Vol. 121, No. 1 Printed in U.S.A.
OF BACTERIOLOGY, Jan. 1975, p. 173-183 Copyright i 1975 American Society for Microbiology
JOURNAL
Temperature-Sensitive Deoxyribonucleic Acid Replication in a dnaC Mutant of Bacillus subtilis JUNE J. ANDERSEN AND A. T. GANESAN* Department of Genetics, Lt. Joseph P. Kennedy, Jr., Laboratories for Molecular Medicine, Stanford, California 94035
Received for publication 30 September 1974
A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenicol, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment. In Bacillus subtilis, genes controlling deoxyribonucleic acid (DNA) metabolism occupy at least nine distinct groups at different segments of the chromosome (12). Mutants in the dnaA linkage group seem to be defective in the synthesis of precursors of DNA replication (1). dnaB and dna-i mutants may be defective in the initiation of DNA replication (14, 19). We describe here a detailed characterization of a mutation, dnaC6, that lies near the origin of the chromosome and belongs to the dnaC linkage group. A similar temperature-sensitive (ts) mutant was described by Bazill and Retief (2). MATERIALS AND METHODS
Materials. Table 1 lists the B. subtilis strains used. Spizizen minimal medium (16) was supplemented with 20 mg of appropriate amino acids per liter or with 0.05% vitamin-free casein hydrolysate plus 150 mg of tryptophan per liter (CH medium). Five milligrams of thymine per liter was added for thymine auxotrophs. Penassay broth (Difco antibiotic medium 3) plus 0.5% glucose was used as enriched medium. Spores were grown on potato agar plates (Difco Bordet Gengou agar base, 30 g/liter; yeast extract, 2 g/liter; MnSO4, 0.1 g/liter) and stored in distilled water at 4 C. Donnellan spore germination medium (8) was modified by reducing the concentration of glutamic acid and asparagine to 0.3 mg/ml. Phage were titered on LTT plates (15 g of tryptone [Difco J, 5 g of NaCl, and 15 g of agar per liter). Chloramphenicol (CAP) was obtained from Sigma Chemical Co. Nalidixic acid was a gift of SterlingWinthrop Research Institute. 6-(p-Hydroxyphenylazo)-uracil (HPUra) was provided by Bernard
Langley of Imperial Chemical Industries, Ltd. Baker spectral-grade toluene was used to make permeable cells. Bromodeoxyuridine triphosphate was kindly supplied by R. Burger, Department of Molecular Biology, University of California, Berkeley, and D. Brutlag, Biochemistry Department, Stanford University. Deoxyribonucleotide triphosphates of adenine, guanine, cytosine, and thymine were from SchwarzMann. Transformation and mapping. Competent cells and DNA were prepared and transformed by the method of Stewart (18), except that temperature-sensitive strains were grown at 30 C. All transformations except those in which derived mutations were transformed to another auxotroph were performed in the linear range of DNA concentration. Approximate map position was located by the method of Borenstein and Ephrati-Elizur (4). The degree of linkage between two loci was determined by using a member of the dnaC linkage group (12), dnaC230, in a two-factor transformation cross (9). Donor DNA was prepared with strains SB1085 (thy, trpC, dnaC230) and SB19 (prototroph). A 250Mliter volume of competent &B1082 (trpC, hisB, pheA, dnaC6) cells was transformed with 5 uliters of DNA by incubation at 30 C for 30 min. Appropriate dilutions were plated on duplicate plates of nutrient agar incubated at 43 C for ts+ and on glucose-minimal agar plus 50 mg of all amino acids except histidine per liter at 30 C for his+. The recombination index for this cross was: RI = dnaC6+ (SB1085 donor) dnaC6+ (SB19 donor) his+ (SB1085 donor) his+ (SB19 donor) No colonies grew on control plates with competent cells only. Transduction was carried out with the
173
174
ANDERSEN AND GANESAN TABLE 1. Bacillus subtilis strains used
Strain 1. 2. 3. 4. 5. 6. 7. 8.
Genotype designation
SB5 trpC, hisA, pyrA SB19 str-2, prototroph SB566 thy, trpC SB920 purA16, Ieu8, metB SB1058 trpC, hisB, pheA SB1080 thy, trpC, pheA SB1081 thy, trpC, pheA, dnaC6 SB1082 trpC, hisB, pheA, dnaC6
9. SB1083
purA16, leu-8, dnaC6
10. SB1084
thy, trpC, dnaC6
11. SB1085
thy, trpC, dnaC230
Sourcea
in the same specific activity of radioactive thymine as at 30 C:
% DNA replicated F. Rothman N. Sueoka F. Gillan°
SB1058 by transformation SB920 by transformation SB1080 by transformation G. Bazill
aAll strains except 3, 4, and 11 are from the Stanford Genetics Department collection. 'Strain SB1081 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment of strain SB1080 followed by 5-bromouracil selection (3). To avoid the possibility of multiple mutations, SB1082 was prepared by transforming the ts phenotype into SB1058. The ts phenotype was transferred by marker exchange from SB1082 to obtain SB1084 for use in experiments requiring uptake of radioactive thymine. generalized transducing phage PBS1. Lysates were prepared by the method of Young et al. (20). The lysates were titered on Bacillus pumilus by mixing 0.5-ml phage dilutions with 1 ml of log-phase B. pumilus (107 cells/ml), incubating the mixture for 5 min at 30 C, then adding 1.5 ml of soft agar at 45 C. Tubes were mixed and spread onto LTT plates. Plaques were scored after 6 to 8 h at 37 C. A multiplicity of infection of about 0.5 gave the highest transduction frequency (2 x 10-6 transductants per phage). Transductants were picked onto nutrient agar and replica-plated to determine phenotype. Growth, labeling, and lysis of cells. Turbidity was measured on a Klett-Summerson colorimeter with a red filter. A Klett unit under these conditions equals 2 x 10" colony-forming units per ml. Synthesis of DNA, ribonucleic acid, and protein was measured by the incorporation of radioactivity into whole cells precipitated with cold 5% trichloroacetic acid. Radioactive labels used for DNA were ["C Jthymine (Schwarz/Mann, 53 mCi/mmol) and ['H Ithymine (Schwarz/Mann, 17.5 Ci/mmol); for ribonucleic acid the label was ["4C]uracil (New England Nuclear, 56.3 mCi/mmol); for protein the label was ["C Jphenylalanine (Calbiochem, 375 mCi/mmol). Samples added to equal volumes of cold 10% trichloroacetic acid were filtered on Whatman GF/C glass-fiber filters. Filters were rinsed three times with cold saline sodium citrate (15 mM NaCl, 1.5 mM sodium citrate) and 0.02% trichloroacetic acid, dried, and counted. The percentage of DNA replicated was determined by uniformly labeling DNA with radioactive thymine for at least five generations. Cultures were growing exponentially during the experiment. A sample was precipitated in cold trichloroacetic acid at the time of transfer to the restrictive temperature for cpm,, and the rest was incubated at the restrictive temperature
J. BACnMOL.
=
cpm - cpmo x 100 cpmo
where cpmo is the radioactivity per milliliter at zero minutes of incubation. The effect of inhibiting DNA synthesis during incubation at 46 C was determined as follows. Exponentially growing cultures in CH medium labeled for at least five generations with 1 MCi of ['H Jthymine per ml (17.5 Ci/mmol) were exposed to 20 gg of nalidixic acid per ml for 10 min to allow inhibition of DNA synthesis before transfer to 46 C. At 0, 15, 30, and 45 min after transfer to 46 C, nalidixic acid was removed by filtering the cultures on a membrane filter (type HA, Millipore Corp.), rinsing with 2 volumes of Spizizen medium plus 0.5% glucose at 46 C, and resuspending the cells in the same volume of CH medium plus 5 jug of thymine per ml at 46 C. The specific activity of radioactive thymine remained the same as at 30 C. A 1-ml sample was removed immediately and precipitated with cold 10% trichloroacetic acid for the initial point (cpmo). At intervals, samples were removed and precipitated with cold 10% trichloroacetic acid. The same experiment was performed using HPUra (175 MM) as the inhibitor of DNA synthesis at 46 C, except that the culture was labeled with 0.25 MCi of ['Hithymine per ml (43.2 Ci/mmol) and the HPUra was added at the time of transfer to 46 C. For the control experiment, the culture was transferred to 46 C without HPUra. After 45 min at 46 C, the culture was filtered and resuspended at 46 C in the same volume of CH medium with the same specific activity of radioactivity. For lysis, cells were concentrated 25-fold by centrifugation at 4 C and resuspended in 1 ml of a solution containing 0.1 M ethylenediaminetetraacetic acid, 0.1 M NaN,, and 100 Mg of lysozyme. After 15 min at 37 C, the addition of sodium lauryl sulfate (0.625% final concentration) caused complete lysis. The products of DNA synthesis in vivo were analyzed by pycnography in CsCl. Cells were density-labeled by growth in medium containing "NH4+ and 2H2O (heavy medium) (10) to give the DNA a buoyant density of 1.756 instead of 1.703 g/ml in CsCl. Toluene treatment. Exponentially growing cells were toluene-treated by the method of Moses and Richardson (15), except that they were agitated in a Vortex mixer at 25 C in the presence of 1% toluene for 4 min. The toluene-treated cells were assayed for DNA synthesis at 30 or 43 C in 2 ml of a solution containing: 0.9 to 1.5 x 10' cells/ml; 20% sucrose; 0.1 M KPO4 buffer at pH 7.6; 75 mM MgCl,; 7.5 mM 0-mercaptoethanol; deoxyadenosine 5'-triphosphate, deoxycytidine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and bromodeoxyuridine triphosphate, at 50 MM each; 2.5 MM ['Hjthymidine 5'-triphosphate (equivalent specific activity, 130 counts/min per pmol); and 1.25 mM adenosine 5'-triphosphate. The reaction was stopped by adding 6 ml of cold buffer containing 0.1 M KPO4, 0.6 M NaCl, and 40 mM
VOL. 121, 1975
175
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
ethylenediaminetetraacetic acid. Native B. subtilis DNA has a bouyant density of 1.703 g/ml in CsCl. The observed densities in CsCl for hybrid molecules with bromouracil fully substituted in one strand and in both strands are 1.758 and 1.813 g/ml, respectively (C. Laird, Ph.D. thesis, Stanford University, Stanford, Calif., 1966).
10 2
10 9
RESULTS Linkage Determination. Germinating spores release DNA into the medium in a sequential fashion from origin to terminus (4), and this DNA could be used to transform a suitable multiple auxotroph. The approximate map position of a gene can be determined by comparing the pattern of appearance of transformants for the gene to that of transformants of auxotrophs that have known map positions. DNA released from germinating SB566 spores was used to transform competent SB1083 cells. The very high frequency of transformants for dnaC6 and purA16 compared to other markers suggested a close location of these two genes. Phage PBS1-mediated transduction confirmed the suggested map position. The cotransduction of dnaC6 with purA16 was measured with strain SB1083 as a recipient. The co-transduction frequencies of ts+ with purA16+ was 0.94 (680/727) and of purA16+ with ts+ was 0.98 (501/600). Since the dnaC linkage group is also closely linked to purA16 (0.96 co-transduction with purA16 [12 ]), a two-factor transformation cross was performed with strain SB1085 carrying dnaC230 as the donor and strain SB1082 carrying dnaC6 as the recipient. Both strains SB1082 and SB1085 can be transformed to ts+ under the selective conditions used (nutrient agar plates at 45 C). A reduced frequency of recombination between mutants of the same phenotype (9), as reflected in a recombination index less than 1, i.e., 3 x 10-s, indicates that dnaC230 and dnaC6 are members of the same linkage group. Growth and macromolecular synthesis at restrictive temperatures. After transfer of strains carrying dnaC6 from 30 to 43 C in CH medium, the cells elongate and divide, but form chains. Cell mass continues to increase for at least 4 h; however, the rate is reduced within 1 h after transfer to 43 C. The turbidity increases to about fourfold the initial turbidity at the time of transfer (Fig. 1). Viable cells increase by 6.4-fold in CH medium during the first 2 h at 43 C, but decrease rapidly thereafter. Cell division continues for 60 min after DNA synthesis stops at 43 C. The primary effect of the lesion is the arrest of DNA synthesis in strain dnaC6 within 30 min at
X
6
40x c
tn
C,
43c/
CL 0_
E
L 0
--
I-
0
U
oi
10l -4
-2
0
2
4
Hour5 at 43C FIG. 1. Survival of strain SB1081 in CH medium after transfer from 30 to 43 C. Turbidity in Klett units (O); colony-forming units (0) on nutrient agar incubated at 30 C.
43 C (Fig. 2) while synthesis of RNA and protein continues. The amount of DNA replicated after transfer to a restrictive temperature varies with the temperature of incubation (Fig. 3). An isogenic ts+ strain incorporates radioactive thymine equivalent to 304% of the template at 40 C and 282% at 49 C in 120 min. Upon incubation at 46 C for 45 min, DNA synthesis ceases, but resumes in less than 30 min after transfer to 30 C even in the presence of CAP (Fig. 4). Different amounts of CAP reduce the rate of DNA synthesis to different degrees; however, an amount of DNA equivalent to 25% of the labeled template was synthesized in the presence of 150 .g of CAP per ml. Pattern of DNA synthesis in vivo. The pattern of residual DNA synthesis in a strain carrying dnaC6 after transfer to 43 C was analyzed by determining the amounts of chromosome
origin (purA16) and terminus (metB)
markers replicated at 43 C compared with the amounts at 30 C. Similar amounts of origin and terminus were synthesized at 30 and 43 C (Table 2). The amounts of origin and terminus replicated were measured by density-labeling the DNA at 30 C, transferring the cells to CH medium at 43 C, and using the newly replicated DNA from the hybrid density region of a CsCl equilibrium gradient for transformation. As a control, an identical sample was incubated at 30 C rather than 43 C after the transfer to CH
ANDERSEN AND GANESAN
176
J. BACTERIOL.
medium. The resulting distribution of radioac- of synthesis at 43 C, 42% of the "C-labeled tivity in the gradient showed that during 90 min DNA was transferred to the intermediate-density stratum (Fig. 5). The pattern of biological transferred is shown in Fig. 5. The activity a biological activity patterns were compared after 0 equivalent amounts of DNA synthesis because the rate of DNA synthesis varied with tempera6 ture. The control results for transfer of biological activity were interpolated to those expected for 40% transfer of "C-labeled template DNA (Table 2). 4 UDNA synthesis ceases during the 90-min Q incubation period at 43 C. If the temperature is then lowered to 30 C, DNA synthesis begins 2) again within 20 min. The pattern of this resumed DNA synthesis at 30 C was also analyzed by pycnography in CsCl. The pattern of radio0 activity resulting from 40 min of DNA synthesis 30 C is shown in Fig. 6a. Assays for biological at lb
:c.
0 I
12
U Q L
U
4t
40 n
0
x 30 £ (j
'2o L-
4 3t
10
0
60 40 Minutes
12'0
FIG. 2. Synthesis of DNA, ribonucleic acid, and protein by strain SB1081. At 30 C, 0.5 MCi of ["CJthymine, 0.1 gCi of ["C]uracil, or 0.5 gCi of ["CJphenylalanine per ml was added to early logphase cells. After 30 min, the cultures were divided and half were transferred to 43 C. Samples (0.1 ml each) were added to cold 10%o trichloroacetic acid at intervals. (a) ["C Ithymine at 30 C (-) and 43 C (0); (b) ["C]uracil at 30 C (C) and 43 C (Q); (c) ["Cjphenylalanine at 30 C (A) and 43 C (A).
0
60 120 Minutes of incubation
FIG. 3. Percentage of DNA replicated, as a function of temperature, by strain SB1084. Cultures were uniformly labeled (1 ACi of [3HJthymine per ml) by exponential growth for at least five generations at 30 C. At 107 colony-forming units per ml, log-phase cultures with 1 ACi of [8H]thymine per ml were transferred to 30 C (0), 40 C (0), 43 C (A), 46 C (A), and 49 C (C), and 1-ml samples were precipitated by addition of 1 ml of cold 10% trichloroacetic acid. cpmO = 531 counts/min.
VOL. 121, 1975
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
160_
_
TABLE 2. Synthesis in vivo by strain SB1082 after transfer from "l NH4+ -2H20 to CH medium
i6z
% Parental label
140
trans-
Growth temp
120 30 C control
100 _
I0
< z
177
Residual synthesis at
% Biological activity in
intermediate
density
ferred to
diate den- purA16 metB gin) minus) 18 30 28
40a
52a
37a
60
72
50
75
82
54
42
45
46
41
89
30
43 C
a
~60-
Restarted synthesis at 30 C after 90 min at 43 C
40-
20
-
aData for 40% transfer of parental label was interpolated linearly from experiments with 18 and 60%
/
transfer.
thus, a second round of initiation must not have occurred. If none of the old growing points had contin0 60 120 ued when the temperature was lowered, one would not expect to see the terminus replicated Minutesat 30C FIG. 4. Effect of CAP on the resumption of DNA within 40 min. (Doubling time at 30 C in CH synthesis. Strain SB1084, prelabeled by exponential medium is 80 min.) Table 2 shows that 30% of growth for at least five generations with 1 MCi of the metB activity has been replicated compared [3Hjthymine per ml, was transferred to 46 C. After a to 37% for the control. Synthesis must resume at 45-min period at 46 C, levels of CAP from 0 to 150 many of the old growing points when the Mg/mI were added and the cultures were returned to 30 C, still in ['HJthymine (1 MCi/ml). Samples (1 ml) temperature is lowered. Effect of the thermosensitive lesion during were precipitated by addition to cold 10%o trichloroacetic acid at intervals and were filtered. Symbols: *, spore germination. There was little DNA synthesized in SB1082 spores germinated at 46 C no CAP; 0, 20 ;g of CAP per ml; A, 50Sug of CAP per ml; 100 Mg of CAPper ml;C, 150Mg of CAPper ml. (Fig. 7), even though outgrowing spores could be cpmO 8,234 counts/min. observed under a phase-contrast microscope. At 2, 3 and 4 h of growth, spores were transferred from 30 to 46 C. The sample transferred to 46 C activity indicate that both the origin and the after 2 h showed no more DNA synthesis than terminus were replicated during this period spores germinated at 46 C. In contrast, the (Fig. 6b). The control for this experiment was germinating spores transferred to 46 C after 3 h the same as for the previous experiment, began to synthesize DNA, and the sample namely, replication at 30 C after transfer from transferred at 4 h continued DNA synthesis for heavy medium to CH medium, but without the 2 h after transfer to 46 C. incubation at 43 C. For a comparable amount of Effects of inhibiting DNA synthesis at the DNA replicated, the resumed synthesis shows a restrictive temperature. The result of an exmuch larger amount of origin marker replicated periment to determine whether the capacity to than the control. As shown in Table 2, 89% of synthesize a residual amount of DNA at 46 C is the purA16 activity has been replicated com- stable or is progressively inactivated in the pared to 52% for the control. This result indi- absence of DNA synthesis in a strain carrying cates that initiation must have occurred at the dnaC6 is shown in Table 3. Both nalidixic acid chromosomal origin after the temperature was (7) and HPUra (5) specifically inhibit DNA lowered from 43 to 30 C. No purA16 activity was synthesis and were used during incubation at found in the fully light DNA density position; 46 C to inhibit residual DNA synthesis by strain 20
,,
=
178
ANDERSEN AND GANESAN
SB1084; then the inhibitors were removed and the amount of residual DNA synthesis at 46 C was measured. Table 3 shows that no further DNA replication took place at 46 C in the control culture. In contrast, cultures inhibited at 46 C retained the capacity to synthesize a residual amount of DNA even after 60 min at 46 C. Toluene-treated cells. Toluene treatment of B. subtilis renders the cells permeable to direct
J. BACTE3RIOL.
precursors of DNA synthesis. In the presence of adenosine 5'-triphosphate and the four deoxyribonucleotide triphosphates, toluene-treated cells synthesize biologically active DNA semiconservatively (13, 15). To determine whether DNA replication in a toluene-treated dnaC6 strain reflects the pattern observed in vivo, cells were assayed for DNA synthesis. The optimal time of toluene-treatment varied with the strain, e.g., strains carrying dnaC6 required
al.
l4C 200
-
100 _
Fraction number
4-
>-~~b
00
0
0
~~~~~000I
20 30 40 Top Fraction number FIG. 5. Pattern of DNA synthesized at 43 C after transfer from 15NH4+-2H20 medium. Strain SB1082 was prelabeled by growth at 30 C in 15NH4+-2H2O medium containing 0.5 tCi of [4C]thymine per ml. Log-phase cells (7 x 109) were filtered and suspended in CH medium containing 5 pCi of [3H]thymine per ml and 250 pg of deoxyadenosine per ml. After 90 min at 43 C, cells were centrifuged at 4 C, lysed, and mixed with CsCI, 0.01 M tris(hydroxymethyl)aminomethane (pH 8.0), and 2 pliters of light marker SB920 DNA to a refractive index of 0
10
1.4005 and a volume of 8 ml. Gradient tubes were centrifuged in a Spinco 50 rotor at 35,000 rpm at 4 C. Samples (0.133 ml) were collected in 60 fractions, and 50-pgliter samples were precipitated with cold 10% trichloroacetic acid. Position of the light marker DNA (arrow) was determined by transformation of competent SB5 cells. (a) Distribution of radioactivity on a CsCI gradient: 14C counts/min (0), 3H counts/min (A). (b) Biological activity was measured by transformation of 0.3% competent SB920 cells to purA+ (0) and metB+ (O)).
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
VOL. 121, 1975
only 4 min at 25 C and the activity decreased upon further treatment. The addition of 20% sucrose to the assay mixture stabilized DNA replication in toluene-treated cells at 43 C both in strain SB1082 (dnaC6) and strain SB1058 (ts+). Figure 8 shows the kinetics of DNA synthesis by toluene-treated cells. Addition of adenosine 5'-triphosphate stimulated incorporation 8- to 10-fold. At an assay temperature of 30 C, the ts+ strain and the mutant synthesized DNA for at least 60 min (Fig. 8a and b). When assayed at 43 C, the ts+ strain synthesized DNA for 40 min, whereas the dnaC6 strain stopped synthe-
179
sis after 20 min. dnaC6 cells incorporated only one-third as much 8H as normal cells before synthesis ceased at 43 C. No synthesis was observed if dnaC6 cells were incubated at 43 C before toluene treatment (Fig. 8c). DNA isolated in the experiments shown in Fig. 8b was analyzed by CsCl pycnography to compare the DNA synthesized in dnaC6 cells at 30 and 43 C. Figure 9 shows the pattern of radioactivity after DNA replication in toluenetreated cells carrying dnaC6 in the presence of a mixture [3H]TTP and BrdUrdTP in the ratio 1:20. Under these conditions, the hybrid molecules have a density of 1.740 g/ml. The parental
al. 400
200
0 10
20
30
Frac
b. > 20 4-
*'~~~~Il' 10
1
30 40 Fraction number FIG. 6. Pattern of DNA synthesized at 30 C after a 90-min incubation at 43 C. Log-phase SB1082 cells were prelabeled by growth at 30 C in 15NH4+-'H,O medium containing 0.5 uCi of [14CJthymine per ml. The tempera-
ture of incubation was raised from 30 to 43 C for 90 min. Cells (7 x 109) were transferred from "NH4+-2H2O to CH medium and from ["4C Jthymine to ['H]thymine when the temperature was lowered from 43 to 30 C. Labeling, lysis, and assays were performed as described in the legend to Fig. 5. (a) Distribution of radioactivity on a CsCI gradient: 14C counts/min (-), 'H counts/min (A). (b) Biological activity: purA16+ (0), metB+ (O9).
180
ANDERSEN AND GANESAN
J. BACTFJOL.
Q
:c ._
r)
I
6 4 Hours of outgrowth FIG. 7. DNA synthesis during spore germination of strain SB1082. Purified spores (4 x 107/ml) were added to 5 uCi of [3H]thymine per ml (17.5 Ci/mmol). Germinating spores were transferred from 30 to 46 C after 2, 3, and 4 h. Samples (0.1 ml) were precipitated by addition to cold 10%o trichloroacetic acid, filtered and counted. DNA synthesis was determined at 30 C (-) and 46 C (0) and transfer from 30 to 46 C at 2 h (O)), 3 h (A), and 4 h
(A). ration of radioactive label into DNA at assay temperatures of 43 and 30 C gives an overestimate of semiconservative DNA synthesis. A larger proportion of the radioactivity incorpoDNA replicateda (%) Time of inhibition Nalidixic rated by toluene-treated dnaC6 cells appears to Control" HPUrad (min) acidc be nonconservative synthesis at 43 C than at 30 C (Fig. 9). 14.2 16.4 -1.2 0 Biologically active DNA molecules synthe22.3 25.9 15 sized at 30 C occupy both hybrid- and inter17.9 20.8 30 mediate-density strata and might reflect semi31.9 13.3 45 conservative synthesis (13) in toluene-treated 22.2 60 cells. Strain SB1082 (dnaC6) transferred only a Within 30 min at 46 C after removal of the 2.3% of the purA16+ activity to a denser posiinhibitor. tion after synthesis at 43 C, compared with 15% b 100% = 4,879 counts/min. after synthesis at 30 C. Thus, DNA synthesis is C 100% = 13,194 counts/min. temperature sensitive in cells carrying dnaC6 d 100% = 4,300 counts/min. both in vivo and after toluene treatment. DNA cosediments with unlabeled, light stanDISCUSSION dard DNA (Fig. 9A and B). After DNA synthesis at 30 C, the 3H-labeled molecules that sediThe above results indicate that the dnaC6 mented at a heavier density than parental DNA locus lies near the origin of the B. subtilis were distributed bimodally. The molecules chromosome and is closely linked to the purA16 found between hybrid (1.740 g/ml) and light gene. In this and another dnaC-group mutant parental DNA (1.703 g/ml) have a density of (2), ribonucleic acid and protein synthesis con1.718 g/ml and might represent density transi- tinues after the arrest of DNA synthesis. Strains tion points. A. comparison of the gross incorpo- carrying dnaC6 or dnaC230 are capable of
TABLE 3. Capacity for DNA synthesis after inhibition at the restrictive temperature in strain SB1084
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
VOL. 121, 1975 ISO
100
b
200
co/ ~0
150
100 _
50
50
C. 0
0
20 60 40 Minutes of assay
FIG. 8. Kinetics of DNA synthesis by toluenetreated cells. Strains SB1082 and SB1058 (the isogenic ts+ strain) were toluenized and assayed in the presence of adenosine 5'-triphosphate, [3H]thymidine 5'-triphosphate (130 counts/min per pmol), and bromodeoxyuridine triphosphate. Incorporation into cold acid-insoluble material by toluene-treated cells was measured for: (a) strain SB1058 grown at 30 C, toluene-treated and assayed at 30 C (0) and 43 C (U); (b) strain SB1082 grown at 30 C, toluene-treated, and assayed at 30 C (A) and 43 C (A); (c) strain SB1082 toluene-treated after 45 min of growth at 43 C and assayed at 30 C (0) and 43 C (0).
beginning DNA synthesis in the presence of CAP after the incubation temperature is lowered from 43 to 30 C. The rate of this synthesis is depressed by CAP, indicating that synthesis of proteins required to achieve a normal rate of DNA synthesis was reduced. This might result from either fewer replication forks synthesizing
181
at the normal rate or from all the replication forks synthesizing at a slower rate. Nevertheless, the fact that some synthesis is observed in the presence of 150 ,ug of CAP per ml argues that either protein synthesis is not required to begin DNA synthesis, or that CAP-resistant protein synthesis can supply the necessary proteins. Viable cell number continued to increase for 60 min after DNA synthesis stopped at 43 C in a dnaC6 strain, suggesting that the process of cell division in this mutant is not coupled directly to DNA chain elongation as in a few known cases (11, 14). Furthermore, the cellular components required for cell division might either be preformed in the cell before the temperature step or their synthesis might proceed normally at 43 C at least for a while. We have confirmed and extended the hypothesis that dnaC mutants are not defective in initiation of new rounds of DNA synthesis (2). Evidence comes from the CsCl gradient analysis, which suggests that during residual DNA synthesis at 43 C the amount of origin marker replicated is similar to the amount replicated at 30 C (Table 2). The majority of initiations continue to occur at 43 C as in other temperature-sensitive dna mutants (19). In contrast, a DNA initiation mutant carrying dna-1 could only transfer 11.7% of purA16 activity to hybrid density when 61% of the template DNA was replicated at 45 C. The dnaC6 cultures also replicated significant amounts (30%) of terminus marker after resuming synthesis at 30 C, in contrast to the initiation mutant carrying dna-1 which transferred only 10.8% of terminus marker activity after 40 min of resumed synthesis (19). Strains carrying dnaC6 initiate a new round of replication in addition to continuing the previous round when the temperature is lowered from 43 to 30 C. This is typical whenever DNA synthesis is inhibited in cells while protein synthesis continues, as in dnaB mutants of Escherichia coli (17). When spores carrying dnaC6 are germinated at 46 C, little synthesis can be detected. These results are similar to B. subtilis 168ts-134, a mutant possibly affected in the initiation of new rounds of DNA replication (14), which was later mapped as a dnaB mutant (12). Since neither mutant begins DNA synthesis when the spores are germinated at 46 C, this result cannot be used to distinguish between an initiator mutant and one that is defective in a component required to sustain DNA synthesis. There are other explanations for the relatively slow cessation of DNA synthesis when the temperature is raised. Here we consider two
182
ANDERSEN AND GANESAN
J. BACTMOL.
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5
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FRACTION NUMBER
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35
TOP
FIG. 9. Products of synthesis in toluene-treated SB1082 (dnaC6) cells assayed at 30 and 43 C. Products of the reactions shown in Fig. 8b were analyzed by CsCl density gradients as described in the legend to Fig. 5. DNA synthesized in toluene-treated cells was labeled with bromodeoxyuridine triphosphate and ['HJthymidine 5'-triphosphate (130 counts/min per pmol). Biological activity of SB920 unlabeled marker DNA was determined by transformation of competent SB5 cells to trpC+ (0). (A) Distribution of radioactivity and biological activity on a CsCI equilibrium gradient after synthesis at 30 C: 'H counts/min (A), purA16+ (0). (B) Distribution of radioactivity and biological activity on a CsCI gradient after synthesis at 43 C: 'H counts/min (A), purA16+
(0). possibilities: first, that the thermosensitive component decays slowly at 46 C and, second, that a component required for DNA synthesis is consumed and not resynthesized at 46 C. Inhibition of DNA synthesis with nalidixic acid or HPUra showed that the capacity to synthesize DNA was stable at 46 C. These results are not consistent with the first possibility, unless a factor required for replication is stabilized somehow in the absence of DNA replication. A simpler explanation for the behavior would be that the factor is depleted only during DNA synthesis. The capacity for DNA synthesis is destroyed within 15 min at 45 C if thymine starvation is used as the DNA synthesis inhibitor with a strain carrying dnaC230 (2). Opposite results obtained here using HPUra or nalidixic acid as the inhibitor of DNA synthesis raised the possibility that secondary effects of inhibition may influence the results. HPUra has almost no secondary effects in 60 min at 75 ug/ml (6). Effects of higher concentrations have not been reported. A 25-,ug amount of nalidixic acid per ml affects protein synthesis and cell viability within 60 min (7). Thymine starvation decreases cell viability within 30 min and might induce latent phage. Thymine starvation, nalidixic acid, and HPUra all cause limited DNA
degradation (6). Part of the incorporation at 46 C after the removal of inhibitors might represent repair synthesis. A toluene-treated dnaC6 strain and its isogenic ts+ strain showed that their pattern of DNA synthesis is similar to that of untreated viable cells. dnaC6 cells, which shut off DNA synthesis at 43 C in vivo, cannot resume DNA synthesis in toluene-treated cells. Pycnographic analysis of DNA synthesized in these cells at 43 C indicates a marked reduction of DNA synthesis. Since DNA replication is also temperature sensitive in toluene-treated cells, the defect is probably not in the synthesis of precursors or cofactors like adenosine 5'-triphosphate. Whether the thermosensitivity of the toluenetreated dnaC6 cells is due to the same component as in vivo or whether it is the result of a secondary effect (e.g., a membrane defect that makes the toluene-treated cells more fragile and, thus, more thermosensitive) has not been
determined. The nature of the ts lesion in strains carrying dnaC6 seems to be in the production of a required factor that is consumed during DNA synthesis. When the temperature is raised, the pool of factor is depleted only if DNA synthesis is proceeding. Lowering the temperature reactivates factor production and DNA synthesis
VOL. 121, 1975
proceeds
even
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
in the absence of protein synthe-
sis.
ACKNOWLEDGMENTS We wish to thank Ann Ganesan and P. C. Cooper for critical readings of the manuscript. JoAnn Katheiser provided excellent technical assistance. This work was aided by Public Health Service grants GM-14108 and 2 T01-GM 295 from the National Institute of General Medical Sciences and by grant GB 8739 from the National Science Foundation. A. T. Ganesan is a recipient of Public Health Service Research Career Program Award GM-50199 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bazill, G. W. 1972. Temperature-sensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis. Mol. Gen. Genet. 117:19-29. 2. Bazill, G., and Y. Retief. 1969. Temperature-sensitive DNA synthesis in a mutant of Bacillus subtilis. J. Gen. Microbiol. 56:87-97. 3. Bonhoeffer, R., and H. Schaller. 1965. A method for selective enrichment of mutants based on the high UV sensitivity of DNA containing 5-Bromouracil. Biochem. Biophys. Res. Commun. 20:93-97. 4. Borenstein, S., and E. Ephrati-Elizur. 1969. Spontaneous release of DNA in sequential genetic order by Bacillus subtilis. J. Mol. Biol. 45:137-152. 5. Brown, N. C. 1970. 6-(p-Hydroxyphenylazo)-uracil: a selective inhibitor of host DNA replication in phageinfected Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 67:1454-1461. 6. Brown, N. C. 1971. Inhibition of bacterial DNA replication by 6-(p-hydroxyphenylazo)-uracil: differential effect on repair and semiconservative synthesis in Bacillus subtilis. J. Mol. Biol. 59:1-16. 7. Cook, T. M., K. G. Brown, J. V. Boyle, and W. A. Goss. 1966. Bactericidal action of nalidixic acid on Bacillus subtilis. J. Bacteriol. 92:1510-1514. 8. Donellan, J., E. Nags, and H. Levinson. 1964. Chemically
9. 10. 11.
12.
13. 14.
15. 16. 17. 18. 19.
20.
183
defined, synthetic media for sporulation and for germination and growth of Bacillus subtilis. J. Bacteriol. 87:332-336. Dubnau, D., C. Goldthwaite, I. Smith, and J. Marmur. 1967. Genetic mapping in Bacillus subtilis. J. Mol. Biol. 27:163-185. Ganesan, A. T. 1968. Studies on the in vitro synthesis of transforming DNA. Proc. Nat. Acad. Sci. U.S.A.61:1058-1065. Gross, J. D., D. Karamata, and P. Hempstead. 1968. Temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Cold Spring Harbor Symp. Quant. Biol. 33:307-312. Karamata, D., and J. Gross, 1970. Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol. Gen. Genet. 108:277-287. Matsushita, T., K. White, and N. Sueoka. 1971. Chromosome replication in toluenized Bacillus subtilis cells. Nature N. Biol. 232:111-114. Mendelson, N., and J. Gross. 1967. Characterization of a temperature-sensitive mutant of Bacillus subtilis defective in deoxyribonucleic acid replication. J. Bacteriol. 94:1603-1608. Moses, R., and C. Richardson. 1970. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc. Nat. Acad. Sci. U.S.A. 67:674-681. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078. Stein, G., and P. Hanawalt. 1969. Initiation of DNA replication cycles of Escherichia coli following DNA synthesis inhibition. J. Mol. Biol. 46:135-144. Stewart, C. 1969. Physical heterogeneity among Bacillus subtilis deoxyribonucleic acid molecules carrying particular genetic markers. J. Bacteriol. 98:1239-1247. White, K., and N. Sueoka. 1973. Temperature-sensitive DNA synthesis mutants of Bacillus subtilis. Genetics 73:185-214. Young, F. E., C. Smith, and B. E. Reilly. 1969. Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. J. Bacteriol. 98:1087-1097.
OF BACTERIOLOGY, Jan. 1975, p. 173-183 Copyright i 1975 American Society for Microbiology
JOURNAL
Temperature-Sensitive Deoxyribonucleic Acid Replication in a dnaC Mutant of Bacillus subtilis JUNE J. ANDERSEN AND A. T. GANESAN* Department of Genetics, Lt. Joseph P. Kennedy, Jr., Laboratories for Molecular Medicine, Stanford, California 94035
Received for publication 30 September 1974
A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenicol, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment. In Bacillus subtilis, genes controlling deoxyribonucleic acid (DNA) metabolism occupy at least nine distinct groups at different segments of the chromosome (12). Mutants in the dnaA linkage group seem to be defective in the synthesis of precursors of DNA replication (1). dnaB and dna-i mutants may be defective in the initiation of DNA replication (14, 19). We describe here a detailed characterization of a mutation, dnaC6, that lies near the origin of the chromosome and belongs to the dnaC linkage group. A similar temperature-sensitive (ts) mutant was described by Bazill and Retief (2). MATERIALS AND METHODS
Materials. Table 1 lists the B. subtilis strains used. Spizizen minimal medium (16) was supplemented with 20 mg of appropriate amino acids per liter or with 0.05% vitamin-free casein hydrolysate plus 150 mg of tryptophan per liter (CH medium). Five milligrams of thymine per liter was added for thymine auxotrophs. Penassay broth (Difco antibiotic medium 3) plus 0.5% glucose was used as enriched medium. Spores were grown on potato agar plates (Difco Bordet Gengou agar base, 30 g/liter; yeast extract, 2 g/liter; MnSO4, 0.1 g/liter) and stored in distilled water at 4 C. Donnellan spore germination medium (8) was modified by reducing the concentration of glutamic acid and asparagine to 0.3 mg/ml. Phage were titered on LTT plates (15 g of tryptone [Difco J, 5 g of NaCl, and 15 g of agar per liter). Chloramphenicol (CAP) was obtained from Sigma Chemical Co. Nalidixic acid was a gift of SterlingWinthrop Research Institute. 6-(p-Hydroxyphenylazo)-uracil (HPUra) was provided by Bernard
Langley of Imperial Chemical Industries, Ltd. Baker spectral-grade toluene was used to make permeable cells. Bromodeoxyuridine triphosphate was kindly supplied by R. Burger, Department of Molecular Biology, University of California, Berkeley, and D. Brutlag, Biochemistry Department, Stanford University. Deoxyribonucleotide triphosphates of adenine, guanine, cytosine, and thymine were from SchwarzMann. Transformation and mapping. Competent cells and DNA were prepared and transformed by the method of Stewart (18), except that temperature-sensitive strains were grown at 30 C. All transformations except those in which derived mutations were transformed to another auxotroph were performed in the linear range of DNA concentration. Approximate map position was located by the method of Borenstein and Ephrati-Elizur (4). The degree of linkage between two loci was determined by using a member of the dnaC linkage group (12), dnaC230, in a two-factor transformation cross (9). Donor DNA was prepared with strains SB1085 (thy, trpC, dnaC230) and SB19 (prototroph). A 250Mliter volume of competent &B1082 (trpC, hisB, pheA, dnaC6) cells was transformed with 5 uliters of DNA by incubation at 30 C for 30 min. Appropriate dilutions were plated on duplicate plates of nutrient agar incubated at 43 C for ts+ and on glucose-minimal agar plus 50 mg of all amino acids except histidine per liter at 30 C for his+. The recombination index for this cross was: RI = dnaC6+ (SB1085 donor) dnaC6+ (SB19 donor) his+ (SB1085 donor) his+ (SB19 donor) No colonies grew on control plates with competent cells only. Transduction was carried out with the
173
174
ANDERSEN AND GANESAN TABLE 1. Bacillus subtilis strains used
Strain 1. 2. 3. 4. 5. 6. 7. 8.
Genotype designation
SB5 trpC, hisA, pyrA SB19 str-2, prototroph SB566 thy, trpC SB920 purA16, Ieu8, metB SB1058 trpC, hisB, pheA SB1080 thy, trpC, pheA SB1081 thy, trpC, pheA, dnaC6 SB1082 trpC, hisB, pheA, dnaC6
9. SB1083
purA16, leu-8, dnaC6
10. SB1084
thy, trpC, dnaC6
11. SB1085
thy, trpC, dnaC230
Sourcea
in the same specific activity of radioactive thymine as at 30 C:
% DNA replicated F. Rothman N. Sueoka F. Gillan°
SB1058 by transformation SB920 by transformation SB1080 by transformation G. Bazill
aAll strains except 3, 4, and 11 are from the Stanford Genetics Department collection. 'Strain SB1081 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment of strain SB1080 followed by 5-bromouracil selection (3). To avoid the possibility of multiple mutations, SB1082 was prepared by transforming the ts phenotype into SB1058. The ts phenotype was transferred by marker exchange from SB1082 to obtain SB1084 for use in experiments requiring uptake of radioactive thymine. generalized transducing phage PBS1. Lysates were prepared by the method of Young et al. (20). The lysates were titered on Bacillus pumilus by mixing 0.5-ml phage dilutions with 1 ml of log-phase B. pumilus (107 cells/ml), incubating the mixture for 5 min at 30 C, then adding 1.5 ml of soft agar at 45 C. Tubes were mixed and spread onto LTT plates. Plaques were scored after 6 to 8 h at 37 C. A multiplicity of infection of about 0.5 gave the highest transduction frequency (2 x 10-6 transductants per phage). Transductants were picked onto nutrient agar and replica-plated to determine phenotype. Growth, labeling, and lysis of cells. Turbidity was measured on a Klett-Summerson colorimeter with a red filter. A Klett unit under these conditions equals 2 x 10" colony-forming units per ml. Synthesis of DNA, ribonucleic acid, and protein was measured by the incorporation of radioactivity into whole cells precipitated with cold 5% trichloroacetic acid. Radioactive labels used for DNA were ["C Jthymine (Schwarz/Mann, 53 mCi/mmol) and ['H Ithymine (Schwarz/Mann, 17.5 Ci/mmol); for ribonucleic acid the label was ["4C]uracil (New England Nuclear, 56.3 mCi/mmol); for protein the label was ["C Jphenylalanine (Calbiochem, 375 mCi/mmol). Samples added to equal volumes of cold 10% trichloroacetic acid were filtered on Whatman GF/C glass-fiber filters. Filters were rinsed three times with cold saline sodium citrate (15 mM NaCl, 1.5 mM sodium citrate) and 0.02% trichloroacetic acid, dried, and counted. The percentage of DNA replicated was determined by uniformly labeling DNA with radioactive thymine for at least five generations. Cultures were growing exponentially during the experiment. A sample was precipitated in cold trichloroacetic acid at the time of transfer to the restrictive temperature for cpm,, and the rest was incubated at the restrictive temperature
J. BACnMOL.
=
cpm - cpmo x 100 cpmo
where cpmo is the radioactivity per milliliter at zero minutes of incubation. The effect of inhibiting DNA synthesis during incubation at 46 C was determined as follows. Exponentially growing cultures in CH medium labeled for at least five generations with 1 MCi of ['H Jthymine per ml (17.5 Ci/mmol) were exposed to 20 gg of nalidixic acid per ml for 10 min to allow inhibition of DNA synthesis before transfer to 46 C. At 0, 15, 30, and 45 min after transfer to 46 C, nalidixic acid was removed by filtering the cultures on a membrane filter (type HA, Millipore Corp.), rinsing with 2 volumes of Spizizen medium plus 0.5% glucose at 46 C, and resuspending the cells in the same volume of CH medium plus 5 jug of thymine per ml at 46 C. The specific activity of radioactive thymine remained the same as at 30 C. A 1-ml sample was removed immediately and precipitated with cold 10% trichloroacetic acid for the initial point (cpmo). At intervals, samples were removed and precipitated with cold 10% trichloroacetic acid. The same experiment was performed using HPUra (175 MM) as the inhibitor of DNA synthesis at 46 C, except that the culture was labeled with 0.25 MCi of ['Hithymine per ml (43.2 Ci/mmol) and the HPUra was added at the time of transfer to 46 C. For the control experiment, the culture was transferred to 46 C without HPUra. After 45 min at 46 C, the culture was filtered and resuspended at 46 C in the same volume of CH medium with the same specific activity of radioactivity. For lysis, cells were concentrated 25-fold by centrifugation at 4 C and resuspended in 1 ml of a solution containing 0.1 M ethylenediaminetetraacetic acid, 0.1 M NaN,, and 100 Mg of lysozyme. After 15 min at 37 C, the addition of sodium lauryl sulfate (0.625% final concentration) caused complete lysis. The products of DNA synthesis in vivo were analyzed by pycnography in CsCl. Cells were density-labeled by growth in medium containing "NH4+ and 2H2O (heavy medium) (10) to give the DNA a buoyant density of 1.756 instead of 1.703 g/ml in CsCl. Toluene treatment. Exponentially growing cells were toluene-treated by the method of Moses and Richardson (15), except that they were agitated in a Vortex mixer at 25 C in the presence of 1% toluene for 4 min. The toluene-treated cells were assayed for DNA synthesis at 30 or 43 C in 2 ml of a solution containing: 0.9 to 1.5 x 10' cells/ml; 20% sucrose; 0.1 M KPO4 buffer at pH 7.6; 75 mM MgCl,; 7.5 mM 0-mercaptoethanol; deoxyadenosine 5'-triphosphate, deoxycytidine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and bromodeoxyuridine triphosphate, at 50 MM each; 2.5 MM ['Hjthymidine 5'-triphosphate (equivalent specific activity, 130 counts/min per pmol); and 1.25 mM adenosine 5'-triphosphate. The reaction was stopped by adding 6 ml of cold buffer containing 0.1 M KPO4, 0.6 M NaCl, and 40 mM
VOL. 121, 1975
175
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
ethylenediaminetetraacetic acid. Native B. subtilis DNA has a bouyant density of 1.703 g/ml in CsCl. The observed densities in CsCl for hybrid molecules with bromouracil fully substituted in one strand and in both strands are 1.758 and 1.813 g/ml, respectively (C. Laird, Ph.D. thesis, Stanford University, Stanford, Calif., 1966).
10 2
10 9
RESULTS Linkage Determination. Germinating spores release DNA into the medium in a sequential fashion from origin to terminus (4), and this DNA could be used to transform a suitable multiple auxotroph. The approximate map position of a gene can be determined by comparing the pattern of appearance of transformants for the gene to that of transformants of auxotrophs that have known map positions. DNA released from germinating SB566 spores was used to transform competent SB1083 cells. The very high frequency of transformants for dnaC6 and purA16 compared to other markers suggested a close location of these two genes. Phage PBS1-mediated transduction confirmed the suggested map position. The cotransduction of dnaC6 with purA16 was measured with strain SB1083 as a recipient. The co-transduction frequencies of ts+ with purA16+ was 0.94 (680/727) and of purA16+ with ts+ was 0.98 (501/600). Since the dnaC linkage group is also closely linked to purA16 (0.96 co-transduction with purA16 [12 ]), a two-factor transformation cross was performed with strain SB1085 carrying dnaC230 as the donor and strain SB1082 carrying dnaC6 as the recipient. Both strains SB1082 and SB1085 can be transformed to ts+ under the selective conditions used (nutrient agar plates at 45 C). A reduced frequency of recombination between mutants of the same phenotype (9), as reflected in a recombination index less than 1, i.e., 3 x 10-s, indicates that dnaC230 and dnaC6 are members of the same linkage group. Growth and macromolecular synthesis at restrictive temperatures. After transfer of strains carrying dnaC6 from 30 to 43 C in CH medium, the cells elongate and divide, but form chains. Cell mass continues to increase for at least 4 h; however, the rate is reduced within 1 h after transfer to 43 C. The turbidity increases to about fourfold the initial turbidity at the time of transfer (Fig. 1). Viable cells increase by 6.4-fold in CH medium during the first 2 h at 43 C, but decrease rapidly thereafter. Cell division continues for 60 min after DNA synthesis stops at 43 C. The primary effect of the lesion is the arrest of DNA synthesis in strain dnaC6 within 30 min at
X
6
40x c
tn
C,
43c/
CL 0_
E
L 0
--
I-
0
U
oi
10l -4
-2
0
2
4
Hour5 at 43C FIG. 1. Survival of strain SB1081 in CH medium after transfer from 30 to 43 C. Turbidity in Klett units (O); colony-forming units (0) on nutrient agar incubated at 30 C.
43 C (Fig. 2) while synthesis of RNA and protein continues. The amount of DNA replicated after transfer to a restrictive temperature varies with the temperature of incubation (Fig. 3). An isogenic ts+ strain incorporates radioactive thymine equivalent to 304% of the template at 40 C and 282% at 49 C in 120 min. Upon incubation at 46 C for 45 min, DNA synthesis ceases, but resumes in less than 30 min after transfer to 30 C even in the presence of CAP (Fig. 4). Different amounts of CAP reduce the rate of DNA synthesis to different degrees; however, an amount of DNA equivalent to 25% of the labeled template was synthesized in the presence of 150 .g of CAP per ml. Pattern of DNA synthesis in vivo. The pattern of residual DNA synthesis in a strain carrying dnaC6 after transfer to 43 C was analyzed by determining the amounts of chromosome
origin (purA16) and terminus (metB)
markers replicated at 43 C compared with the amounts at 30 C. Similar amounts of origin and terminus were synthesized at 30 and 43 C (Table 2). The amounts of origin and terminus replicated were measured by density-labeling the DNA at 30 C, transferring the cells to CH medium at 43 C, and using the newly replicated DNA from the hybrid density region of a CsCl equilibrium gradient for transformation. As a control, an identical sample was incubated at 30 C rather than 43 C after the transfer to CH
ANDERSEN AND GANESAN
176
J. BACTERIOL.
medium. The resulting distribution of radioac- of synthesis at 43 C, 42% of the "C-labeled tivity in the gradient showed that during 90 min DNA was transferred to the intermediate-density stratum (Fig. 5). The pattern of biological transferred is shown in Fig. 5. The activity a biological activity patterns were compared after 0 equivalent amounts of DNA synthesis because the rate of DNA synthesis varied with tempera6 ture. The control results for transfer of biological activity were interpolated to those expected for 40% transfer of "C-labeled template DNA (Table 2). 4 UDNA synthesis ceases during the 90-min Q incubation period at 43 C. If the temperature is then lowered to 30 C, DNA synthesis begins 2) again within 20 min. The pattern of this resumed DNA synthesis at 30 C was also analyzed by pycnography in CsCl. The pattern of radio0 activity resulting from 40 min of DNA synthesis 30 C is shown in Fig. 6a. Assays for biological at lb
:c.
0 I
12
U Q L
U
4t
40 n
0
x 30 £ (j
'2o L-
4 3t
10
0
60 40 Minutes
12'0
FIG. 2. Synthesis of DNA, ribonucleic acid, and protein by strain SB1081. At 30 C, 0.5 MCi of ["CJthymine, 0.1 gCi of ["C]uracil, or 0.5 gCi of ["CJphenylalanine per ml was added to early logphase cells. After 30 min, the cultures were divided and half were transferred to 43 C. Samples (0.1 ml each) were added to cold 10%o trichloroacetic acid at intervals. (a) ["C Ithymine at 30 C (-) and 43 C (0); (b) ["C]uracil at 30 C (C) and 43 C (Q); (c) ["Cjphenylalanine at 30 C (A) and 43 C (A).
0
60 120 Minutes of incubation
FIG. 3. Percentage of DNA replicated, as a function of temperature, by strain SB1084. Cultures were uniformly labeled (1 ACi of [3HJthymine per ml) by exponential growth for at least five generations at 30 C. At 107 colony-forming units per ml, log-phase cultures with 1 ACi of [8H]thymine per ml were transferred to 30 C (0), 40 C (0), 43 C (A), 46 C (A), and 49 C (C), and 1-ml samples were precipitated by addition of 1 ml of cold 10% trichloroacetic acid. cpmO = 531 counts/min.
VOL. 121, 1975
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
160_
_
TABLE 2. Synthesis in vivo by strain SB1082 after transfer from "l NH4+ -2H20 to CH medium
i6z
% Parental label
140
trans-
Growth temp
120 30 C control
100 _
I0
< z
177
Residual synthesis at
% Biological activity in
intermediate
density
ferred to
diate den- purA16 metB gin) minus) 18 30 28
40a
52a
37a
60
72
50
75
82
54
42
45
46
41
89
30
43 C
a
~60-
Restarted synthesis at 30 C after 90 min at 43 C
40-
20
-
aData for 40% transfer of parental label was interpolated linearly from experiments with 18 and 60%
/
transfer.
thus, a second round of initiation must not have occurred. If none of the old growing points had contin0 60 120 ued when the temperature was lowered, one would not expect to see the terminus replicated Minutesat 30C FIG. 4. Effect of CAP on the resumption of DNA within 40 min. (Doubling time at 30 C in CH synthesis. Strain SB1084, prelabeled by exponential medium is 80 min.) Table 2 shows that 30% of growth for at least five generations with 1 MCi of the metB activity has been replicated compared [3Hjthymine per ml, was transferred to 46 C. After a to 37% for the control. Synthesis must resume at 45-min period at 46 C, levels of CAP from 0 to 150 many of the old growing points when the Mg/mI were added and the cultures were returned to 30 C, still in ['HJthymine (1 MCi/ml). Samples (1 ml) temperature is lowered. Effect of the thermosensitive lesion during were precipitated by addition to cold 10%o trichloroacetic acid at intervals and were filtered. Symbols: *, spore germination. There was little DNA synthesized in SB1082 spores germinated at 46 C no CAP; 0, 20 ;g of CAP per ml; A, 50Sug of CAP per ml; 100 Mg of CAPper ml;C, 150Mg of CAPper ml. (Fig. 7), even though outgrowing spores could be cpmO 8,234 counts/min. observed under a phase-contrast microscope. At 2, 3 and 4 h of growth, spores were transferred from 30 to 46 C. The sample transferred to 46 C activity indicate that both the origin and the after 2 h showed no more DNA synthesis than terminus were replicated during this period spores germinated at 46 C. In contrast, the (Fig. 6b). The control for this experiment was germinating spores transferred to 46 C after 3 h the same as for the previous experiment, began to synthesize DNA, and the sample namely, replication at 30 C after transfer from transferred at 4 h continued DNA synthesis for heavy medium to CH medium, but without the 2 h after transfer to 46 C. incubation at 43 C. For a comparable amount of Effects of inhibiting DNA synthesis at the DNA replicated, the resumed synthesis shows a restrictive temperature. The result of an exmuch larger amount of origin marker replicated periment to determine whether the capacity to than the control. As shown in Table 2, 89% of synthesize a residual amount of DNA at 46 C is the purA16 activity has been replicated com- stable or is progressively inactivated in the pared to 52% for the control. This result indi- absence of DNA synthesis in a strain carrying cates that initiation must have occurred at the dnaC6 is shown in Table 3. Both nalidixic acid chromosomal origin after the temperature was (7) and HPUra (5) specifically inhibit DNA lowered from 43 to 30 C. No purA16 activity was synthesis and were used during incubation at found in the fully light DNA density position; 46 C to inhibit residual DNA synthesis by strain 20
,,
=
178
ANDERSEN AND GANESAN
SB1084; then the inhibitors were removed and the amount of residual DNA synthesis at 46 C was measured. Table 3 shows that no further DNA replication took place at 46 C in the control culture. In contrast, cultures inhibited at 46 C retained the capacity to synthesize a residual amount of DNA even after 60 min at 46 C. Toluene-treated cells. Toluene treatment of B. subtilis renders the cells permeable to direct
J. BACTE3RIOL.
precursors of DNA synthesis. In the presence of adenosine 5'-triphosphate and the four deoxyribonucleotide triphosphates, toluene-treated cells synthesize biologically active DNA semiconservatively (13, 15). To determine whether DNA replication in a toluene-treated dnaC6 strain reflects the pattern observed in vivo, cells were assayed for DNA synthesis. The optimal time of toluene-treatment varied with the strain, e.g., strains carrying dnaC6 required
al.
l4C 200
-
100 _
Fraction number
4-
>-~~b
00
0
0
~~~~~000I
20 30 40 Top Fraction number FIG. 5. Pattern of DNA synthesized at 43 C after transfer from 15NH4+-2H20 medium. Strain SB1082 was prelabeled by growth at 30 C in 15NH4+-2H2O medium containing 0.5 tCi of [4C]thymine per ml. Log-phase cells (7 x 109) were filtered and suspended in CH medium containing 5 pCi of [3H]thymine per ml and 250 pg of deoxyadenosine per ml. After 90 min at 43 C, cells were centrifuged at 4 C, lysed, and mixed with CsCI, 0.01 M tris(hydroxymethyl)aminomethane (pH 8.0), and 2 pliters of light marker SB920 DNA to a refractive index of 0
10
1.4005 and a volume of 8 ml. Gradient tubes were centrifuged in a Spinco 50 rotor at 35,000 rpm at 4 C. Samples (0.133 ml) were collected in 60 fractions, and 50-pgliter samples were precipitated with cold 10% trichloroacetic acid. Position of the light marker DNA (arrow) was determined by transformation of competent SB5 cells. (a) Distribution of radioactivity on a CsCI gradient: 14C counts/min (0), 3H counts/min (A). (b) Biological activity was measured by transformation of 0.3% competent SB920 cells to purA+ (0) and metB+ (O)).
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
VOL. 121, 1975
only 4 min at 25 C and the activity decreased upon further treatment. The addition of 20% sucrose to the assay mixture stabilized DNA replication in toluene-treated cells at 43 C both in strain SB1082 (dnaC6) and strain SB1058 (ts+). Figure 8 shows the kinetics of DNA synthesis by toluene-treated cells. Addition of adenosine 5'-triphosphate stimulated incorporation 8- to 10-fold. At an assay temperature of 30 C, the ts+ strain and the mutant synthesized DNA for at least 60 min (Fig. 8a and b). When assayed at 43 C, the ts+ strain synthesized DNA for 40 min, whereas the dnaC6 strain stopped synthe-
179
sis after 20 min. dnaC6 cells incorporated only one-third as much 8H as normal cells before synthesis ceased at 43 C. No synthesis was observed if dnaC6 cells were incubated at 43 C before toluene treatment (Fig. 8c). DNA isolated in the experiments shown in Fig. 8b was analyzed by CsCl pycnography to compare the DNA synthesized in dnaC6 cells at 30 and 43 C. Figure 9 shows the pattern of radioactivity after DNA replication in toluenetreated cells carrying dnaC6 in the presence of a mixture [3H]TTP and BrdUrdTP in the ratio 1:20. Under these conditions, the hybrid molecules have a density of 1.740 g/ml. The parental
al. 400
200
0 10
20
30
Frac
b. > 20 4-
*'~~~~Il' 10
1
30 40 Fraction number FIG. 6. Pattern of DNA synthesized at 30 C after a 90-min incubation at 43 C. Log-phase SB1082 cells were prelabeled by growth at 30 C in 15NH4+-'H,O medium containing 0.5 uCi of [14CJthymine per ml. The tempera-
ture of incubation was raised from 30 to 43 C for 90 min. Cells (7 x 109) were transferred from "NH4+-2H2O to CH medium and from ["4C Jthymine to ['H]thymine when the temperature was lowered from 43 to 30 C. Labeling, lysis, and assays were performed as described in the legend to Fig. 5. (a) Distribution of radioactivity on a CsCI gradient: 14C counts/min (-), 'H counts/min (A). (b) Biological activity: purA16+ (0), metB+ (O9).
180
ANDERSEN AND GANESAN
J. BACTFJOL.
Q
:c ._
r)
I
6 4 Hours of outgrowth FIG. 7. DNA synthesis during spore germination of strain SB1082. Purified spores (4 x 107/ml) were added to 5 uCi of [3H]thymine per ml (17.5 Ci/mmol). Germinating spores were transferred from 30 to 46 C after 2, 3, and 4 h. Samples (0.1 ml) were precipitated by addition to cold 10%o trichloroacetic acid, filtered and counted. DNA synthesis was determined at 30 C (-) and 46 C (0) and transfer from 30 to 46 C at 2 h (O)), 3 h (A), and 4 h
(A). ration of radioactive label into DNA at assay temperatures of 43 and 30 C gives an overestimate of semiconservative DNA synthesis. A larger proportion of the radioactivity incorpoDNA replicateda (%) Time of inhibition Nalidixic rated by toluene-treated dnaC6 cells appears to Control" HPUrad (min) acidc be nonconservative synthesis at 43 C than at 30 C (Fig. 9). 14.2 16.4 -1.2 0 Biologically active DNA molecules synthe22.3 25.9 15 sized at 30 C occupy both hybrid- and inter17.9 20.8 30 mediate-density strata and might reflect semi31.9 13.3 45 conservative synthesis (13) in toluene-treated 22.2 60 cells. Strain SB1082 (dnaC6) transferred only a Within 30 min at 46 C after removal of the 2.3% of the purA16+ activity to a denser posiinhibitor. tion after synthesis at 43 C, compared with 15% b 100% = 4,879 counts/min. after synthesis at 30 C. Thus, DNA synthesis is C 100% = 13,194 counts/min. temperature sensitive in cells carrying dnaC6 d 100% = 4,300 counts/min. both in vivo and after toluene treatment. DNA cosediments with unlabeled, light stanDISCUSSION dard DNA (Fig. 9A and B). After DNA synthesis at 30 C, the 3H-labeled molecules that sediThe above results indicate that the dnaC6 mented at a heavier density than parental DNA locus lies near the origin of the B. subtilis were distributed bimodally. The molecules chromosome and is closely linked to the purA16 found between hybrid (1.740 g/ml) and light gene. In this and another dnaC-group mutant parental DNA (1.703 g/ml) have a density of (2), ribonucleic acid and protein synthesis con1.718 g/ml and might represent density transi- tinues after the arrest of DNA synthesis. Strains tion points. A. comparison of the gross incorpo- carrying dnaC6 or dnaC230 are capable of
TABLE 3. Capacity for DNA synthesis after inhibition at the restrictive temperature in strain SB1084
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
VOL. 121, 1975 ISO
100
b
200
co/ ~0
150
100 _
50
50
C. 0
0
20 60 40 Minutes of assay
FIG. 8. Kinetics of DNA synthesis by toluenetreated cells. Strains SB1082 and SB1058 (the isogenic ts+ strain) were toluenized and assayed in the presence of adenosine 5'-triphosphate, [3H]thymidine 5'-triphosphate (130 counts/min per pmol), and bromodeoxyuridine triphosphate. Incorporation into cold acid-insoluble material by toluene-treated cells was measured for: (a) strain SB1058 grown at 30 C, toluene-treated and assayed at 30 C (0) and 43 C (U); (b) strain SB1082 grown at 30 C, toluene-treated, and assayed at 30 C (A) and 43 C (A); (c) strain SB1082 toluene-treated after 45 min of growth at 43 C and assayed at 30 C (0) and 43 C (0).
beginning DNA synthesis in the presence of CAP after the incubation temperature is lowered from 43 to 30 C. The rate of this synthesis is depressed by CAP, indicating that synthesis of proteins required to achieve a normal rate of DNA synthesis was reduced. This might result from either fewer replication forks synthesizing
181
at the normal rate or from all the replication forks synthesizing at a slower rate. Nevertheless, the fact that some synthesis is observed in the presence of 150 ,ug of CAP per ml argues that either protein synthesis is not required to begin DNA synthesis, or that CAP-resistant protein synthesis can supply the necessary proteins. Viable cell number continued to increase for 60 min after DNA synthesis stopped at 43 C in a dnaC6 strain, suggesting that the process of cell division in this mutant is not coupled directly to DNA chain elongation as in a few known cases (11, 14). Furthermore, the cellular components required for cell division might either be preformed in the cell before the temperature step or their synthesis might proceed normally at 43 C at least for a while. We have confirmed and extended the hypothesis that dnaC mutants are not defective in initiation of new rounds of DNA synthesis (2). Evidence comes from the CsCl gradient analysis, which suggests that during residual DNA synthesis at 43 C the amount of origin marker replicated is similar to the amount replicated at 30 C (Table 2). The majority of initiations continue to occur at 43 C as in other temperature-sensitive dna mutants (19). In contrast, a DNA initiation mutant carrying dna-1 could only transfer 11.7% of purA16 activity to hybrid density when 61% of the template DNA was replicated at 45 C. The dnaC6 cultures also replicated significant amounts (30%) of terminus marker after resuming synthesis at 30 C, in contrast to the initiation mutant carrying dna-1 which transferred only 10.8% of terminus marker activity after 40 min of resumed synthesis (19). Strains carrying dnaC6 initiate a new round of replication in addition to continuing the previous round when the temperature is lowered from 43 to 30 C. This is typical whenever DNA synthesis is inhibited in cells while protein synthesis continues, as in dnaB mutants of Escherichia coli (17). When spores carrying dnaC6 are germinated at 46 C, little synthesis can be detected. These results are similar to B. subtilis 168ts-134, a mutant possibly affected in the initiation of new rounds of DNA replication (14), which was later mapped as a dnaB mutant (12). Since neither mutant begins DNA synthesis when the spores are germinated at 46 C, this result cannot be used to distinguish between an initiator mutant and one that is defective in a component required to sustain DNA synthesis. There are other explanations for the relatively slow cessation of DNA synthesis when the temperature is raised. Here we consider two
182
ANDERSEN AND GANESAN
J. BACTMOL.
36
~30
U
24
-J '(
18
N 0
a.x
° 12
C)
0
co
(E
at 6
a
pLr
1 0
5
10
15
20
25
FRACTION NUMBER
30
0 35 TOP
5
10
15
20
25
FRACTION NUMBER
30
35
TOP
FIG. 9. Products of synthesis in toluene-treated SB1082 (dnaC6) cells assayed at 30 and 43 C. Products of the reactions shown in Fig. 8b were analyzed by CsCl density gradients as described in the legend to Fig. 5. DNA synthesized in toluene-treated cells was labeled with bromodeoxyuridine triphosphate and ['HJthymidine 5'-triphosphate (130 counts/min per pmol). Biological activity of SB920 unlabeled marker DNA was determined by transformation of competent SB5 cells to trpC+ (0). (A) Distribution of radioactivity and biological activity on a CsCI equilibrium gradient after synthesis at 30 C: 'H counts/min (A), purA16+ (0). (B) Distribution of radioactivity and biological activity on a CsCI gradient after synthesis at 43 C: 'H counts/min (A), purA16+
(0). possibilities: first, that the thermosensitive component decays slowly at 46 C and, second, that a component required for DNA synthesis is consumed and not resynthesized at 46 C. Inhibition of DNA synthesis with nalidixic acid or HPUra showed that the capacity to synthesize DNA was stable at 46 C. These results are not consistent with the first possibility, unless a factor required for replication is stabilized somehow in the absence of DNA replication. A simpler explanation for the behavior would be that the factor is depleted only during DNA synthesis. The capacity for DNA synthesis is destroyed within 15 min at 45 C if thymine starvation is used as the DNA synthesis inhibitor with a strain carrying dnaC230 (2). Opposite results obtained here using HPUra or nalidixic acid as the inhibitor of DNA synthesis raised the possibility that secondary effects of inhibition may influence the results. HPUra has almost no secondary effects in 60 min at 75 ug/ml (6). Effects of higher concentrations have not been reported. A 25-,ug amount of nalidixic acid per ml affects protein synthesis and cell viability within 60 min (7). Thymine starvation decreases cell viability within 30 min and might induce latent phage. Thymine starvation, nalidixic acid, and HPUra all cause limited DNA
degradation (6). Part of the incorporation at 46 C after the removal of inhibitors might represent repair synthesis. A toluene-treated dnaC6 strain and its isogenic ts+ strain showed that their pattern of DNA synthesis is similar to that of untreated viable cells. dnaC6 cells, which shut off DNA synthesis at 43 C in vivo, cannot resume DNA synthesis in toluene-treated cells. Pycnographic analysis of DNA synthesized in these cells at 43 C indicates a marked reduction of DNA synthesis. Since DNA replication is also temperature sensitive in toluene-treated cells, the defect is probably not in the synthesis of precursors or cofactors like adenosine 5'-triphosphate. Whether the thermosensitivity of the toluenetreated dnaC6 cells is due to the same component as in vivo or whether it is the result of a secondary effect (e.g., a membrane defect that makes the toluene-treated cells more fragile and, thus, more thermosensitive) has not been
determined. The nature of the ts lesion in strains carrying dnaC6 seems to be in the production of a required factor that is consumed during DNA synthesis. When the temperature is raised, the pool of factor is depleted only if DNA synthesis is proceeding. Lowering the temperature reactivates factor production and DNA synthesis
VOL. 121, 1975
proceeds
even
DNA REPLICATION IN A B. SUBTILIS dnaC MUTANT
in the absence of protein synthe-
sis.
ACKNOWLEDGMENTS We wish to thank Ann Ganesan and P. C. Cooper for critical readings of the manuscript. JoAnn Katheiser provided excellent technical assistance. This work was aided by Public Health Service grants GM-14108 and 2 T01-GM 295 from the National Institute of General Medical Sciences and by grant GB 8739 from the National Science Foundation. A. T. Ganesan is a recipient of Public Health Service Research Career Program Award GM-50199 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bazill, G. W. 1972. Temperature-sensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis. Mol. Gen. Genet. 117:19-29. 2. Bazill, G., and Y. Retief. 1969. Temperature-sensitive DNA synthesis in a mutant of Bacillus subtilis. J. Gen. Microbiol. 56:87-97. 3. Bonhoeffer, R., and H. Schaller. 1965. A method for selective enrichment of mutants based on the high UV sensitivity of DNA containing 5-Bromouracil. Biochem. Biophys. Res. Commun. 20:93-97. 4. Borenstein, S., and E. Ephrati-Elizur. 1969. Spontaneous release of DNA in sequential genetic order by Bacillus subtilis. J. Mol. Biol. 45:137-152. 5. Brown, N. C. 1970. 6-(p-Hydroxyphenylazo)-uracil: a selective inhibitor of host DNA replication in phageinfected Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 67:1454-1461. 6. Brown, N. C. 1971. Inhibition of bacterial DNA replication by 6-(p-hydroxyphenylazo)-uracil: differential effect on repair and semiconservative synthesis in Bacillus subtilis. J. Mol. Biol. 59:1-16. 7. Cook, T. M., K. G. Brown, J. V. Boyle, and W. A. Goss. 1966. Bactericidal action of nalidixic acid on Bacillus subtilis. J. Bacteriol. 92:1510-1514. 8. Donellan, J., E. Nags, and H. Levinson. 1964. Chemically
9. 10. 11.
12.
13. 14.
15. 16. 17. 18. 19.
20.
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defined, synthetic media for sporulation and for germination and growth of Bacillus subtilis. J. Bacteriol. 87:332-336. Dubnau, D., C. Goldthwaite, I. Smith, and J. Marmur. 1967. Genetic mapping in Bacillus subtilis. J. Mol. Biol. 27:163-185. Ganesan, A. T. 1968. Studies on the in vitro synthesis of transforming DNA. Proc. Nat. Acad. Sci. U.S.A.61:1058-1065. Gross, J. D., D. Karamata, and P. Hempstead. 1968. Temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Cold Spring Harbor Symp. Quant. Biol. 33:307-312. Karamata, D., and J. Gross, 1970. Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol. Gen. Genet. 108:277-287. Matsushita, T., K. White, and N. Sueoka. 1971. Chromosome replication in toluenized Bacillus subtilis cells. Nature N. Biol. 232:111-114. Mendelson, N., and J. Gross. 1967. Characterization of a temperature-sensitive mutant of Bacillus subtilis defective in deoxyribonucleic acid replication. J. Bacteriol. 94:1603-1608. Moses, R., and C. Richardson. 1970. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc. Nat. Acad. Sci. U.S.A. 67:674-681. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078. Stein, G., and P. Hanawalt. 1969. Initiation of DNA replication cycles of Escherichia coli following DNA synthesis inhibition. J. Mol. Biol. 46:135-144. Stewart, C. 1969. Physical heterogeneity among Bacillus subtilis deoxyribonucleic acid molecules carrying particular genetic markers. J. Bacteriol. 98:1239-1247. White, K., and N. Sueoka. 1973. Temperature-sensitive DNA synthesis mutants of Bacillus subtilis. Genetics 73:185-214. Young, F. E., C. Smith, and B. E. Reilly. 1969. Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. J. Bacteriol. 98:1087-1097.
