Deoxyribonucleic Acid Synthesis and Deoxynucleotide Metabolism ...

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

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

Deoxyribonucleic Acid Synthesis and Deoxynucleotide Metabolism During Bacterial Spore Germination PETER SETLOW'

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

Received for publication 12 March 1973

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

Spore germination in Bacillus is an attractive were from Sigma Chemical Co. [Methyl-'HJdeoxysystem for studying control of deoxyribonucleic thymidine monophosphate (3H-dTMP), a-32P-deoxyacid (DNA) replication since initiation of chro- thymidine triphosphate (32P-dTTP), [5-'H]deoxmosome replication is quite synchronous (31, ycytidine, [5-3H]cytidine monophosphate (3H-CMP), and [8-_HJdeoxyadenosine 32). Although ribonucleic acid (RNA) and pro- [8-8HJdeoxyguanosine, obtained from Schwarz BioResearch; and tein synthesis begin within the first minutes of were [methyl-1HJdeoxythymidine ( 3H-TdR), [1-_C ]uridine spore germination, DNA replication, and in one ("IC-UR), and [8- 4C]deoxyadenosine monophosphate case all DNA synthesis, does not begin for 1 to 2 ("4C-dAMP) were purchased from New England Nuh (23, 26, 29). Since the initiation of DNA clear Corp. The radiochemical purity of the nucleoreplication is sensitive to chloramphenicol (18, tides and nucleosides was checked by paper chroma31), it was suggested that the synthesis of tography (Schleicher and Schuell, no. 589 orange proteins such as initiators, membrane proteins, ribbon) by using an isopropanol: water: ammonia solor polymerases turn on chromosome replication vent (7:1:2) (system I). The 3H-TdR was purified this chromatographic system. Failure to do so and net DNA synthesis. However, attention using in spurious results. resulted must also be given to the capacity of the Other chemicals. Nalidixic acid was purchased to the germinating spore produce deoxyribonu- from Calbiochem. Chloramphenicol, mitomycin C, cleotides essential for net DNA synthesis. The reduced triphosphopyridine nucleotide (TPNH), and inability to synthesize ribonucleotides early in dithiothreitol were purchased from Sigma Chemical germination due to the absence of nucleotide Co. Diphenylamine was the product of Eastman biosynthetic enzymes (23) suggests that deox- Organic Chemicals and was recrystallized from yribonucleotide biosynthesis may also be im- ethanol. Polyethylenimine-coated, thin-layer chromatography sheets were purchased from Brinkmann Inpaired. Actinomycin D was a gift of Merck, We have found that DNA synthesis during struments. and an alternating copolymer of Sharpe Dohme, germination (see Fig. 8) is correlated with the deoxyadenylate and and deoxythymidylate [poly d(AT) ] deoxynucleotide pool size, and this in turn with for the ligase assays was prepared as described by the level of ribonucleotide reductase. Modrich and Lehman (14). MATERIALS AND METHODS Nucleotides and nucleosides. Unlabeled ribo- and deoxyribonucleotides were purchased from P-L Biochemicals. Unlabeled deoxyribonucleosides were obtained from Calbiochem, and adenosine and uridine ' Present address: Department of Biochemistry, University of Connecticut Health Center, Farmington, Conn. 06032.

Growth of spores and cells. All work described here was carried out with Bacillus megaterium QM B1551, originally obtained from H. S. Levinson (U.S. Army Natick Laboratory, Natick, Mass.). Spores and 32P-labeled spores were obtained from cultures grown in supplemented nutrient broth as previously described (22), lyophilized, and stored at room temperature. The specific radioactivity of 32P-labeled spores

1099

1100

SETLOW

was also determined as previously described (22). Spores containing 3H-TdR in their DNA were grown as described by Donnellan and Setlow (7) with 0.4 mCi of 3H-TdR per 200 ml of supplemented nutrient broth. Germination of spores. Unless otherwise noted the standard conditions for spore germination were as follows: spores (20 mg [dry wt ] per ml) were heat shocked in water for 10 min at 60 C and cooled prior to initiation of germination. The germination medium was that of Spizizen (24) containing glucose, citrate, and salts, and supplemented with 0.1% casein hydrolysate. Germination experiments were initiated by addition of spores to 0.4 mg (dry wt) per ml, and cultures were agitated in a gyratory water bath at 30 C. The rate of initiation of germination, and that of subsequent growth was measured by following the optical density at 660 nm. Within 10 min of starting germination >90% of the spores appeared dark in the phase constrast microscope. Enzyme assays. DNA polymerase (EC 2.7.7.7) was assayed with activated calf thymus DNA as the template as described by Okazaki and Kornberg (17), and DNA ligase was assayed as described by Modrich and Lehman (14). Deoxyribonucleoside kinases, deoxyadenosine monophosphate (dAMP), and deoxythymidine monophosphate (dTMP) kinase (EC 2.7.4.c) were assayed essentially as described by Grav and Smellie (9). The reaction mixture (0.5 ml) contained 100 mM tris(hydroxymethyl)aminomethane (Tris) (pH 8.0), 5 mM MgCl2, 5 mM adenosine triphosphate (ATP), and 0.1 mM ,-mercaptoethanol. Labeled deoxyribonucleosides were present at 0.5 mM; dAMP and dTMP at 0.4 mM. The assay was started by addition of extract (0.1-1.0 mg of protein), and after 15 min at 37 C the reaction was halted by boiling for 5 min. After centrifugation of coagulated material a sample of the supernatant fluid (50 Mliters) was spotted on Whatman no. 1 paper together with appropriate markers. The chromatogram was developed for 24 h (descending) in isobutyric acid:ammonia:0.1 M ethylenediaminetetraacetic acid (EDTA) (100:60:1.6). After drying, the marker spots were located under ultraviolet light, cut out, and counted. Kinase activity is given as the total nucleoside or nucleotide phosphorylated. Ribonucleotide reductase was assayed by a modification of the method of Bertani et al. (2). Reaction mixtures contained 50 mM Tris-hydrochloride (pH 8.0), 20 mM MgCl2, 10 mM ATP, 10 mM dithiothreitol, 1.3 mM TPNH, 0.2 mM 3H-CMP, and enzyme (0.5-2.5 mg [dry wt] spores) in a volume of 50 jl.iters. After 30 min at 37 C the reaction was stopped by addition of 1 ml of 1 M HCI containing 25 nmol of cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP). After centrifugation the supernatant fluid was boiled for 15 min to hydrolyze pyrophosphate bonds and then flash evaporated and redissolved in 25 Mliters of water. A 20-,gliter amount was analyzed by paper chromatography as described above by using ethanol-saturated sodium tetraborate-5 M ammonium acetate-0.5 M EDTA (220:80:20:0.5 by vol) as the solvent (28). Spots corresponding to CMP and dCMP were then cut out

J. BACTERIOL.

and counted. The assay was linear for 30 min when less than 4% conversion of CMP to dCMP occurred. Within the latter limitation, the assay was also linear with spore concentration from 0.15 to 2.5 mg per assay. Initial experiments indicated that it was difficult to detect enzyme activity in extracts from B. megaterium spores or cells. I therefore resorted to spores made permeable by repeated freezing and thawing as a source of enzyme (25) (see below). Assay of RNA and DNA synthesis. RNA and DNA synthesis were measured by incorporation of "C-UR or 3H-TdR, respectively, into acid-insoluble material. Both '4C-UR and 8H-TdR were added to 25 MM and were present from the start of germination. Samples (0.4 ml) were taken at various times and diluted fivefold with 6.7% cold trichloroacetic acid. After 30 mnin at 4 C the sanmples were filtered through glass fiber filters, washed five times with 5 ml of 5% trichloroacetic acid containing 1 mM UR or 1 mM TdR, and then washed four times with absolute ethanol. The filters were then dried and counted in a scintillation counter. DNA synthesis was also assayed by measuring the incorporation of 14C-UR into alkali-stable, acidinsoluble material. Spores were germinated in the presence of "4C-UR (25 AtM), and samples were withdrawn and diluted 1:1 with 2 M NaOH. After incubation in a closed tube for 18 h at 37 C the solution was neutralized with HCI, 0.1 mg of salmon sperm DNA was added as carrier, and the solution was made 0.5 M in HC104. After 30 min at 4 C the precipitate was collected by filtration, washed, and counted as described above. DNA levels were determined colorimetrically with diphenylamine using calf thymus DNA as the standard (21). Extraction of nucleotides. Nucleotides were extracted from germinating spores with cold 5% trichloroacetic acid. Samples of germinating spores (500 Aliters) were passed through a membrane filter (Millipore Corp., 0.45 um), and the filter was immediately (90%). (ii) The material into which TdR was incororated banded in alkaline cesium chloride with the cellular DNA (P. Setlow, unpublished experiments, 1972). (iii) Incorporation of TdR was blocked by mitomycin C, and incorporation of both TdR and UR was blocked by nalidixic acid (Fig. 3). Enzymes of DNA and deoxynucleotide metabolism in spores and cells. IncQrporation of both UR and TdR into DNA was blocked not only by inhibitors of DNA synthesis, but also by chloramphenicol (Fig. 3a, b), which not only blocked all DNA synthesis if added at zero time, but also prevented the initiation of rapid DNA synthesis if added at 45 min (Fig. 3b). This requirement of protein synthesis for DNA syn-

TIbAE IN MINUTES

FIG. 2. Extent of incorporation of TdR or UR into DNA during spore germination in the presence of 1 mM AdR.

B

A

l I

NO ADDTlON5

IU

'.i I NO

ADDITIONS s, / //

C.lNC

NAtlDIXIC ACIO

/

WC OACl IN

/AlII A PAC 'I

\.ol.

/

AW0HtOlAV*f#NIOt/L o

4O

1200 0

No

TIME

IN

X

.,

aO@ =O

6=6-4

_ +'

20

40

60

O

80

MINUTES

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 D were present at 100 gg/ml and mitomycin C and nalidixic acid at 10 Ag/ml.

J. BACTERIOL.

thesis, also reported by others (11, 18, 31), suggests that proteins involved in DNA synthesis were deficient in the germinating spore. The spore does contain levels of DNA ligase and DNA polymerase similar to those in the vegetative cell (Table 1) (8). However, it is possible that the polymerase assay did not detect the replicative polymerase. Other proteins possibly needed for DNA synthesis could be initiator proteins, membrane proteins or nucleases. Another important consideration is the ability of the germinating spore to produce deoxyribonucleotides. Spores are incapable of synthesizing ribonucleotides during the first minutes of germination (23). Dormant spores did, however, contain a number of enzymes of deoxyribonucleotide metabolism, including kinases for dAMP, dTMP, AdR, and TdR (EC 2.7.1.21) (Table 1), and the levels of these enzymes in dormant spores were in most cases similar to those in vegetative cells. Even the lower level of TdR kinase in the dormant spore is sufficient to permit DNA synthesis at a rate at least fivefold higher than that observed in the first 55 min of germination. The specific activities of the kinases for dAMP, dTMP, and TdR were also similar to those determined in spores of B. subtilis SB-133 (8). Deoxycytidine kinase was not detected in either spores or cells, whereas deoxyguanosine kinase was found only in cells. Ribo- and deoxyribonucleotides in dormant and germinating spores. Despite the presence of a number of deoxyribonucleoside and deoxyribonucleotide kinases, no deoxyribonucleotides and no TdR were detected in dormant spores (Table 2). This is in contrast to the significant ribonucleotide levels (Table 2). Indeed the levels of total ribonucleotide are known to be almost identical in both dormant spores and vegetative cells (23). Deoxyribonucleotide triphosphates (dXTPs) were detectable after 10 min of germination but made up only 4% of the total nucleoside triphosphates (XTPs) (Fig. 4, note different scales). Furthermore, during the next 40 min the ribonucleoside triphosphates (rXTPs) increased 5.5-fold, whereas the dXTPs increased only 2-fold and thus comprised only 1.6% of the total XTP 50 min after the start of germination (Fig. 4). However, at this time the dXTP level began to increase rapidly, reaching a value of 10% that of the total XTPs: this level was maintained throughout further growth (Fig. 4). The rapid increase in the dXTP level was due to an increase in all four dXTPs (Fig. 5), with all but dGTP increasing almost in parallel. In comparison, the increases in individual rXTPs from 10 to 50 min were of greatly different magnitudes,

VOL. 114, 1973

DNA SYNTHESIS AND DEOXY(NUCLEOTIDE METABOLISM

TABLE 1. Enzyme levels in cells and spores of B. megaterium Unitsa/mg protein Enzyme

DNA polymerase DNA ligase dAMP kinase5 dTMP kinase TdR kinase GdR kinased CdR kinaseb, d AdR kinaseb

Cells

Spores

17.2 157 830C 110c 30 7 < 1.0 16

13.6 163 230c 90C 4