigated in the phage 2C-Bacillus subtilis system. The latter differs from T4-infected E. coli in that formation of cell components, which is halted by infection with T4, continues after the attachment of phage 2C to the host cell (6, 38). Although the genome of phage 2C is a double-stranded DNA molecule similar in size to T4 DNA, it contains hydroxymethyluracil in the place of thymine (33, 46). In addition, 2C DNA has two strands of different buoyant densities that are transcribed to different extents during the replication cycle (18). Because of the peculiar features and functions of 2C DNA, it was thought that a study of conservation and transfer of 2C DNA would furnish data on the regulatory systems which catalyze DNA duplication and recombination in virus-infected gram-positive organisms.
breakdown of input viral genomes and reutilization of the degradation products were excluded (9). It was then shown that T. DNA, whereas replicating in a semiconservative fashion, undergoes a fragmentary transfer (23, 30). Pieces of parental phage DNA, relatively homogeneous in size and comprising 5 to 10% of the genome, are inserted into newly formed DNA molecules by the process of genetic recombination (5, 25, 26). The latter is also responsible for MATERIALS AND METHODS the production of very large replicative intermediates at late stages of the replication cycle (8). Growth of bacteria and bacteriophage. Virus 2C These strips, 4 to 5 phage equivalent units long, was multiplied in the 168/2 strain of B. subtilis (leuapparently are formed by DNA molecules cova- tryp-). The composition of synthetic YS medium for 718
VOL. 17, 1976
growth of this microorganism (7), as well as the techniques for concentration and titration of virus 2C (6), has been previously described. Labeling of parental phage. 32P-labeled phage were obtained by infecting bacteria (multiplicity of infection of 5) in low-phosphate YS medium (7) containing 0.25 mCi of [32Pjorthosphosphate and 20 ,gg of [31P]orthophosphate for 2.5 x 107 cells per ml. The multiplication cycle was continued to complete lysis. Virions with [3H Juracil-labeled DNA were produced by lytic infection of B. subtilis in YS medium containing 0.1 mCi of [3H]uracil and 2.5 x 107 cells/ml. "Heavy" labeled parental particles were prepared by infecting overnight cultures of B. subtilis 168/2 in YS medium containing 0.1 mCi of [3H]uracil, 5 x 107 cells/ml and with D20 (99.75%) replacing H2O. Infected cells were shaken at 37 C until lysis was complete. Centrifugal analysis of phage particles and viral DNA. Phage particles, concentrated by ultracentrifugation (15,000 rpm, 50 min, 4 C) were purified by equilibrium banding in CsCl (p at 23 C = 1.52 g/cm3, 33,000 rpm, SW50.1 Spinco rotor, 4 C, 16 h). For density gradient analysis of DNA, CsCl was added to DNA solutions in SSC buffer (0.15 M NaCl plus 0.015 M sodium citrate) (p at 23 C = 1.75 g/cm3), and centrifugation was performed either in an angular rotor (R65, Spinco) or in a swing-out rotor (SW 50.1, Spinco) (33,000 rpm, 19 C, 65 h). Apparent buoyant densities in CsCl were 1.742 g/cm3 for native DNA, 1.762 g/cm3 for the heavy (H) strand, and 1.752 g/cm 3 for the light (L) strand. Sonic treatment and shearing of viral DNA. Sonic treatment of viral DNA (10 Mg/ml in SSC) was accomplished in a Raytheon ultrasonic vibrator at 10,000 cycles/min for 2 min at 15 C. Controlled shear degradation of 2C DNA was achieved by stirring DNA solutions for 15 min at 4 C, at different speeds, with a stainless-steel blade (5 cm in diameter) driven by a Cole-Parmer stirrer coupled with a solid-state speed controller. Concentration of DNA in 0.1 M NaCl-0.003 M EDTA-0.05 M Tris buffer (pH 7.4) was 1 ug/ml (3). Molecular weight measurements of sheared DNA. For sedimentation velocity evaluation of molecular weight, 0.2 ml of sheared DNA solution was layered on top of a 4-ml linear 5 to 20% sucrose gradient in 1.0 M NaCl-0.02 M Tris-hydrochloride buffer (pH 7.4), and samples were centrifuged in a SW50.1 rotor (Spinco) for 120 min at 35,000 rpm at 19 C. Fractions were collected on Whatmann 3MM filter papers, and radioactivity was measured by liquid scintillation spectrometry. Molecular weights were calculated by the formula of Burgi and Hershey (3): D2/Dj = (M2Ml) 035, where D is the distance from the meniscus and M is the molecular weight. Intact 2C DNA used as marker has a molecular weight of 1.08 x
108.
Denaturation and separation of DNA strands. Phages, suspended in SSC containing 0.01 M EDTA, were extracted with water-saturated redistilled phenol and 1% sodium dodecyl sulfate at 18 C. DNA (2 optical density at 260 nm units/ml) was mixed with a solution of poly (G) (50 Mg) pretreated at 95 C for 2 min. After incubation at 95 C for 6 min, the mixture
PHAGE 2C REPLICATION IN B. SUBTILIS
719
was cooled rapidly in ice, CsCl was added to a density of 1.76 g/cm3, and the mixture was centrifuged at 33,000 rpm for 70 h at 19 C in a Spinco SW 50.1 rotor. Fractions collected were analyzed by radioactivity or spectrophotometric (absorbance at 260 nm) measurements. After binding of poly (G), the density of the H strand was shifted to 1.810 g/cm3, and that of the L strand was shifted to 1.762. Hydrolysis of viral DNA and determination of nucleotide composition. Enzymatic hydrolysis of 2C DNA was accomplished as follows. To a solution of radioactive DNA (2 optical density at 260 nm units/ ml) exhaustively dialyzed against H20 at 4 C, 50,umol of Tris-hydrochloride (pH 7.4), 10 mol of MgCl2, and 100 Mg of DNase were added, and the mixture was incubated for 1 h at 37 C. The pH was shifted to 9 with NaOH, snake venom phosphodiesterase (20 Mg) was added, and digestion was pursued for 2 h at 37 C. After heating (95 C, 2 min) and centrifugation, the supernatant containing free nucleotides was applied to polyethyleneimine-cellulose plates for thin-layer chromatography. Development was achieved with two solvent systems, the first containing 1 N acetic acid and the second containing 3 M LiCl plus 1 N acetic acid (1:9, vol/vol). In control experiments, column chromatography (Dowex 1 x 8, 100 to 200 mesh) was used. Stepwise elution of nucleotides was accomplished with 0.01 M HCOOH (dCMP), 0.1 M HCOOH (dAMP), 0.1 M HCOOH plus 0.05 M HCOONH4 (dHMUMP), and 0.1 M HCOOH plus 0.2 M HCOONH4 (dGMP) (11). Additional controls were done by use of a microanalytical procedure of lowvoltage electrophoresis on cellulose acetate strips (10). Fractionation was obtained with citrate buffer (pH 3.15) and 150-min runs at 300 V and 20 C. Spots of nucleotides were cut and eluted in 0.05 N KOH for 3 h at 37 C, and radioactivity was measured by scintillation spectrometry. Radioisotopes and chemicals. [32P]orthophosphate (20 mCi/mg) and [6-3HJuracil (28 Ci/mmol) were obtained from IRE (Mol, Belgium). Deuterium oxide (99.75% purity), CsCl (analytical grade), and snake venom phosphodiesterase I were products from Merck (Darmstadt, West Germany). Poly (G), 5-hydroxymethyluracil, and DNase C (1 x crystallized) were obtained from Sigma Chemical Co. (St. Louis, Mo.). L broth, Casamino Acids (acid hydrolysate of casein), and yeast extract were purchased from Difco Laboratories (Detroit, Mich.). EDTA, trichloroacetic acid, and sodium dodecyl sulfate (SDS) were products from BDH (Poole, England). SSC buffer was at pH 7. Polyethyleneimine-impregnated cellulose plates were obtained from Macherey-Nagel (Duren, West Germany). Radioactivity measurements. For evaluation of 32p_ and 3H-labeled DNA, bovine serum albumin (40 Mg) and ice-cold 0.5 M trichloroacetic acid were added, and samples were filtered through 0.45-Mm micropore membranes (Schleicher and Schull, Dassel, West Germany). Precipitates were washed with ice-cold 0.5 M trichloroacetic acid, dried, and counted in a liquid scintillation spectrometer. Alternatively, ultracentrifugal fractions collected on 3MM filters (Whatman, Springfield Mill, England) were dried and counted in glass vials. Toluene containing 4 Mg of
720
HOET, FRASELLE, AND COCITO
J. VIROL.
Omnifluor [98% 2,5-diphenyloxazole and 2% 1,4-bis(5-phenyloxazol)-2-benzene] per ml was used as scintillation fluid.
RESULTS (i) Analysis of native DNA in parental and progeny phage particles. Data from the literature indicate that some viral nucleic acids are transferred to progeny virions and others are not. To find out whether 2C DNA is conserved during the replication cycle and incorporated into offspring phage, virions carrying 3H-labeled DNA were prepared by growing infected B. subtilis in the presence of [8H]uracil. Since 2C DNA contains hydroxymethyluracil, labeled uracil is a specific marker for viral DNA (8). One aliquot of purified labeled virions was extracted with phenol, and the remaining part was used to infect exponential cells in the presence of unlabeled uracil. After termination of the multiplication cycle, viral particles were purified, and DNA was extracted. Solutions of parental and progeny viral DNA, after the addition of 32P-labeled 2C DNA as reference, were centrifuged at equilibrium in CsCl gradients. Figure 1A and B show overlapping peaks of 3H and 32P tracings, thus indicating conservation and transfer of parental DNA to progeny
virions. In these experiments, about 40% of the radioactivity of the adsorbed parental phages was present as acid-precipitable counts in DNase-treated lysates and 20 to 30% in purified progeny phages. These values are comparable to the ones reported for T-even phages (9). It can still be argued, however, that an unusual base such as hydroxymethyluracil, hypothetically derived from DNA degradation, would be preferentially incorporated into newly made viral chromosomes. To rule out such a possibility, 32P-labeled phage was used to infect unlabeled B. subtilis in high- [31P]phosphate medium, and progeny viral DNA was analyzed. Identical results were obtained (Fig. 1C). (ii) Analysis of denatured viral DNA. The presence in the 2C genome of two strands of different buoyant densities offers the possibility of comparing the efficiency of transfer of single strands. This was done by using 3H-labeled purified phage particles to infect unlabeled cells. Virions liberated by lysis of host cells were harvested and purified. DNA extracted from parental and progeny phage was denatured in the presence of poly (G) and submitted to equilibrium centrifugation. Figure 2 shows the two strands of 2C DNA
B
A
C \ 1.4050 c
\1.4040 X)
32P 3H x
2 1.4030 z)
~~3H32P
32P 2-
2-10
E
4-8 5.~
5-
~
~
~
~
-
I,
~~~~~ U-
3
4~~~2 FRACT IONS FIG. 1. Conservation of parental label and its transfer to progeny. DNA extracted from purified parental and progeny virus particles was centrifuged in CsCI gradient after the addition of reference phage DNA. (A) Parental DNA labeled with [3Hjuracil and reference DNA labeled with [32Pjphosphate; (B) progeny DNA labeled with [3HJuracil and reference DNA labeled with [(2Plphosphate; (C) progeny DNA labeled with [32Plphosphate. Symbols: 0, 3H;-0, 32P.
VOL. 17, 1976
~~~~PHAGE 2C
REPLICATION IN B. SUB TILIS
721
B
A
C~~~~~~~~~~~~~~~~~~~~~~~~~~C,
¶~ ~ ~ 4~ ~ ~ ~Q ~1 75
z -
0~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I E
0
FRACTIONS
FIG. 2. Efficiency of transfer of 2C DNA strands. Unlabeled bacteria
were
infected
with
Hjuracil-labeled
phage, and offspring particles were purified. DNA extracted from parental and progeny virions was denatured in the presence of poly (G) and centrifuged in CsCl gradients. (A) Parental DNA strands (B) progeny DNA strands.
widely separated after CsCl centrifugation. Integration of radioactivity within the two peaks gave similar values for parental and progeny
TABLE 1. Nucleotide composition of the two strands of parental and progeny phage DNAa
DNA. This indicates that the two strands of 2C are transferred with equal efficiency to offspring phage particles. (iii) Nucleotide composition of the two DNA strands of parental and offspring phage. Although data in Fig. 1 and 2 point to
DNA
Composition of the DNA digest (%)
GenSource
era-
tion
C
HMU
A
G
G+ C
L strand
1 2
19.9 20.2
27.1 26.7
31.6 33.3
21.2 19.7
41.1 39.9
H strand
1 2
23.3 21.8
32.0 32.2
27.8 28.9
16.8 17.1
40.1 38.9
Native DNA
1
19.8
30.7
31.2
18.1
38.0
the conservation and transfer of 2C DNA at the
they cannot hypothetical degradation of parental chromosomes and use of the building blocks for progeny DNA formation. This possibility was checked by nucleotide analysis of parental and level of both molecules and strands, rule out
progeny
a
DNA strands. For this purpose
32P-
phage was used to infect unlabeled high-phosphate medium. DNA extracted from purified virions of the first and DNA second generations was denatured. strands, separated in CsCl gradients, were hydrolyzed to nucleotides, which were then analyzed either by thin-layer or column chromatography or by electrophoresis. labeled
bacteria in
Data in Table 1 indicate very similar nucleo-
tide composition for the H strands of parental and progeny virions. The
same
held true for the
L strands. versus dispersive transAlthough data presented above with a transfer without degrada-
(iv) Conservative fer of 2C DNA. are
tion
consistent of 2C
between
DNA,
the two
we
were
unable
to
decide
models of conservative
fragmentary transfer. This problem
was
and
investi-
a Progeny (second generation) virions were produced by infection of unlabeled cells in unlabeled medium with 32P-labeled parental (first generation)
phage. 'C, Deoxycytidylic acid; HMU, hydroxymethyldeoxyuridylic acid; A, deoxyadenylic acid; G, deoxyguanylic acid. Data are average values of four experiments of the same kind made with three separation techniques.
gated by double labeling experiments. Heavy labeled virions were obtained by multiplication of 2C in B. subtilis in medium containing (3H uracil and with D,0 replacing water. Purified phages were used to infect cells in H20 medium containing unlabeled uracil. Native DNA extracted from parental and progeny virions was analyzed in CsCl density gradients after the addition of H20 2C DNA as density marker.
722
J. VIROL.
HOET, FRASELLE, AND COCITO
Figure 3 shows the banding of the first (Fig. broad heterogeneous peaks of DNA molecules of 3A) and second (Fig. 3B) generation virions: the densities ranging from that of completely light latter type formed a large band at a density to that of the hybrid species. A considerable slightly higher than that of light phage. portion of 2C DNA overlapped the light 82P The [ lH]uracil tracings in Fig. 4B show very band of newly formed phage DNA, but most of A
REFRACTIV
(120)
\~ ~
B
INDEX
3850 3840
3830 3820 3
3H 32p
A32p E.0
5-2
0.5
C4 w
z
0Q25 4110 I
2
4~~~~~~~c
FRACTIONS
FIG. 3. Buoyant density of progeny phage derived from heavy and radioactive parental virions. Purified phage particles which had incorporated [3Hluracil and D20 (see Materials and Methods) were used to infect bacteria in H20 medium containing 100 ug of uracil/ml. Parental (A) and progeny (B) virions were centrifuged in CsCI (p at 23 C = 1.52 g/cm2) after the addition of a small number of 32P-labeled light phage and of an excess of unlabeled light phage (B only) as density markers. Symbols: ---, optical density at 260 nm; *, 3H; 0, 92p.
B
A
1.4060 1.4050 1.4040
t s t . Ix 3H32i
3H 32p
E
.43 '-
0
U. .
4~ ~ ~ ~ ~ ~
Il
w~~~~~~~~z
a-.
