JOURNAL OF BACTERIOLOGY, June 1973, p. 1099-1107Copyright 0 1973 American Society for Microbiology

Vol. 114, No. 3Printed in U.S.A.

Deoxyribonucleic Acid Synthesis andDeoxynucleotide Metabolism During Bacterial

Spore GerminationPETER SETLOW'

The Department of Biochemistry, Stanford University School ofMedicine, Stanford, California 94305, and theDepartment of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032

Received for publication 12 March 1973

Deoxyribonucleic acid (DNA) synthesis during germination of Bacillusmegaterium spores takes place in two stages. In stage I (0-56 min) DNA synthesisis slow and there is no detectable net synthesis, whereas in stage II (from 65 minon) the rate of synthesis is much faster and net DNA synthesis occurs.Deoxyribonucleotide pool sizes match the rates of DNA synthesis in stages I andII. The level of deoxyribonucleotide triphosphates is not correlated with the levelof deoxyribonucleotide kinases, but rather with that of ribonucleotide reductaseactivity.

Spore germination in Bacillus is an attractivesystem for studying control of deoxyribonucleicacid (DNA) replication since initiation of chro-mosome replication is quite synchronous (31,32). Although ribonucleic acid (RNA) and pro-tein synthesis begin within the first minutes ofspore germination, DNA replication, and in onecase all DNA synthesis, does not begin for 1 to 2h (23, 26, 29). Since the initiation of DNAreplication is sensitive to chloramphenicol (18,31), it was suggested that the synthesis ofproteins such as initiators, membrane proteins,or polymerases turn on chromosome replicationand net DNA synthesis. However, attentionmust also be given to the capacity of thegerminating spore to produce the deoxyribonu-cleotides essential for net DNA synthesis. Theinability to synthesize ribonucleotides early ingermination due to the absence of nucleotidebiosynthetic enzymes (23) suggests that deox-yribonucleotide biosynthesis may also be im-paired.We have found that DNA synthesis during

germination (see Fig. 8) is correlated with thedeoxynucleotide pool size, and this in turn withthe level of ribonucleotide reductase.

MATERIALS AND METHODSNucleotides and nucleosides. Unlabeled ribo- and

deoxyribonucleotides were purchased from P-L Bio-chemicals. Unlabeled deoxyribonucleosides were ob-tained from Calbiochem, and adenosine and uridine

' Present address: Department of Biochemistry, Univer-sity of Connecticut Health Center, Farmington, Conn. 06032.

were from Sigma Chemical Co. [Methyl-'HJdeoxy-thymidine monophosphate (3H-dTMP), a-32P-deoxy-thymidine triphosphate (32P-dTTP), [5-'H]deox-ycytidine, [5-3H]cytidine monophosphate (3H-CMP),[8-8HJdeoxyguanosine, and [8-_HJdeoxyadenosinewere obtained from Schwarz BioResearch; and[methyl-1HJdeoxythymidine ( 3H-TdR), [1-_C ]uridine("IC-UR), and [8- 4C]deoxyadenosine monophosphate("4C-dAMP) were purchased from New England Nu-clear Corp. The radiochemical purity of the nucleo-tides and nucleosides was checked by paper chroma-tography (Schleicher and Schuell, no. 589 orangeribbon) by using an isopropanol: water: ammonia sol-vent (7:1:2) (system I). The 3H-TdR was purifiedusing this chromatographic system. Failure to do soresulted in spurious results.

Other chemicals. Nalidixic acid was purchasedfrom Calbiochem. Chloramphenicol, mitomycin C,reduced triphosphopyridine nucleotide (TPNH), anddithiothreitol were purchased from Sigma ChemicalCo. Diphenylamine was the product of EastmanOrganic Chemicals and was recrystallized fromethanol. Polyethylenimine-coated, thin-layer chroma-tography sheets were purchased from Brinkmann In-struments. Actinomycin D was a gift of Merck,Sharpe and Dohme, and an alternating copolymer ofdeoxyadenylate and deoxythymidylate [poly d(AT) ]for the ligase assays was prepared as described byModrich and Lehman (14).Growth of spores and cells. All work described

here was carried out with Bacillus megaterium QMB1551, originally obtained from H. S. Levinson (U.S.Army Natick Laboratory, Natick, Mass.). Spores and32P-labeled spores were obtained from cultures grownin supplemented nutrient broth as previously de-scribed (22), lyophilized, and stored at room tempera-ture. The specific radioactivity of 32P-labeled spores

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was also determined as previously described (22).Spores containing 3H-TdR in their DNA were grownas described by Donnellan and Setlow (7) with 0.4mCi of 3H-TdR per 200 ml of supplemented nutrientbroth.Germination of spores. Unless otherwise noted the

standard conditions for spore germination were asfollows: spores (20 mg [dry wt ] per ml) were heatshocked in water for 10 min at 60 C and cooled prior toinitiation of germination. The germination mediumwas that of Spizizen (24) containing glucose, citrate,and salts, and supplemented with 0.1% casein hy-drolysate. Germination experiments were initiatedby addition of spores to 0.4 mg (dry wt) per ml, andcultures were agitated in a gyratory water bath at 30C. The rate of initiation of germination, and that ofsubsequent growth was measured by following theoptical density at 660 nm. Within 10 min of startinggermination >90% of the spores appeared dark in thephase constrast microscope.Enzyme assays. DNA polymerase (EC 2.7.7.7) was

assayed with activated calf thymus DNA as thetemplate as described by Okazaki and Kornberg (17),and DNA ligase was assayed as described by Modrichand Lehman (14). Deoxyribonucleoside kinases, de-oxyadenosine monophosphate (dAMP), and deoxy-thymidine monophosphate (dTMP) kinase (EC2.7.4.c) were assayed essentially as described by Gravand Smellie (9). The reaction mixture (0.5 ml) con-tained 100 mM tris(hydroxymethyl)aminomethane(Tris) (pH 8.0), 5 mM MgCl2, 5 mM adenosinetriphosphate (ATP), and 0.1 mM ,-mercaptoethanol.Labeled deoxyribonucleosides were present at 0.5mM; dAMP and dTMP at 0.4 mM. The assay wasstarted by addition of extract (0.1-1.0 mg of protein),and after 15 min at 37 C the reaction was halted byboiling for 5 min. After centrifugation of coagulatedmaterial a sample of the supernatant fluid (50 Mliters)was spotted on Whatman no. 1 paper together withappropriate markers. The chromatogram was devel-oped for 24 h (descending) in isobutyric acid:am-monia:0.1 M ethylenediaminetetraacetic acid(EDTA) (100:60:1.6). After drying, the marker spotswere located under ultraviolet light, cut out, andcounted. Kinase activity is given as the total nucleo-side or nucleotide phosphorylated.

Ribonucleotide reductase was assayed by a modifi-cation of the method of Bertani et al. (2). Reactionmixtures contained 50 mM Tris-hydrochloride (pH8.0), 20 mM MgCl2, 10 mM ATP, 10 mM dithio-threitol, 1.3 mM TPNH, 0.2 mM 3H-CMP, andenzyme (0.5-2.5 mg [dry wt] spores) in a volume of 50jl.iters. After 30 min at 37 C the reaction was stoppedby addition of 1 ml of 1 M HCI containing 25 nmol ofcytidine monophosphate (CMP) and deoxycytidinemonophosphate (dCMP). After centrifugation thesupernatant fluid was boiled for 15 min to hydrolyzepyrophosphate bonds and then flash evaporated andredissolved in 25 Mliters of water. A 20-,gliter amountwas analyzed by paper chromatography as describedabove by using ethanol-saturated sodium tetrabo-rate-5 M ammonium acetate-0.5 M EDTA(220:80:20:0.5 by vol) as the solvent (28). Spotscorresponding to CMP and dCMP were then cut out

and counted. The assay was linear for 30 min whenless than 4% conversion of CMP to dCMP occurred.Within the latter limitation, the assay was also linearwith spore concentration from 0.15 to 2.5 mg perassay. Initial experiments indicated that it was dif-ficult to detect enzyme activity in extracts from B.megaterium spores or cells. I therefore resorted tospores made permeable by repeated freezing andthawing as a source of enzyme (25) (see below).

Assay of RNA and DNA synthesis. RNA andDNA synthesis were measured by incorporation of"C-UR or 3H-TdR, respectively, into acid-insolublematerial. Both '4C-UR and 8H-TdR were added to 25MM and were present from the start of germination.Samples (0.4 ml) were taken at various times anddiluted fivefold with 6.7% cold trichloroacetic acid.After 30 mnin at 4 C the sanmples were filtered throughglass fiber filters, washed five times with 5 ml of 5%trichloroacetic acid containing 1 mM UR or 1 mMTdR, and then washed four times with absoluteethanol. The filters were then dried and counted in ascintillation counter.DNA synthesis was also assayed by measuring the

incorporation of 14C-UR into alkali-stable, acid-insoluble material. Spores were germinated in thepresence of "4C-UR (25 AtM), and samples werewithdrawn and diluted 1:1 with 2 M NaOH. Afterincubation in a closed tube for 18 h at 37 C thesolution was neutralized with HCI, 0.1 mg of salmonsperm DNA was added as carrier, and the solutionwas made 0.5 M in HC104. After 30 min at 4 C theprecipitate was collected by filtration, washed, andcounted as described above. DNA levels were deter-mined colorimetrically with diphenylamine using calfthymus DNA as the standard (21).

Extraction of nucleotides. Nucleotides were ex-tracted from germinating spores with cold 5% tri-chloroacetic acid. Samples of germinating spores (500Aliters) were passed through a membrane filter (Mil-lipore Corp., 0.45 um), and the filter was immediately(<5 s) placed in 5 ml of cold 5% trichloroacetic acidalong with 100 nmol of each of the eight commonnucleoside triphosphates. After 1 h at 4 C the solutionwas centrifuged and the supernatant fluid was ex-tracted five times with diethyl ether, bringing the pHup to about 5.0. The solution was then flash evapora-ted, and the residue was suspended in 100 Aliters ofwater prior to chromatography. This procedure wasshown to give good recoveries of ribonucleoside tri-phosphates (3), and control experiments in whicha-32P-dTTP or a-32P-deoxyadenosine triphosphate(32P-dATP) were added to the initial trichloroaceticacid extract also gave 80 to 90% recovery of thedeoxynucleoside triphosphates. That the trichloroa-cetic acid extraction procedure was indeed providingan accurate value for ribo- and deoxyribonucleosidetriphosphate pools was further substantiated by ex-traction of a few samples of germinating spores byusing boiling 80% n-propanol as described by Bieleski(3). Previous work has shown that both methods givesimilar values (within 10%) for ribonucleoside triphos-phate levels in germinating B. megaterium spores(22), and in this work I also found that ribo- anddeoxyribonucleoside triphosphate levels determined

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DNA SYNTHESIS AND DEOXYNUCLEOTIDE METABOLISM

using both extraction procedures gave similar results(within 12%). The trichloroacetic acid extractionprocedure was routinely used because 32P-labeledmaterial in n-propanol extracts sometimes interferedwith accurate determination of dGTP.

Nucleotides were extracted from 32P-labeled dor-mant spores using boiling 80% n-propanol (22), asoriginally described by Bieleski (3). The nucleotideswere further purified by using activated charcoal.82P-labeled spores (6 mg dry wt) were boiled for 10min in 4 ml of 80% n-propanol. The solution was flashevaporated, the residue was suspended in 1 ml ofwater containing 50 nmol of each common nucleosidemonophosphate, and after 1 h at 4 C the suspensionwas centrifuged. The supernatant fluid was made to0.33 M HCI, 0.033 M sodium pyrophosphate, and 330,ug of bovine serum albumin per ml (final volume, 3ml) and then diluted 1:1 with acid-washed Norit (20%by weight). After 20 min at 4 C the solution wascentrifuged, and the charcoal was washed three timeswith water. Nucleotides were eluted with two washesof 7 ml of alkaline (pH 11, NH4OH)-50% ethanol, andthe eluates were pooled, filtered, flash evaporated,and dissolved in 100 ILliters of water prior to chroma-tography. Recoveries of 'H-dTMP, 'IC-AMP, and32P-ATP added to the initial n-propanol extract inparallel experiments were 75%, 82%, and 80%, respec-tively.

Extraction of enzymes. Enzymes were extractedfrom germinated spores by using lysozyme as previ-ously described (23), except that KCN was omitted,and phenylmethylsulfonyl fluoride was present at10-4 M to prevent proteolysis. The enzyme comple-ment of dormant spores was determined by germinat-ing spores for 20 min under standard conditions, butin the presence of KCN (10 mM) and KF (10 mM) toblock protein synthesis (22). These spores were har-vested by centrifugation and lysed. All extracts werenot centrifuged, but were vortexed vigorously toreduce their viscosity.

Ribonucleotide reductase was assayed in wholespores made permeable by the freeze-thaw techniqueof Steinberg and Halvorson (25). The germinatedspores were centrifuged and frozen (this took about 3min), and just prior to assays the frozen pellets weresuspended at 75 mg/ml in 50 mM KPO4 (pH 7.4)containing 100 Mg of chloramphenicol per ml andfrozen and thawed five times.

Chromatography. Nucleotides were separated bytwo-dimensional, thin-layer chromatography onpolyethylenimine-impregnated plastic sheets (Brink-mann Instruments). Ribo- and deoxyribonucleosidemonophosphates were separated with the system ofRanderath and Randerath (20) (system II), and theribo- and deoxyribonucleoside triphosphates wereseparated with the system of Neuhard (15) as modi-fied by Colby and Edlin (6) (system III). The latterseparation was improved if the plate was prerun to 12cm above the origin in methanol:water (1:2) afterspotting the sample. This prerun was perpendicular tothe direction of the first solvent. Nucleoside mono-,di-, and tri-phosphates were separated by a secondsystem described by Randerath and Randerath (19)(system IV). Chromatography markers were identi-

fled under ultraviolet light, and radioactive com-pounds were detected by autoradiography. Radioac-tive spots were then cut out and counted in a gas-flowcounter. A sample of the growth medium or germina-tion medium was counted at the same time todetermine the specific radioactivity of the phosphate.

RESULTSDNA level during spore germination. Dur-

ing germination of Bacillus spores there is noincrease in DNA content for 1 to 3 h (26, 30).Our results with B. megaterium also show nonet increase in DNA content until about 60 minafter the start of germination (Fig. 1). At thistime the DNA content increased rapidly, dou-bling by 120 niin. The first cell division tookplace between 155 and 180 min (data notshown).DNA synthesis during spore germination.

DNA synthesis followed by incorporation oflabeled UR showed a pattern similar to theDNA content (Fig. 2). Rapid DNA synthesiswhich began only after 55 min of germinationwas preceded by a very low level of synthesis;DNA synthesis measured with labeled TdR(plus deoxydenosine [AdR]) also showed aninitial slow rate, followed at about 60 min by afaster rate (Fig. 2). The TdR incorporation wasgreatly stimulated by the addition of deox-yadenosine as in Escherichia coli (4). The AdRpresumably provides deoxyribose-1-phosphateto utilize the thymine generated by TdR break-down by thymidine phosphorylase (EC 2.3.2.4)(1 1). As with E. coli, incorporation of exogenousTdR into DNA during rapid DNA synthesisaccounted for only about 40% of the dTMPresidues polymerized (Fig. 2). However, during

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FIG. 1. DNA level during spore germination. Sam-ples (25 ml) were withdrawn from germinating cul-tures at the indicated times, adjusted to 5% withtrichloroacetic acid, and centrifuged. The pellet waswashed with 10 ml of cold 5% trichloroacetic acid andrecentrifuged, and the pellet was lyophilized. The dryspores were then suspended in 1.5 ml of 0.5M HCIO4for 15 min at 70 C. After centrifugation the superna-tant fluid was analyzed for DNA.

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slow DNA synthesis, there was little or nodilution of exogenous TdR by endogenoussources (Fig. 2, inset).That the initial slow incorporation of TdR

and UR was indeed into DNA was shown by thefollowing criteria. (i) The label was acid insolu-ble and alkali stable (>90%). (ii) The materialinto which TdR was incororated banded inalkaline cesium chloride with the cellular DNA(P. Setlow, unpublished experiments, 1972).(iii) Incorporation of TdR was blocked by mi-tomycin C, and incorporation of both TdR andUR was blocked by nalidixic acid (Fig. 3).Enzymes of DNA and deoxynucleotide me-

tabolism in spores and cells. IncQrporation ofboth UR and TdR into DNA was blocked notonly by inhibitors ofDNA synthesis, but also bychloramphenicol (Fig. 3a, b), which not onlyblocked all DNA synthesis if added at zero time,but also prevented the initiation of rapid DNAsynthesis if added at 45 min (Fig. 3b). Thisrequirement of protein synthesis for DNA syn-

TIbAE IN MINUTES

FIG. 2. Extent of incorporation of TdR or UR intoDNA during spore germination in the presence of 1

mM AdR.

IU

A

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NAtlDIXIC ACIO

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NO ADDITIONS s,

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// C.lNC/AlIIPAC\.ol.A .,

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_+'aO@O6=6-4 =O

4O No 12000 20 40 60 80

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FIG. 3. Effect of inhibitors on the uptake of TdR or

UR into DNA during spore germination measured (a)with thymidine plus deoxyadenosine (1 mM), or (b)with uridine. Chloramphenicol and actinomycin Dwere present at 100 gg/ml and mitomycin C andnalidixic acid at 10 Ag/ml.

thesis, also reported by others (11, 18, 31),suggests that proteins involved in DNA synthe-sis were deficient in the germinating spore. Thespore does contain levels of DNA ligase andDNA polymerase similar to those in the vegeta-tive cell (Table 1) (8). However, it is possiblethat the polymerase assay did not detect thereplicative polymerase. Other proteins possiblyneeded for DNA synthesis could be initiatorproteins, membrane proteins or nucleases.Another important consideration is the abilityof the germinating spore to produce deox-yribonucleotides. Spores are incapable of syn-thesizing ribonucleotides during the first min-utes of germination (23). Dormant spores did,however, contain a number of enzymes of deox-yribonucleotide metabolism, including kinasesfor dAMP, dTMP, AdR, and TdR (EC 2.7.1.21)(Table 1), and the levels of these enzymes indormant spores were in most cases similar tothose in vegetative cells. Even the lower level ofTdR kinase in the dormant spore is sufficient topermit DNA synthesis at a rate at least fivefoldhigher than that observed in the first 55 min ofgermination. The specific activities of the ki-nases for dAMP, dTMP, and TdR were alsosimilar to those determined in spores of B.subtilis SB-133 (8). Deoxycytidine kinase wasnot detected in either spores or cells, whereasdeoxyguanosine kinase was found only in cells.Ribo- and deoxyribonucleotides in dormant

and germinating spores. Despite the presenceof a number of deoxyribonucleoside and deox-yribonucleotide kinases, no deoxyribonucleo-tides and no TdR were detected in dormantspores (Table 2). This is in contrast to thesignificant ribonucleotide levels (Table 2). In-deed the levels of total ribonucleotide are knownto be almost identical in both dormant sporesand vegetative cells (23).

Deoxyribonucleotide triphosphates (dXTPs)were detectable after 10 min of germination butmade up only 4% of the total nucleoside triphos-phates (XTPs) (Fig. 4, note different scales).Furthermore, during the next 40 min the ribo-nucleoside triphosphates (rXTPs) increased5.5-fold, whereas the dXTPs increased only2-fold and thus comprised only 1.6% of the totalXTP 50 min after the start of germination (Fig.4). However, at this time the dXTP level beganto increase rapidly, reaching a value of 10% thatof the total XTPs: this level was maintainedthroughout further growth (Fig. 4). The rapidincrease in the dXTP level was due to anincrease in all four dXTPs (Fig. 5), with all butdGTP increasing almost in parallel. In compari-son, the increases in individual rXTPs from 10to 50 min were of greatly different magnitudes,

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VOL. 114, 1973 DNA SYNTHESIS AND DEOXY

TABLE 1. Enzyme levels in cells and spores of B.megaterium

Unitsa/mg proteinEnzyme

Cells Spores

DNA polymerase 17.2 13.6DNA ligase 157 163dAMP kinase5 830C 230cdTMP kinase 110c 90CTdR kinase 30 4GdR kinased 7 <0.5CdR kinaseb, d < 1.0 <0.5AdR kinaseb 16 5

aUnits are nanomoles of product formed per hourexcept for DNA ligase which is picomoles of nickssealed per hour.

"The values for these enzymes may be low due topresence of deaminases.

c 60 to 75% of the products were the triphosphates.d Abbreviations: deoxyguanosine, GdR; and deox-

ycytidine, CdR.

TABLE 2. Nucleotide and nucleoside levels indormant spores

Nanomoles/gramCompounda (dry wt)

Spores Cells

AMPb, c 1080CMPb. c 275GMPb c 230UMPb c 505ADP + UDPC 385dAMP + dCMP + dGMP + < 25dTMPb

dCDP + dGDPb <10ATP + CTP + GTP + UTPd <10 6100dATP + dCTP + dGTP + <5 590dTTpd

TdRe <5

a Abbreviations: adenosine monophosphate, AMP;cytidine monophosphate, CMP; guanosine mono-phosphate, GMP; uridine monophosphate, UMP;adenosine diphosphate, ADP; uridine diphosphate,UDP; deoxycytidine monophosphate, dCMP; deox-yguanosine monophosphate, dGMP; deoxycytidinediphosphate, dCDP; deoxyguanosine diphosphate,dGDP; cytidine triphosphate, CTP; guanosine tri-phosphate, GTP; uridine triphosphate, UTP; deox-ycytidine triphosphate, dCTP; deoxyguanosine tri-phosphate, dGTP.

b Determined by thin-layer chromatography in sys-tem II of extracts of 32P-labeled dormant spores.

c Determined by thin-layer chromatography in sys-tem IV of extracts of 32P-labeled dormant spores.

d Determined by.thin-layer chromatography in sys-tem III of extracts of 32P-labeled dormant spores.

e Determined by paper chromatography in system Iof extracts (80% n-propanol) of 3H-TdR-labeled dor-mant spores.

(NUCLEOTIDE METABOLISM 1103

and ATP levels actually decreased between 60and 80 min (Fig. 6).A number of compounds were tested for their

effect on rXTP and dXTP pools after 10 or 30min of germination (Table 3). Inhibition ofDNA synthesis by nalidixic acid under condi-tions where RNA synthesis was unaffected hadno effect on either the rXTP or dXTP levels.However, chloramphenicol lowered both thedXTP and rXTP levels, probably by blockingsynthesis of ribonucleotide biosynthetic en-zymes (22). However, some dXTPs were de-tected in spores germinated in chloramphenicol.Addition of exogenous ribonucleosides elevatedboth rXTP and dXTP pools, and these in-creases were not blocked by chloramphenicol(Table 3).Ribonucleotide reductase activity during

spore germination. Although the low levels ofdeoxyribonucleotides from 0 to 50 min sug-gested low or absent ribonucleotide reductaseactivity, the chloramphenicol-insensitive pro-duction of deoxyribonucleotides from exogenousribonucleosides indicated that some ribonucleo-tide reductase was indeed present during thistime. This was further suggested by the incorpo-ration of labeled UR into DNA from 0 to 50 min(Fig. 2). Indeed, ribonucleotide reductase activ-ity was found in dormant spores (Fig. 7),although the specific activity was only 1/15ththat of the vegetative cell (Setlow, P., unpub-lished results, 1972). The enzyme level re-mained constant until about 35 min of germina-tion, at which time it increased about sixfold

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FIG. 4. Total ribonucleoside triphosphate anddeoxynucleoside triphosphate levels during spore ger-mination. The Spizizen medium contained 'Ao theamount of phosphate normally present, and thespecific radioactivity was 5.104 counts per min pernmol. This lower phosphate concentration did notaffect the time required for the first vegetative dou-bling to occur. Triphosphate levels were calculatedassuming 3 mol of phosphate per mol of triphosphate,except for the 10-min point in which 2 mol ofphosphate per mol of triphosphate was used sincenucleotide biosynthesis has not yet begun at this time(23).

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DNA synthesis in stage I. Calculation of the/,,^ amount ofDNA synthesized in stage I, using the

/<dCTP value for UR incorporation (Fig. 2), gives a//

dvalue of 0.05 mg of DNA per g of dry spores, or3.7% of the total DNA in the dormant spore,

/,(dTTP - -~ and of this small amount, one-half was synthe-dTP sized in the last 10 min of stage I. Clearly thev,T' small amount of DNA synthesis in stage I is not

difGd6TP detectable as a net increase in DNA content..U- The exact nature and importance of the DNA

synthesized in stage I is at present unclear.Several laboratories have observed a slow rate ofDNA synthesis early in spore germination corre-sponding to our stage I (11, 18, 31). This early

zosphate levels dur- synthesis has been suggested to be of the repairfns were identical to type (18, 31). However, it was inhibited by

6- (p-hydroxyphenylazo)-uracil, which isthought to inhibit replicative, but not repair,DNA synthesis (5, 11). Furthermore, other

3000 workers have been unable to detect any early, DNA synthesis during spore germination (27,

GTP 29). The reasons for these differing results areUTP 2000 not clear, and more work is needed on the/~/XOnature and significance of the DNA synthesis in

stage I._t\CTP 000 ° Source of deoxynucleotides in stages I and

TABLE 3. Effect of several compounds on nucleosidetriphosphate levels of germinating sporesa

20 40 60 100 120

TIME IN MIlNUTES

FIG. 6. Ribonucleoside triphosphate levels duringspore germination. Conditions were identical to thosein Fig. 4.

just prior to the rapid increase in the dXTPlevels (Fig. 4). This increase in ribonucleotidereductase activity was abolished by the addi-tion of chloramphenicol. The ribonucleotidereductase of germinating spores required ATPand TPNH or dithiothreitol for full activity,suggesting that this enzyme system is similar tothat of E. coli, and utilizes reduced thioredoxinas a source of reducing power (10) (Table 4).The use of CMP as the substrate does notindicate that reduction is at this level ratherthan the diphosphate.

DISCUSSIONDNA synthesis during spore germination can

be divided into two stages (Fig. 8): stage I whereDNA synthesis is slow, and stage II where DNAsynthesis is more rapid ( > 15-fold) and contin-ues on into vegetative growth. The values forisotope incorporation into DNA during stages Iand II appear to reflect only the rate of DNAsynthesis, since little or no DNA breakdown was

detected (Table 3; Setlow, P., unpublishedresults, 1972).

Nanomoles/g (dry spores)c

Total ribo- Total deoxy-Additionb nucleoside nucleoside

triphosphate triphosphate

lod 30 10 30

None 1,820 3,170 84 127Nalidixic acid (7 sg/ml)e 1,700 2,930 87 120Chloramphenicol (100 1,784' 39'

Ag/ml)Inosine (10-' M) + 5,785' 227'

uridine (10-' M)Inosine (10-' M) + 7,271' 198'

uridine (10-' M) +chloramphenicol (100slg/ml)aGermination conditions and the calculation of

nucleotide levels were as described in the legend toFig. 4.

b All additions were present from the start ofgermination.

c All values are averages of two separate determina-tions which differed by less than 20%.

d Time of germination (minutes).eThis concentration of nalidixic acid caused no

inhibition of RNA synthesis by 30 min.'The pools of all nucleotides were lower than the

control.' The pools of all nucleotides were higher than the

control.

1104

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a:

E

IE

FIG. 5. Deoxynucleoside tripling spore germination. Conditiothose in Fig. 4.

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DNA SYNTHESIS AND DEOXYNUCLEOTIDE METABOLISMVOL. 114, 1973

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FIG. 7. Ribonucleotide reductaspore germination. Samples, 40indicated times, centrifuged and jand assayed as described in MateChloramphenicol was added to 10

TABLE 4. Requirements for ribonLby germinating spi

Assay conditions

Complete .......................Complete - ATP ...............Complete - TPNH .............Complete - dithiothreitol .......Complete - dithiothreitol - TPNComplete + dCMP (0.5 mM) ....

a Spores were harvested after;tion, and 0.5 mg were assayed.

II. The deoxyribonucleotidesand II are derived almostribonucleotide reduction. Thstage II, where the large amcsynthesis shows an overt requnucleotide reduction. Althouglyribonucleotide is required insence of detectable deoxyribordormant spore and the presennucleotide reductase also suitides as the principal source oftides. This contention is furtby the absence of significantbreakdown during stage I. Ifwas supplying a significantdeoxyribonucleotides for DISstage I, then dXTPs mightaccumulate under conditionsthesis was blocked. However, aacid which inhibited DNA sRNA synthesis had no effe4dXTP or rXTP levels in stsuggesting that breakdown of Iwas minimal, and provided les

deoxyribonucleotides in stage I. This is in sharpcontrast to the almost total dependence of earlyRNA synthesis on RNA degradation to providea source of ribonucleotides (23).The most striking change in deoxyribonucleo-

tide levels during stage I is from <25 nmol/g at0 min (Table 2) to 80 nmol/g at 10 min (Fig. 4).This initial rapid increase is probably also dueto ribonucleotide reduction and not DNA break-

Il ADDED

down since nalidixic acid addition did notelevate the dXTP level at 10 min despite

EQ X0 12 inhibiting DNA synthesis at this time (Setlow,P., unpublished results, 1972) (Table 3). The

se activity during reason for the rapidity of this initial increaseml, were taken at may be the absence from the dormant spore offrozen, and treated deoxyribonucleotides, which might themselvesIrials and Methods. inhibit ribonucleotide reductase (Table 4).00 Ag/mLs Deoxyribonucleotide levels in stages I and

II. The low level of dXTP in stage I and theucleotide reduction rapid increase in stage II are extremely striking.ores This difference is magnified further when one

dCMP formed calculates the rates of deoxyribonucleotide ac-

(nmoV30 cumulation (DNA synthesis plus increasedmin)a dXTP level) (Fig. 2, 4) in stages I and II. There

is a 15- to 25-fold increase in the rate of0.24 deoxyribonucleotide synthesis in going from

<0.005 stage I to be the first 30 min of stage II. Ob-0.16 viously, we would like to know the reason for

H 0.01 this dramatic change in the rate of deoxyribo-0.13 nucleotide formation.

One factor which does not appear to bei0 min of germina- involved in controlling deoxyribonucleotide ac-

cumulation is levels of deoxyribonucleotide ki-nases, since these enzymes are present in the

in both stages I dormant spore at levels similar to those in thecompletely from,is is clearest in STAG I)unt of net DNA RNA SYNTHESIS -50iirement for ribo- 30'DNASYNTHtSIo L}0h much less deox- 4 tOTEIN SYNTHESIS/stage I, the ab- . IttNCUOTIDE REDUCTASE

iucleotides in the RI2ONCLE-OTIDA

ice of active ribo- 4ggest ribonucleo- ICo TAt dXTP LEVELdeoxyribonucleo- z

her strengtheneddetectable DNA O,

0 20 *0 60 0 100 120

DNA breakdown TIME IN AAINUTES

amount of the FIG. 8. DNA and deoxynucleotide metabolism inIA synthesis in spore germination. Data for the DNA level and rate ofbe expected to DNA synthesis are taken from Fig. 1 and 2. Thewhere DNA syn- dXTP level is from Fig. 4 except for the point at zero

llevel of nalidixic time which is the maximum value for deoxynucleotideynthesis but not in the dormant spore (Table 2) if it were 80% in thect on either the triphosphate form. The time for the initiation of theage

on iThler te increase in ribonucleotide reductase is taken from Fig.age I (Table 3), 7, and the times for initiation of protein, RNA, andpre-existing DNA ribonucleotide synthesis are taken from previous work3s than 10% of the (23).

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J. BACTERIOL.

vegetative cell (Table 1). This is in contrast tothe work of Tanooka et al. (27) who found nodTMP kinase in dormant spores of B. subtilis168. We have no explanation for this discrep-ancy.A possible step which might be involved in

the increased rate of deoxynucleotide synthesisin stage II is ribonucleotide reduction, since therates of synthesis of all four common deoxynu-cleotides increase at the same time (Fig. 5). Thelarge increase in ribonucleotide reductase activ-ity just prior to stage II (Fig. 7) supports thishypothesis, and this experiment further sug-gests (but does not prove) that synthesis of someprotein or proteins of the ribonucleotide reduc-tase system may cause the increased level ofdeoxynucleotide accumulation in stage II. How-ever, by analogy with the regulation of theribonucleotide reductase of Escherichia coli (12,13), it is also possible that the dramatic changesin rXTP levels in stage I may also play a role inregulating ribonucleotide reduction.

Possible involvement of dXTP levels incontrol of rate of DNA synthesis. The lowdXTP levels during the slow DNA synthesis instage I and the increased dXTP levels duringrapid synthesis in stage II invite speculationthat dXTP levels might in some way be in-volved in regulating the rate of DNA synthesis.Indeed, it is known that in E. coli the rate ofDNA synthesis can be decreased up to 10-foldif the dXTP level is decreased by either hy-droxyurea treatment of wild-type bacteria, orby lowering the thymine levels for thymine-re-quiring mutants (1, 16). Whether dXTP levelsdo indeed play some role in regulating the rateof DNA synthesis during early spore germina-tion is not shown by our experiments, since thelow dXTP levels in stage I may be an effect or acorrelary rather than the cause of the slowDNA synthesis.

ACKNOWLEDGMENTSIt is a great pleasure to acknowledge the advice and

support of Arthur Komberg in whose laboratory much of thiswork was carried out. I am also indebted to Paul Modrich forthe assays of DNA ligase.

This work was supported by grants from the NationalInstitutes of Health and the National Science Foundation toArthur Kornberg, and a grant from the University of Connect-icut Research Foundation to Dr. Peter Setlow.

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