JOURNAL OF BACTERIOLOGY, Sept. 1978, p. 1158-1161 0021-9193/78/0135-1158$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 135, No. 3 Printed in U.S.A.

DNA Synthesis in Competent Bacillus subtilis Cells KENNETH S. LOVEDAYt Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 27 May 1978

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells. Previous work has shown that cultures of competent Bacillus subtilis can be separated into two fractions and that only a minor fraction (10%) is capable of taking up and integrating exogenous DNA (3, 8, 11, 13, 15). The rates of RNA and protein synthesis of the bacteria in the minor fraction (competent bacteria) are substantially lower than those of the bacteria in the major fraction (noncompetent bacteria) (3, 5, 7, 13, 14). The level of DNA synthesis in competent bacteria has been a matter of concern for some time. Newly transformed bacteria do not begin to multiply until several hours after the DNA has been taken up (11, 13, 14), and, if they require thymine, are resistant to thymineless death, suggesting a substantial reduction in the rate of DNA synthesis (11). Using competent cultures fractionated on Renografin gradients, Dooley et al. (5) reported a sixfold reduction in the rate of DNA synthesis in bacteria found in the competent fraction. However, this fractionation procedure does not assure the removal of all of the noncompetent bacteria from the competent fraction (3, 8). It remains possible that the residual DNA synthesis observed in the competent fraction occurs in the contaminating noncompetent bacteria. McCarthy and Nester (11) examined the amount of DNA synthesis in newly transformed cells by following the kinetics of viability loss resulting from disintegration of incorporated 3H after labeling cellular DNA with [3H]thymine. They found that transformants lost viability 15 times more slowly than the nontransformed cells. Since the loss of viability of transformed bacteria was marginal, these observations can only set an upper limit of 7% for the relative rate of DNA synthesis in competent bacteria. Although none of the investigators claims that t Present address: Department of Clinical Genetics, Children's Hospital Medical Center, Boston, MA 02115.

DNA synthesis is completely absent in competent cells, examination of the question seems

worthwhile. Bacterial DNA synthesis is in fact evident in bacteria that have taken up bacteriophage Oe DNA, and this synthesis appears to be semiconservative. CsCl density gradients were used to analyze lysates of competent cells (SB291) that had taken up transfecting DNA isolated from the virulent phage oe (10). Since se DNA has a higher buoyant density in CsCl than B. subtilis DNA, the two DNA types were readily separated on CsCl density gradients. Most of the label present in the double-stranded transfecting DNA that had been taken up by the competent cells is found as single-stranded phage DNA in lysates (10). In addition, a substantial amount (15 to 20%) is found in bacterial DNA (10). Since the transfecting DNA was free of bacterial DNA (phage were purified using a CsCl step gradient and an equilibrium gradient), this label must represent the incorporation into bacterial DNA of nucleotides resulting from degradation of the transfecting DNA. The label does not represent trapping of 4e DNA in bacterial DNA since shearing and recentrifuging the DNA from the bacterial DNA peak does not release any material of phage DNA density. Either the presence of 6- (p-hydroxyphenylazo)-uracil (HPUra) or prior infection with helper phage blocks further bacterial DNA replication (1, 2, 4, 12) and also prevents this incorporation of label from phage DNA into the bacterial DNA, suggesting that the incorporation is due to semiconservative replication. The drug HPUra has been shown to inhibit polymerase III of B. subtilis, but does not interfere with repair synthesis (1, 2, 4). In the absence of HPUra, the amount of incorporation into bacterial DNA of 3"P from labeled transfecting DNA is about 10% (Fig. 1A). The addition of HPUra to the culture reduced the amount of incorporation to less than 1% (Fig. 1B).

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O00

n~~~~~~~~~~~ 20 + HPUra

B 0-~~~~~~~~~~

PB

0

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80-~~~~~~

401 0~~~~~~~ 10

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FIG. 1. Effect ofHPUra on incorporation ofphage DNA label into bacterial DNA. The competent bacteria from 50 ml of culture fractionated on a Renografin gradient were divided into two parts, one of which received HPUra (100 pg/ml). After both cultures were incubated with :P-labeled phage DNA (2 x 10' cpm/pg of DNA) for 60 min at 37°C, 25 pg of deoxyribonuclease I per ml was added for 2 min. The cells were washed, lysed, and treated with 5 M NaClO4 as described in Table 1. Aliquots were counted to determine the level of DNA uptake (without HPUra, 3,(000 cpm; with HPUra, 5,X000 cpm) which confirmed previous reports that HPUra does not affect uptake of DNA (6, 9). Phage DNA and bacterial DNA labeled with ;H were added as density markers, and the lysates were centrifuged to equilibrium in CsCL. The gradients were collected on filters and washed with 5% trichloroacetic acid and ethanol. The fractions from (A) were counted on a gas flow Nuclear Chicago counter to determine ;P radioactivity. The background of this counter (which has been subtracted) is 1 cpm, and the efficiency of counting :P is 40% that detected by liquid scintillation counting. The fractions from (B) were counted in the liquid scintillation counter which has a background of 10 cpm (also subtracted). The arrows mark the positions of native phage DNA (P) at 1. 74 g/cm ' and native bacterial DNA (B) at 1.70 g/cm'. The total amount of radioactivity on gradient A is 910 cpm with 90 cpm in bacterial DNA; the total amount on gradient B is 3,780 cpm with less than 40 cpm in bacterial DNA. The recovery of radioactivity distributed on the gradients was more than 90%.

In an experiment using density-laibeled transfecting DNA labeled with 32P, the amount of 3P incorporated into the bacterial DNA was 22% (Fig. 2A). Prior infection with helper phage reduced the amount of incorporation to 3 to 4%

(Fig. 2B). The helper phage, 4e s27, is a mutant defective in an early gene affecting phage DNA replication, but remains effective in stopping bacterial DNA replication (12). To prevent the incorporation of phage DNA label into bacterial

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J. BACTERIOL.

r_ w 1

100

B

W HP

P

100 90 80

7060 504030 20-

10 _ 10

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FIG. 2. Effect of helper phage on incorporation ofphage DNA label into bacterial DNA. The competent bacteria from 50 ml of culture fractionated on a Renografin gradient were divided into two parts, one of which was preinfected with helper phage, 4e s27, for 10 min at a multiplicity of infection of 5. After both cultures were incubated for 30 min at 37° with density-labeled, 32P-labeled phage DNA (0.5 pg/ml and lt cpm/p,g of DNA) (10), deoxyribonuclease I was added (25 pg/ml) for 2 min. The cells were washed, lysed, treated with 5 M NaC1O4, and centrifuged to equilibrium in CsCI. The gradients were collected and counted in a liquid scintillation counter. (A) Without phage; (B) with phage. The arrows mark the positions of densitylabeled, native phage DNA (HP) at 1.78 g/cm:3, native phage DNA (P) at 1.74 g/cm3, and native bacterial DNA (B) at 1. 70 g/cm'. Total amount of radioactivity: gradient (A), 1,930 cpm with 420 cpm in bacterial DNA; gradient (B), 1,590 cpm with 60 cpm in bacterial DNA. Recovery of radioactivity distributed on the gradients was more than 90%o.

DNA, the helper phage must preinfect the competent cells (Table 1). Addition of the helper phage after transfecting DNA had already been taken up by the cells (superinfection) did not reduce the amount of incorporation. Since the presence of HPUra prevents the incorporation of degraded products of transfecting DNA into bacterial DNA, I conclude that competent cells can replicate their chromosomes in a semiconservative manner. Similarly, prior infection of competent bacteria prevents incor-

poration of phage DNA label into bacterial DNA, presumably as a result of the phage-induced inhibition of bacterial DNA replication. In the absence of competent cells, no transforming DNA or transfecting DNA label is incorporated by the culture. Therefore, the presence of phage DNA label in bacterial DNA cannot be the result of extracellular degradation and subsequent utilization of the degraded products by noncompetent cells. Furthermore, the noncompetent bacteria do not use nucleotides

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TABLE 1. Effect of helper phage on incorporation of phage DNA label into bacterial DNAa Sample

Total cpm on gradient

Cpm in bacterial DNA

Fraction in DNA

e DNA 600 95 0.16 Preinfection 0e 280

DNA synthesis in competent Bacillus subtilis cells.

JOURNAL OF BACTERIOLOGY, Sept. 1978, p. 1158-1161 0021-9193/78/0135-1158$02.00/0 Copyright © 1978 American Society for Microbiology Vol. 135, No. 3 P...
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