JouRNAL OF BACTURIOLOGY, June 1977, p. 1030-1037 Copyright C) 1977 American Society for Microbiology
Vol. 130, No. 3 Printed in U.S.A.
Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB (Ts)134 Mutation HEATHER CALLISTER, SUE LE MESURIER, AND R. G. WAKE* Biochemistry Department, University of Sydney, Sydney, N.S.W., Australia
Received for publication 14 March 1977
The nature of the deoxyribonucleic acid synthesis reported by others to occur at 450C in germinating spores ofthe temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 450C. At a slightly higher temperature, 490C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts+ strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 450C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions. The Bacillus subtilis spore contains only completed chromosomes (3, 15). Upon germination, specialized transcription of the chromosome is initiated, and this is soon followed by translation (8). Only after a period of 60 to 120 min does the first round of deoxyribonucleic acid (DNA) replication commence (8, 19), the order of gene replication being the same as in vegetative rounds (15). Various mutants of B. subtilis temperaturesensitive for initiation of vegetative rounds of replication have been described (6, 7, 9, 12, 13, 16, 18). The earliest one described, TsB134, a derivative of strain 168, has been used in many types of study. Initiation of new rounds of replication are very effectively blocked upon transfer of TsB134 to 450C, and existing rounds terminate (2). Although TsB134 has been classified as a dnaB group mutant, it is not known how many cistrons comprise this group (7). When spores of TsB134 are germinated at the temperature restrictive for initiation of vegetative rounds of replication (450C), some DNA synthesis occurs (11). The nature of this DNA synthesis has not been clarified. If it results from the initiation of a normal round of replica-
tion, the possibility that the first round of replication during spore outgrowth is unique, in that its initiation is determined prior to germination, is raised (N. H. Mendelson, private communication). In other words, the action of the dnaB134 gene product might not be needed during the time between the start of germination and initiation of the first round. The first part of this paper describes the results of an investigation into the nature of the DNA synthesis occurring in spores of the original TsB134 strain germinating at 45°0. It will be shown that a normal round of replication can occur in some of the germinating spores under such conditions. In the second part, the dependence of initiation of this first round of replication on the action of the dnaB134 gene product is specifically investigated. MATERIALS AND METHODS Bacterial strains and spore preparations. The B. subtilis strains used and their origin were as follows: 168 trpC2 dnaB (Ts)134 thyA thyB (Mendelson, referred to as TsB134), 168 trpC2 phe thyA thyB (Ganesan, referred to as TTP), and 168 purA16 leuA8 metB5 ilvAl (Marmur, referred to as BD72). The
1030
GERMINATING SPORES
VOL. 130, 1977 isogenic strains SU43 and SU45 were obtained by transformation of strain TTP with DNA from TsB134. They have the genotypes 168 trpC2 dnaB(Ts)134 thyA thyB and 168 trpC2 thyA thyB, respectively. Spores were prepared as described previously (3). Spore germination and incorporation of [3H]thymine. Spores were germinated at a concentration of 2 x 108/ml. For the studies with TsB134, the enriched minimal medium of Mendelson and Gross (12) was used. Spores were transferred immediately to medium at the final temperature. Either thymine (10 ,ug/ml) or a mixture of 5-bromouracil (25 ,Lg/ml) plus thymine (2.5 ,ug/ml) was present. When [methyl-3H]thymine (Radiochemical Centre, Amersham, England) was used, in both the absence and presence of 5-bromouracil, it was present at a specific activity of 2 to 4 mCi/mg. For studies with the isogenic SU45(Ts+) and SU43(Ts) strains, spores were germinated at 340C in the Spizizen glucose-salts medium (1) supplemented with Casamino Acids (0.05%), L-asparagine (0.3%), L-glutamic acid (0.3%), and thymine (30 ,ug/ml). After 45 min, L-tryptophan (50 ,ug/ml) was added and the mixture was shifted to the desired temperature. The time of tryptophan addition was recorded as zero time. For density labeling, a mixture of 5bromouracil (25 ,ug/ml) plus thymine (2.5 ,ug/ml) was used in place of thymine alone. [3H]thymine, in the absence of 5-bromouracil, was used at a specific activity of 2 mCi/mg; when 5-bromouracil was present, it was increased to 6 mCi/mg. For measurements of incorporation of radioactivity during germination, 0.1-ml portions were pipetted into 1.0 ml of ice-cold 10% trichloroacetic acid containing thymine (0.5 mg/ml). After the addition of calf thymus DNA (0.05 ml, 1 mg/ml), the mixtures were filtered after 30 min on glass fiber disks (Whatman GF/C), washed, and prepared for counting as described previously (17). Extraction of DNA, analytical and preparative density gradient equilibrium, and transformation. Details of these procedures have been described in previous publications (3, 4, 16, 17).
RESULTS Extent of replication of DNA in spores of TsB134 germinating at 450C. The DNA synthesis reported to occur in spores of TsB134 germinating at 45°C (11) could have resulted from replication and/or repair of DNA. A direct measure of the extent of replication can be obtained by analytical density gradient sedimentation of suitably prepared extracts after the incorporation of a mixture of 5-bromouracil plus thymine in place of thymine alone. In the original experiments, synthesis of DNA was detected after as little as 90 min of incubation at 450C. Table 1 shows the results of experiments in which germination of TsB134 spores in the presence of 5bromouracil was allowed to proceed for 120 to 240 min at 450C in the same medium used previously. From a total of eight experiments, six
1031
TABLE 1. Extent of DNA replication in spores of TsB134 germinating in medium containing 5bromouracil plus thymine at various temperatures Replication (%)a Round 1 Round 2
Expt
Temp (OC)
Time (min (mi)
1 2 3 4 5 6
45 45 45 45 45 45
7
45
240 240 120 120 240 120 240 120
0 0 22 41 58 13 23 29
240
46
0
240 240 240 240
82 40 67 0
0 0 8 0
8
9
43 45 43 47
0 0 0 0 0 0 0
0
a The overall extents of first- and second-round replication were obtained from the relative amounts of unreplicated (LL), light-heavy (LH), and heavyheavy (HH) density species identified by analytical sedimentation equilibrium in CsCl or Cs2SO4. See reference 17 for details of the calculation.
showed significant amounts of replicated (LH) material, corresponding to 23 to 58% of a round of replication after 240 min. Replication at 450C was observed with three different preparations of spores, but there was variability in the extent of replication between experiments using the same spore preparation (experiments 1, 2, 7, and 8). There was no significant reversion to temperature insensitivity in the spore preparations. It is noteworthy that at 45°C the replicated material is exclusively first-round product. Table 1 also shows that at 430C (two experiments) there was a greater extent of first-round replication (67 to 82%), and it proceeded into the second round to a slight extent (8%) by 240 min in one case. A single experiment at 470C showed no significant replication after 240 min. It is important to point out that initiation of germination of B. subtilis spores is inhibited at high temperatures (14). This was confirmed with the TsB134 spores used here. Thus, after 60 min at 300C, germination (phase darkening) had occurred in one preparation to an extent of >95%, but at 43 and 450C this was reduced to 90 and 40%, respectively. In the last case, 40% of the spores were still phase-bright after 200 min, the remainder having grown out into rods. On the assumption that ungerminated spores are resistant to the extraction procedure, it would follow that the extents of replication shown in Table 1 would relate to the fraction of the population that had germinated. Nature of the replication and extent of re-
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J. BACTICRIOL.
CALLISTER, LE MESURIER, AND WAKE
pair of DNA in spores of TsB134 germinating at 450C. Additional experiments to those described earlier (see reference 3) have confirmed that the chromosomes in TsB134 spores are completed in their replication (H. Callister and R. G. Wake, unpublished data). The results just presented show that significant amounts of replication can occur when TsB134 spores are germinated at 450C. Does this replication result from initiation at the chromosome origin, and is it accompanied by repair of DNA? To answer the first part of this question, germination was allowed to proceed for 80 min at 450C in the presence of a mixture of 5-bromouracil plus thymine. Analytical density gradient analysis showed first-round replication to have proceeded to an average extent of 13%. After preparative density gradient fractionation, 29% of the purAl6 and 80%; at 45 and 490C this was reduced to
Vol. 130, No. 3 Printed in U.S.A.
Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB (Ts)134 Mutation HEATHER CALLISTER, SUE LE MESURIER, AND R. G. WAKE* Biochemistry Department, University of Sydney, Sydney, N.S.W., Australia
Received for publication 14 March 1977
The nature of the deoxyribonucleic acid synthesis reported by others to occur at 450C in germinating spores ofthe temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 450C. At a slightly higher temperature, 490C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts+ strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 450C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions. The Bacillus subtilis spore contains only completed chromosomes (3, 15). Upon germination, specialized transcription of the chromosome is initiated, and this is soon followed by translation (8). Only after a period of 60 to 120 min does the first round of deoxyribonucleic acid (DNA) replication commence (8, 19), the order of gene replication being the same as in vegetative rounds (15). Various mutants of B. subtilis temperaturesensitive for initiation of vegetative rounds of replication have been described (6, 7, 9, 12, 13, 16, 18). The earliest one described, TsB134, a derivative of strain 168, has been used in many types of study. Initiation of new rounds of replication are very effectively blocked upon transfer of TsB134 to 450C, and existing rounds terminate (2). Although TsB134 has been classified as a dnaB group mutant, it is not known how many cistrons comprise this group (7). When spores of TsB134 are germinated at the temperature restrictive for initiation of vegetative rounds of replication (450C), some DNA synthesis occurs (11). The nature of this DNA synthesis has not been clarified. If it results from the initiation of a normal round of replica-
tion, the possibility that the first round of replication during spore outgrowth is unique, in that its initiation is determined prior to germination, is raised (N. H. Mendelson, private communication). In other words, the action of the dnaB134 gene product might not be needed during the time between the start of germination and initiation of the first round. The first part of this paper describes the results of an investigation into the nature of the DNA synthesis occurring in spores of the original TsB134 strain germinating at 45°0. It will be shown that a normal round of replication can occur in some of the germinating spores under such conditions. In the second part, the dependence of initiation of this first round of replication on the action of the dnaB134 gene product is specifically investigated. MATERIALS AND METHODS Bacterial strains and spore preparations. The B. subtilis strains used and their origin were as follows: 168 trpC2 dnaB (Ts)134 thyA thyB (Mendelson, referred to as TsB134), 168 trpC2 phe thyA thyB (Ganesan, referred to as TTP), and 168 purA16 leuA8 metB5 ilvAl (Marmur, referred to as BD72). The
1030
GERMINATING SPORES
VOL. 130, 1977 isogenic strains SU43 and SU45 were obtained by transformation of strain TTP with DNA from TsB134. They have the genotypes 168 trpC2 dnaB(Ts)134 thyA thyB and 168 trpC2 thyA thyB, respectively. Spores were prepared as described previously (3). Spore germination and incorporation of [3H]thymine. Spores were germinated at a concentration of 2 x 108/ml. For the studies with TsB134, the enriched minimal medium of Mendelson and Gross (12) was used. Spores were transferred immediately to medium at the final temperature. Either thymine (10 ,ug/ml) or a mixture of 5-bromouracil (25 ,Lg/ml) plus thymine (2.5 ,ug/ml) was present. When [methyl-3H]thymine (Radiochemical Centre, Amersham, England) was used, in both the absence and presence of 5-bromouracil, it was present at a specific activity of 2 to 4 mCi/mg. For studies with the isogenic SU45(Ts+) and SU43(Ts) strains, spores were germinated at 340C in the Spizizen glucose-salts medium (1) supplemented with Casamino Acids (0.05%), L-asparagine (0.3%), L-glutamic acid (0.3%), and thymine (30 ,ug/ml). After 45 min, L-tryptophan (50 ,ug/ml) was added and the mixture was shifted to the desired temperature. The time of tryptophan addition was recorded as zero time. For density labeling, a mixture of 5bromouracil (25 ,ug/ml) plus thymine (2.5 ,ug/ml) was used in place of thymine alone. [3H]thymine, in the absence of 5-bromouracil, was used at a specific activity of 2 mCi/mg; when 5-bromouracil was present, it was increased to 6 mCi/mg. For measurements of incorporation of radioactivity during germination, 0.1-ml portions were pipetted into 1.0 ml of ice-cold 10% trichloroacetic acid containing thymine (0.5 mg/ml). After the addition of calf thymus DNA (0.05 ml, 1 mg/ml), the mixtures were filtered after 30 min on glass fiber disks (Whatman GF/C), washed, and prepared for counting as described previously (17). Extraction of DNA, analytical and preparative density gradient equilibrium, and transformation. Details of these procedures have been described in previous publications (3, 4, 16, 17).
RESULTS Extent of replication of DNA in spores of TsB134 germinating at 450C. The DNA synthesis reported to occur in spores of TsB134 germinating at 45°C (11) could have resulted from replication and/or repair of DNA. A direct measure of the extent of replication can be obtained by analytical density gradient sedimentation of suitably prepared extracts after the incorporation of a mixture of 5-bromouracil plus thymine in place of thymine alone. In the original experiments, synthesis of DNA was detected after as little as 90 min of incubation at 450C. Table 1 shows the results of experiments in which germination of TsB134 spores in the presence of 5bromouracil was allowed to proceed for 120 to 240 min at 450C in the same medium used previously. From a total of eight experiments, six
1031
TABLE 1. Extent of DNA replication in spores of TsB134 germinating in medium containing 5bromouracil plus thymine at various temperatures Replication (%)a Round 1 Round 2
Expt
Temp (OC)
Time (min (mi)
1 2 3 4 5 6
45 45 45 45 45 45
7
45
240 240 120 120 240 120 240 120
0 0 22 41 58 13 23 29
240
46
0
240 240 240 240
82 40 67 0
0 0 8 0
8
9
43 45 43 47
0 0 0 0 0 0 0
0
a The overall extents of first- and second-round replication were obtained from the relative amounts of unreplicated (LL), light-heavy (LH), and heavyheavy (HH) density species identified by analytical sedimentation equilibrium in CsCl or Cs2SO4. See reference 17 for details of the calculation.
showed significant amounts of replicated (LH) material, corresponding to 23 to 58% of a round of replication after 240 min. Replication at 450C was observed with three different preparations of spores, but there was variability in the extent of replication between experiments using the same spore preparation (experiments 1, 2, 7, and 8). There was no significant reversion to temperature insensitivity in the spore preparations. It is noteworthy that at 45°C the replicated material is exclusively first-round product. Table 1 also shows that at 430C (two experiments) there was a greater extent of first-round replication (67 to 82%), and it proceeded into the second round to a slight extent (8%) by 240 min in one case. A single experiment at 470C showed no significant replication after 240 min. It is important to point out that initiation of germination of B. subtilis spores is inhibited at high temperatures (14). This was confirmed with the TsB134 spores used here. Thus, after 60 min at 300C, germination (phase darkening) had occurred in one preparation to an extent of >95%, but at 43 and 450C this was reduced to 90 and 40%, respectively. In the last case, 40% of the spores were still phase-bright after 200 min, the remainder having grown out into rods. On the assumption that ungerminated spores are resistant to the extraction procedure, it would follow that the extents of replication shown in Table 1 would relate to the fraction of the population that had germinated. Nature of the replication and extent of re-
1032
J. BACTICRIOL.
CALLISTER, LE MESURIER, AND WAKE
pair of DNA in spores of TsB134 germinating at 450C. Additional experiments to those described earlier (see reference 3) have confirmed that the chromosomes in TsB134 spores are completed in their replication (H. Callister and R. G. Wake, unpublished data). The results just presented show that significant amounts of replication can occur when TsB134 spores are germinated at 450C. Does this replication result from initiation at the chromosome origin, and is it accompanied by repair of DNA? To answer the first part of this question, germination was allowed to proceed for 80 min at 450C in the presence of a mixture of 5-bromouracil plus thymine. Analytical density gradient analysis showed first-round replication to have proceeded to an average extent of 13%. After preparative density gradient fractionation, 29% of the purAl6 and 80%; at 45 and 490C this was reduced to
