JouRNAL OF BACTKRIOLoGY, Apr. 1976, p. 108-121 Copyright ©) 1976 American Society for Microbiology

Vol. 126, No. 1 Printed in U.SA.

Effect of Deoxyribonucleic Acid Replication Inhibitors on Bacterial Recombination UMBERTO CANOSI,* ANTONIO G. SICCARDI,' ARTURO FALASCHI, AND GIORGIO MAZZA Istituto di Genetica, Universitd di Pavia,* and Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy Received for publication 14 October 1975

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction.

The recombination process is thought to involve, in most of the proposed models, a certain amount of deoxyribonucleic acid (DNA) synthesis (for a review, see reference 27). This is currently thought to be more akin to repair-type synthesis than to the processes occurring at the growing point during chromosome replication. The existence of a great number of mutants, in several species, in which a single mutation inpairs recombination and repair without affecting DNA replication, strengthens this view. Nonetheless, the data indicating unambigously which molecules are involved in the DNA synthesis occurring in recombination are scarce. The possibility of a certain overlapping of functions, rather than of a complete and clear-cut separation, cannot be ruled out. For instance, the DNA polymerase (Pol) Ill of Escherichia coli plays a function both in replication and repair (20, 21, 52, 56); the T4 DNA polymerase is required both for replication and recombination (40). It is thus conceivable that also in Bacillus subtilis the Pol III or other molecules are involved in the two types of DNA synthesis. We approached the problem of identifying the molecules involved in the DNA synthesis required for recombination by trying to determine the effect of two inhibitors of bacterial

DNA replication on the recombination processes and on related repair activities. These inhibitors are nalidixic acid (NAL) and 6-(phydroxyphenylazo)-uracil (HPUra). For both of these agents, the prompt inhibition of bacterial DNA replication is well demonstrated. NAL exerts its action on all bacterial-type DNA replication systems, including those of the cell organelles (2, 11, 22, 34, 36); HPUra seems specific for gram-positive bacteria (7, 8, 9). The target of NAL is unknown (Pedrini et al., Proc. VIII Int. Cong. Chemother., 1974; 11, 22) but most probably resides in a still undescribed component of the growing point apparatus (45); the target of HPUra is well recognized and corresponds to the Pol III of B. subtilis, which is essential to the DNA replication of this organism (4, 5, 13, 20). The experiments that we report here indicate that the target of NAL is specific for replication, since no effect on recombination or repair was observed. The target of HPUra, however, seems to be involved in recombination as well.

MATERIALS AND METHODS Bacterial strains and bacteriophages. The origin and the description of the strains used in this work are reported in Table 1. The phage PBS-1 was used I Present address: Cattedra di Microbiologia II, Facolta for transduction experiments (55). For transfection, SPP1 (47) and SPO-1 (43) phage DNAs were used. di Medicina, Universitt degli Studi di Pavia, Italy. 108

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109

TABLE 1. List of strains of Bacillus subtilis used Strain Genotypea Origin SB 19 Prototroph J. Lederberg SB 202 tyrAl hisB2 trpC2 aroB2 J. Lederberg PB 3357 adeA16 leu-8 metB5 Mu8u5ul6, N. Sueoka C14 cysA14 I. Takahashi PB 3409 adeA16 cysA14 leu-8 PB 3357 + DNAC14 PB 566/2 thy arg SB 566 thy trpC2 (J. Lederberg) transformed to trp+ and then mutagenized with NTG CU 479 trpC2 ctrAl S. A. Zahler BD 92 trpC2 hisAl cysB3 J. A. Lepesant QB 2 sacA321 purA16 J. A. Lepesant GSY 1027 trpC2 metB4 uvr-1 C. Anagnostopoulos PB 1642 tyrAl hisB2 trpC2 aroB2 polA42 G. Villani PB 3292 thy trpC2 glyC from SB 566 mutagenized with UV PB 1706 tyrAl hisB2 trpC2 aroB2 nal-3 from SB 202 mutagenized with NTG PB 1728 tyrAl hisB2 trpC2 aroB2 azp-80 from SB 202 mutagenized with NTG PB 1731 thy trpC2 glyC azp-80 from PB 3292 + DNA PB 1728 BR 151 lys-3 trpC2 metBlO F. Young BD 54 azp-12 metB5 ile-1 spcB N. Brown PB 1729 lys-3 trpC2 azp-12 from BR 151 + DNA BD 54 a Symbols: ade, arg, aroB, ctr, cys, gly, his, leu, lys, met, thi, thy, trp, and tyr indicate, respectively, requirements for adenine, arginine, shikimic acid, cytidine in absence of ammonium, cysteine, glycine, histidine, leucine, lysine, methionine, thiamine, thymidine, tryptophan, and tyrosine. sacA, Inability to utilize sucrose as carbon source; azp, resistance to azo-pyrimidines; nal, resistance to nalidixic acid; polA, deficiency in DNA polymerase I; rec, deficiency in recombination; spc, resistance to spectinomycin; and uvr, sensitivity to UV light.

Culture media. Spizizen minimal medium MT (50) was used to prepare competent cells. Medium Y (55), Penassay broth (antibiotic medium no. 3, Difco), and tryptose blood agar base (Difco) were used in transduction experiments. The minimal medium of Davis and Mingioli (14) was used for selection of transformants and transductants. TY medium (6) was used for counting infective centers. Bouillon Nutritif Complet (Bio Kar) solidified with agar (nutrient agar) was used to determine cell titer and drug sensitivity, which was assayed according to the procedure of Villani et al. (53). Reagents. NAL was purchased from Serva Feinbiochemica, Heidelberg; HPUra was obtained from B. W. Langley (Imperial Chemical Industries) and, when necessary, reduced by the procedure described by Gass et al. (20). Caffeine was purchased from BDH Chemical Ltd., England. DNA preparation. DNA was prepared according to the method described by Marmur (37). DNA from SPP1 and SPO-1 phages was extracted from purified phages as described by Riva et al. (47). Spore preparation. Spores were prepared on solidified Bouillon Nutritif Complet, supplemented with 10-5 M MnCl2. After incubation at 37 C for 3 days, the spores were collected, washed, and resuspended with water. Spores were then treated with 1 mg of lysozyme per ml at 37 C for 30 min, followed by a 10min treatment with 1 mg of Duponol per ml at room temperature. The spores were collected by centrifugation at 10,000 rpm for 10 min, washed three times with water, resuspended in water, and stored at 4 C. UV irradiation. A Philips 15-W germicidal lamp was used for ultraviolet (UV) irradiation. For host

cell reactivation (HCR) experiments, 5-ml samples containing SPO-1 or SPP1 phages, 108 plaque-forming units (PFU)/ml, suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris; pH 7.2) and 0.005 M MgSO4, or 0.05 M Tris (pH 7.2) and 0.01 M MgCl2, respectively, were irradiated with shaking in 9-cm-diameter plates with different UV doses. For transfection with UV-irradiated phage DNA, 1-ml aliquots of SPP1 DNA, diluted at 2 jig/ ml in 1/10 x SSC (SSC = 0.15 M NaCl plus 0.015 M sodium citrate), were irradiated under agitation in 5-cm-diameter plates, at a dose of 150 erg/mm2. SPO-1 DNA, diluted in 1/10 x SSC at the concentration of 100 ,ug/ml, was irradiated as above at a dose of 900 erg/mm2. Host cell reactivation (HCR). (i) SPO-1. To the irradiated phage, stationary-phase cells of SB 202 or PB 1642 were added to a final titer of 5 x 107 and, after 10 min of incubation at 37 C for phage absorption, plated by the soft-agar method. To test the effect of HPUra on the HCR of SPO-1, SB 202 or PB 1642 cells were pretreated for 10 min with 30 ,ug of HPUra per ml, before mixing with dilutions of irradiated SPO-1. After 10 min of incubation at 37 C, cells of PB 1728 (HPUra-resistant strain) were added to a final titer of 5 x 107, and the mixture was plated on TY plates containing 30 Atg of HPUra per ml. (ii) SPP1. The HCR with SPP1 phage follows essentially the same scheme as for SPO-1, except that spores were used instead of stationary-phase cells. To test the effect of NAL, the spores were pretreated, before being mixed with the phage suspension, with 50 ,ug of the drug per ml for 10 min. As

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an indicator strain, we used spores of strain PB 1706 Distances between markers are expressed as per(NAL-resistant strain) and plating was done on TY centages of recombination according to the convenplates containing 50 ,g of NAL per ml. tion: % recombination = (1 - cotransfer) x 100. Transformation, transfection, and transduction. RESULTS AND DISCUSSION Competent cells were prepared as described by Stewart (51). The transformation and transfection Effect of NAL on transfection. Previous procedures were those described by Mazza et al. (38). work from this laboratory demonstrated that PBS-1 transduction was performed according to the the transformation process shows a lack of senmethod of Hoch et al. (26). DNA synthesis. DNA synthesis was measured by sitivity to NAL (44). This conclusion was based following the incorporation of [3H]deoxyribosyl-thy- on experiments in which the drug was added to mine (dThd) into acid-insoluble material. Strain PB competent cells before the addition of DNA 566/2 thy arg was grown overnight on Bouillon Nu- from a NAL-resistant strain; the drug was prestritif Complet supplemented with 50 ,ug of thymine ent during the incubation with DNA and in the per ml. The cells were collected by centrifugation for selective plates. The frequency of colonies 8 min at 7,000 rpm and suspended at an absorbance transformed to resistance to NAL was found at 560 nm (Amm) of 0.2 or at a turbidity of 40 Klett equal to that of a control experiment without units in 10 ml of prewarmed medium supplemented with 2 UCi of [3H]dThd per ml and 4 ug of drug. This technique was advantageous in that the cells remained in the presence of the drug cold thymine per ml. One-tenth-milliliter samples were added to 2 ml for all the time during which recombination of cold 5% trichloroacetic acid. After standing on ice could take place. Thus, the DNA synthesis necfor 15 min, the samples were collected on glass fiber essary to this event was not affected by the paper (Whatman GF/C) and washed three times drug. Other experiments (54), in which the conwith 10-ml samples of cold trichloroacetic acid and tact with NAL was continued for a longer durafinally with 2 ml of cold 95% ethyl alcohol. The tion before plating, indicated a reduction in radioactivity was determined in a liquid scintilla- transformation proficiency. In these cases, the tion counter. could cause a secondary SPP1 phage DNA synthesis. Cells of strain PB prolonged incubation to damage to differeffect, leading generalized 566/2 were grown in TY broth contai,ning 2 x 10-3 M CaCl2 and 50 jig of thymine per ml. At an A560 of ent cellular processes. We chose to study whether another process 1.5 to 1.7, the culture was centrifuged, washed with TY broth, and then resuspended in the same volume requiring recombination was affected by NAL. of TY medium containing 2 x 10-3 M CaCl2 and 4 ,tg The transfection process with DNA from a of cold thymine per ml. To the samples of cell sus- phage like SPP1 seems a good candidate for this pension, NAL (50 ,ug/ml) or HPUra (30 ,ug/ml) were study. In fact, the production of this phage (like added. After 10 min of incubation at 37 C, [3H]dThd that of SPO-1 (18), SP82, SP50, and 029 [11) is was added to a final concentration of 2 ,ICi/ml, and insensitive to NAL concentrations able to block the cultures were infected with SPP1, to a multiplic- completely bacterial DNA replication (44). We ity of infection of 6 or 7. At intervals, samples were removed for A560 readings and for DNA synthesis also measured the DNA synthesis after SPP1 determination, performed as described above. Phage infection in the presence of NAL. In the experiproduction was determined when the cell turbidity ment reported in Fig. 1, NAL was added at a had reached a minimum. Lysis was completed by concentration of 50 ,g/ml, 10 min before addition of a few drops of chloroform. phage - sufficient time to bring host DNA repliGenetic mapping. The linkage of nal and azp cation to a total stop (44). The results confirmed mutations with other markers of known location on that the infection and phage production are not the genetic map was determined by PBS-1 transduc- impaired by NAL and showed that phage DNA tion. Nalr phenotype (nal-3 marker) was tested on nutrient agar plates containing 50 t&g of NAL per synthesis proceeds at a substantial rate in presml. Azpr phenotype (azp-80) was tested on the same ence of the drug. Another advantage of transfection with SPP1 plates containing 20 ,ug of HPUra per ml. Recombinants for auxotrophic markers were se- DNA is the fact that this process utilizes the lected on minimal medium supplemented with 0.5% same DNA uptake apparatus involved in transglucose and the appropriate auxotrophic require- formation (46). Finally, the production of phage ments (at 25 Ag/ml). When required, glycine was requires a recombination event, as demonadded at a concentration of 50 ,ug/ml. Selection for strated by the 2-hit kinetics of the response to Ctr+ recombinants was made on MT medium where DNA concentration, which is analogous to the ammonium was replaced with glutamate. Suc(sacA marker) and Uvrr (uvr-I marker) phenotypes transfection of several other DNA phages of B. were tested according to the procedure of Lepesant subtilis (49). The recombination machinery reet al. (31). After 2 days of incubation at 37 C, recom- quired is the same as that required for transforbinants were picked, reisolated, and tested for their mation and involves only bacterial genes; rec phenotype. mutants of B. subtilis are impaired at the same

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TIME

(MINUTES)

FIG. 1. Effect of NAL on SPP1 DNA synthesis and phage lysis. Cells of strain PB 566/2 were infected as described in Materials and Methods in presence of NAL. DNA synthesis was followed by [3H]dThd incorporation (a), and lysis was monitored by measuring A560 nm (b). Symbols: Uninfected culture without NAL (a); infected culture without NAL (0); infected culture in presence of 50 pg ofNAL per ml (U); uninfected culture with NAL, 50 jig/ml (A). The final phage yield was 7.5 x 109 PFUlml in the control experiment and 1.1 x 1010 PFUlml in the presence of NAL.

time in transformation and transfection (39) and in no case has it been possible to discriminate these two types of recombination. The phage enzymes partially involved in phage recombination are irrelevant to this phenomenon (49). We could thus take advantage of these properties of SPP1 phage and determine whether the transfection of this phage is affected or not by NAL. The effect of the drug on SPP1 transfection was measured by the experimental scheme reported in Fig. 2. Transfection was performed in presence of NAL (added 10 min before DNA) at a dose (25 ug/ml) that completely blocks DNA synthesis within this time period. After 30 min of incubation and deoxyribonuclease treatment, appropriate dilutions of the cultures and plating for PFU centers were done in TY medium containing the same concentration of NAL. As the indicator strain, spores of strain PB 1706, a NAL-resistant mutant previously described (10), were used. On this strain, the efficiency of plating of SPP1 is the same as on the parental strain and it is not affected by the presence of 25 ,ug of nalidixate per ml. The results are reported in Table 2. From the data, the multi-hit dependence of transfection on DNA concentration is apparent, particularly at low concentrations; NAL did not affect the

111

production of infective centers at neither high nor low DNA concentrations. The same type of experiments were also performed with the DNA of SPO-1 phage. Also, synthesis of the DNA of this phage is insensitive to NAL (18), and the transfection follows a multi-hit kinetics (43). As the data of Table 2 show, NAL had no effect on transfection proficiency, with conditions analogous to those used for SPP1 transfection. All these data demonstrated further that the bacterial recombination system involved in the transfection process, as well as in transformation, is insensitive to NAL; a fortiori, the DNA synthesis required to this purpose is probably not a target of the drug; the possibility is still open that the NAL target is required for recombination, but at a much lower level than for the bacterial DNA replication. Effect of NAL on repair of UV damage. The same approach can be used to investigate the action of NAL on the DNA synthesis involved in repair from UV damage. Pol I, and, to a lesser extent, Pol II, and Pol III are demonstrably involved in this process in E. coli (52). All three enzymes of B. subtilis are insensitive in vitro to the drug (53). In the literature, conflicting data are available. Driedger and Grayston (15) showed a lack of effect of NAL on the repair of X-ray damage in Micrococcus radiodurans. Eberle and Masker (17) in E. coli showed that at early times a good amount of repair synthesis occurs in UV-treated cells; repair synthesis is also seen in the presence of NAL. Only after prolonged incubation with the drug is a reduction of repair synthesis observed. Since the repair of UV damage to SPP1 DNA relies on the host cell enzymes (the HCR of SPP1 is impaired, e.g., in polA mutants of B. subtilis [30, 531, as well as in several other UVsensitive mutants [39], we studied whether the repair of UV-damaged SPP1 DNA was affected by NAL under conditions in which DNA replication is over 95% inhibited. In the first place, we determined HCR of UVirradiated phage in the presence of NAL by the procedure reported in Materials and Methods. UV irradiation of the phage to 2,500 erg/mm2, which gave a residual survival of 5 x 10-3, did not show any further variation of plating efficiency if the phage was plated on NAL (data not shown). Thus the host repair process, including the amount of DNA synthesis necessary to it, does not seem to be sensitive to the drug. The use of a polA strain as host causes a quite lower efficiency of plating of irradiated SPP1 phage in the absence of NAL (53). Thus the removal of a major component of the repair apparatus (most

112

CANOSI ET AL.

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SPP1 DNA

DNase Plating I P81706 spores (-NAL)

SB 202 competent

cells

\

Plating II

1 10' SPP1 DNA NAL

30'

I DNase

spores PB1706 (+NAL

25ug/mi

)

25 ug/mi

FIG. 2. Scheme of the experiment used to test the effect of NAL on SPP1 transfection. TABLE 2. Effect of NAL on SPP1 and SPO-1 transfection" DNA

Plating 1 (-NAL)

ml)cnFUAgl PFU/ml concnl)yg/

Transfection frequency (A)

Plating 2 (+NAL, 25 jLg/ml)

frePF/lTransfection PFU/ml quency (B)

B/A ratio

SPP1

2.28 x 10-1.13 x 10-3 1.05 x 10-2

6 x 103 4.06 x 10' 2.6 x 10"

2 x 10-' 1.35 x 10-3 8.6 x 10-3

0.87 1.19 0.82

3.0 x 104 1.0 x 10-4 1.64 x 10-3 4.91 x 103 3.1 x 10-3 9.25 x 102 a Titer of competent cells was 3 x 108 cells/ml.

3.0 x 104 5.0 x 10 8.5 x 10}

1.0 X 10-4 1.67 x 10-3 2.83 x 10-

1.00

0.01 0.10 1.00

6.84 x 103 3.4 x 10: 3.15 x 106;

SPO-1 5 10 30

probably, of excision repair) could allow the detection of a minor effect of NAL on repair synthesis. The addition of NAL does not cause any further decrease in plating efficiency of irradiated DNA on a polA strain (polA42 [53]; data not shown). Also, the effect of NAL was studied on the infection of UV-irradiated DNA. In this case, in view of the essential requirement of recombination for transfection to occur, the damaged DNA may utilize the recombination-repair pathway more efficiently than the whole phage. The scheme of the experiment was the same as described above (Fig. 2), but in this case, UV-irradiated SPP1 DNA was used. SB 202 competent cells were transfected with SPP1 DNA irradiated with 150 erg/mm2 to a residual transfecting activity of 10% in the presence and absence of NAL. The data are reported in Table 3. NAL (25 ,ug/ml) did not affect the repair mechanisms of transfecting DNA. Here too, the use of a polA strain reduced per se the efficiency of repair, probably by abolishing the excision repair pathway. Nevertheless, we did not observe any effect of NAL on the residual survival. Sensitivity of the experimental system to an inhibitor of recombination and repair. In view

1.02 0.91

of the negative results with NAL, one could surmise that the approach used in these experiments is not apt to demonstrate in a sensitive way an effect on the recombination or repair processes. We could rule out this possibility by the use of caffeine, which is a drug whose depressing activity on recombination and repair has been demonstrated (38). In Table 4 we report the effect of caffeine and/ or NAL on transfection of SB 202 competent cells treated with unirradiated or irradiated (200 erg/mm2) SPP1 DNA (0.01 jig/ml). The scheme of the experiment was similar to that previously reported (Fig. 2). The low dose of transfecting DNA was required to enhance the effect of caffeine on transfection. Caffeine was used at 1.5 mg/ml (a concentration that reduces transfection to 20%), NAL was used at 25 ,ug/ ml. Either caffeine or NAL was present during the whole time course of the experiment, but only NAL was present at the same concentration (25 ,ug/ml) in the TY plates used to titrate the infective centers. The results reported in Table 4 demonstrate that the experimental system used in our work is quite adequate to show the inhibitory effect of caffeine, both on recombination (SPP1 DNA transfection) and on repair (UV-irradiated SPP1 DNA). Under the same conditions, NAL gave no effect on either

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113

TABLE 3. Effect of NAL on transfection with UV-irradiated SPP1 DNAa Plating 2 (+NAL, 25 ,ug/ml)

Plating 1 (-NAL)

Control DNA (A)

B/A ratio

UV-irradiated DNA (B)

Control DNA (C)

UV-irradiated DNA (D)

7 x 10-3 8.7 x 10-4 0.12 9 X 10-4 7.4 x 10-3 SB 202 (parental) 1.4 x 10-4 4.2 x 10-3 0.03 1.5 x 10-4 4.1 x 10-3 PB 1642 (polA 42) a Final concentration of irradiated and unirradiated SPP1 DNA was 0.1 ,ug/ml.

TABLE 4. Effect of caffeine (caff.) and NAL on transfection with irradiated and unirradiated SPPJ DNAa Condition of expt

SPP1 DNA SPP1 DNA (UV irradiated) SPP1 DNA + caff. SPP1 DNA (UV irradiated + caff.) SPP1 DNA + NAL SPP1 DNA (UV iiTadiated + NAL) SPP1 DNA + caff. + NAL SPP1 DNA (UV irradiated + caff. + NAL) a

PFU/ml

% of

103 102 102 10'

100

2.30 x 102 1.30 x 102

89

4.60 x 102 1.50 x 10'

17.5 0.50

2.62 1.25 6.00 1.00

x x x x

D/B ra-

Transfection frequency

Transfection frequency

Competent cells

4.7 23 0.38

4.9

The DNA concentration was 0.01 ,ug/ml; cell titer (4 x

102 cells/ml) is not affected by the presence of caffeine or NAL.

process, and did not cause any further decrease of plating efficiency when added to caffeine. We can thus conclude that in all probability the still unidentified NAL target is involved specifically and exclusively in DNA replication, and is not utilized in the recombination or repair processes. Effect of HPUra on transformation. HPUra (and the related drug 6-(p-hydroxyphenylazo)isocytosine) specifically inhibits DNA replication and Pol III of gram-positive organisms (4, 8, 9, 13, 20). Best studied is the interaction of HPUra with the Pol III of B. subtilis, which is based on the competition of the drug with deoxyguanosine 5'-triphosphate (12, 20, 35). The essential role of Pol III for chromosome replication in microorganisms is well established from the availability of temperature-sensitive mutants of E. coli (21). In B. subtilis, the Pol III overall molecular and catalytic properties are analogous to those of the enzyme with the same denomination in E. coli. Also, in B. subtilis this polymerase is essential to DNA replication, as shown by the inhibition by HPUra (7), by the observation that an HPUra-resistant strain has a Pol III insensitive in vitro to the drug (13, 19), and by the description of a temperature-sensi-

D/C ratio

tio

0.12

0.96

0.03

0.93

tive mutant of this enzyme, which is also thermosensitive in DNA replication (4, 19). In E. coli, Pol III seems important also for repair-type synthesis, whether in excision repair, or in postreplication repair (56). The data obtained in B. subtilis tend to rule out an essential involvement of Pol III in repair of UV damage (7). Two previous reports (16, 33) indicate a lack of sensitivity of the B. subtilis transformation process to HPUra; in both cases, the drug was present only during the incubation of the competent cells with DNA, and one cannot rule out the possibility that Pol III intervenes at a later stage of the process. We therefore decided to study the possible effect of HPUra in transformation by an experimental scheme analogous to the one used previously for NAL (Fig. 2). In the first place, we determined the HPUra dose that completely inhibits DNA replication, and we isolated an HPUra-resistant strain to use as donor DNA. The HPUra concentration of 25 ug/ml rapidly produced complete inhibition of DNA replication (see Fig. 3). The increase in cell turbidity was not appreciably affected by this treatment, and the cell titer was only slightly reduced after 1 h. The same inhibition of DNA replication was observed whether the drug was used in its oxidized or reduced forms. The reduced form is the only one active on the enzyme in vitro (4, 20, 35, 42); the two forms are both active in vivo, and the oxidized form is probably reduced in vivo to the active molecule by a bacterial enzyme (4, 42). In fact, in some instances, the oxidized form is more active in vivo than the reduced one, (8) probably because of easier penetration into the cells. Since DNA replication in our experiments was completely inhibited after a few minutes by at least 25 ,ug of HPUra per ml, the use as donor DNA in transformation of a strain carrying the azp-12 mutation isolated by Brown and identified with the structural gene of Pol III (N. Brown, personal communication) was not possible; this mutation in fact conferred resistance only to 20 ,g of HPUra per ml (as measured by

114

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aI Ilo.

MI -I J

ISO TIME

0

60

(MINUTES)

FIG. 3. Effect of the reduced derivative of HPUra on DNA synthesis and turbidity in PB 566/2 cultures. Incorporation of [3H]dThd into trichloroacetic acid-insoluble material was determined as described in Materials and Methods. Cell turbidity (a), and [3H]dThd incorporation (b) in presence ofHPUra concentrations: 0 pAglml (A.), 10 ug/ml (@), and 25 Aglml (U), respectively.

the ability to form colonies on solid media). We have therefore isolated a strain that is resistant to a higher level of the drug. From NTG (N-

methyl-N-nitro-N'-nitrosoguanidine)-mutagenized SB 202 cells we have obtained several independent HPUra-resistant mutants. One of these (PB 1728) is resistant to a HPUra concentration of 80 ,g/ml, versus 3 ,ug/ml for the parental strain; the mutation was denominated azp-80, and this strain was used as donor DNA in transformation experiments. Competent cells of strain BR 151 were treated with HPUra for 10 min before addition of transforming DNA (0.1 ,ug/ml) from strain PB 1728 (azp-80) and, after the 30-min incubation with DNA, plated on HPUra-containing plates (experimental scheme analogous to that of Fig. 2). The effect of HPUra or its reduced derivative was measured. Table 5 reports, first, the results obtained in two different experiments in which the drug was used without any previous treatment. Transformation to HPUra resistance was reduced to about 20% with 20 or 30 ,ug of the drug per ml (experiments 1 and 2). With the reduced derivative of the drug, the inhibitory effect was observed only when a higher concentration was used during the incubation for transformation (experiments 3, 4, and 5). The effect on the transformation for a biochemical marker was less pronounced (reduction to approximately 40%), but in this case the drug was not present during the incubation in the selective media, and the integration process could partly take place at this time. The inhibition observed suggests a role in

transformation of the HPUra target, i.e., Pol III; the different effects of the reduced or oxidized drugs were probably due to differing permeability between the two forms. Lack of effect of HPUra on transformation in a resistant strain. If the molecule on which HPUra exerts its action to inhibit transformation were the Pol III, we would expect that a mutant in which this enzyme is resistant to the drug should be resistant to the inhibitory effect on transformation. The mutant we have utilized to extract the donor DNA in the experiments reported in Table 5 cannot be utilized for this purpose, since this mutation does not concern Pol III: Ciarrocchi and Fortunato (personal communication) have purified the Pol III of this organism and found that it is as sensitive to HPUra as the wild-type polymerase. On the other hand, the azp-12 mutant isolated by Brown contains Pol III molecules that are insensitive to the drug in vitro; if the effect of HPUra on transformation were aspecific, and due to the interaction with a different molecule, it should be exerted also in the latter mutant. We transferred the azp-12 mutation into another strain (Table 1), and we used it as a recipient in the experiment reported in Table 6. The results showed that the transformation for an auxotrophy marker was not affected by HPUra in this mutant, as expected assuming that the interaction of the drug with Pol Ill is the basis of the reduction of transformation in the strain containing the HPUra-sensitive form of the enzyme. It is worth noticing that the efficiency of

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TABLE 5. Effect ofHPUra and its reduced derivative on transformation Conditions in the expV

HPUra Expt 1 Plating 1 (control) Plating 2 (+HPUra, 30

ml

Frequency transforma-of tion Met+

Transformants Azpr/ mlb

Frequency transforma-of

vival

Transformants Met+/ ml

1.91 X 106

6.6

x

10-3

2.5

x

103

8.6

x

10-6

10"

2.6

x

10-3

5.2

x

102

2.2

x

10-6

Sur-

Viable cells/

2.9

x

108

1

2.4

x

1O#

0.82

6.2

x

tion

Azpr

,ug/ml)

Plating 2 to plating 1 ratio Expt 2 Plating 1 (control) Plating 2 (+HPUra, 20 ,ug/ml) Plating 2 to plating 1 ratio

1.9

x

10

1

1.1

x

10"

5.7

x

10-3

4.9

x

102

3.7

x

10-6

1.7

x

1O"

0.89

4.7

x

108

2.7

x

10-3

1.1

x

102

6.4

x

10-7

Reduced derivative of HPUra Expt 3 Plating 1 (control) Plating 2 (+HPUra, 30

2.97

x

10"

1

1.72

x

106

5.8

x

10-3

1.52

x

104

5.1

x

10-'

2.79

x

108

0.94

1.62

x

106

5.8

x

10-3

1.24

x

104

4.4

x

10-i

0.47

jig/ml)

Plating 2 to plating 1 ratio Expt 4 Plating 1 (control) Plating 2 (+HPUra, 45 ,g/ml) Plating 2 to plating 1 ratio Expt 5 Plating 1 (control) Plating 2 (+HPUra, 100

0.25

0.39

0.17

0.86

1.0

4.18

x

108

1

1.51

X

106

3.6

x

10-3

1.02

x

104

2.44

x

10-'

3.65

x

101

0.87

7.7

x

10'5

2.1

x

10-3

6.3

x

103

1.7

x

10-}

0.69

0.58 2.62

x

10"

1

2.74

x

10"

1.05 X 10-2

1.58

1.94

x

108

0.74

2.64

x

104

1.36

1.69

x

10-4

x

x

104

6.0

x

10--'

10-3

8.7

x

10-6

jig/ml)

0.14 0.013 Plating 2 to plating 1 ratio a See Fig. 2 for the scheme of the experiment. b Transformants for HPUra resistance (Azpr) were selected on nutrient agar plates containing 30 ,ug/ml in experiments no. 1, 3, 4, and 5, and 20 gg/ml in experiment no. 2.

TABLE 6. Effect of HPUra on the transformation of the strain PB 1729 (azp-12)a Condition of expt PB 1729 (control [11)

Viable cells/ml

1.66

x

108

Transformants/ml

Lys+ 9.5

x

103

Transformation frequency

5.7

x

Plating 2

to plating 1 ratio

10-O

0.96 5.5 x 10-; 1.43 x 10" PB 1729 (+HPUra [2]) Lys+ 7.9 x 103 a HPUra in the treated samples was added (at the concentration of 25 jig/ml) 10 min before DNA was added. Lys+ transformants were selected in presence of 10 ,zg of HPUra per ml.

116

CANOSI ET AL.

TIME

(MINUTES)

FIG. 4. Effect of HPUra on SPP1 DNA synthesis and phage lysis. Cells of strain PB 566/2 were infected as described in Materials and Methods in the presence of HPUra. DNA synthesis was followed by [3H]dThd incorporation (a), and cell lysis was monitored by measuring Aw40 nm (b). Symbols: Uninfected culture without HPUra (@); infected culture, without HPUra (0); infected culture in presence of 30 jAg of HPUra per ml (U); uninfected culture in presence of 30 jAg of HPUra per ml (A). The final phage yield was 3.4 x 109 PFUIml in the control experiment, and 1.7 x 108 in the presence of HPUra.

transformation of the azp-12 strain was reduced to approximately 5% with respect to that of azp-12+ strains (data not shown); this is a further indication that an alteration of the Pol III molecule causes an impairment of the transformation process. Effect of HPUra on transfection. The observed reduction of transformation by HPUra action on Pol III indicates a direct involvement of this molecule in the recombination process. It could be argued that the effect is secondary to the interaction of the drug with the growing point apparatus, the slow movement of which is demonstrated in the competent cells during transformation (3); alternatively, the expression of transformants could require cell DNA replication, and only for this reason would the HPUra interfere with transformation. This possibility can be ruled out by studying the effect on another recombination process not involving the B. subtilis genome, but utilizing its recombination machinery, i.e., the multi-hit transfection with phage DNA (see above). SPP1 phage was not fit for this purpose, since its DNA synthesis turned out to be sensitive to HPUra. Phage production was strongly inhibited by the drug, whereas it was unaffected by NAL (see legends to Fig. 1 and 4); the reduction

was

synthesis,

due to an inhibition of SPP1 DNA as shown in Fig. 4.

J. BACTERIOL.

Instead, SPO-1 DNA transfection is adequate for studying the effect of HPUra, since its DNA synthesis and phage production are insensitive to the drug (48, and our unpublished data) and, as mentioned above, the transfection follows a multi-hit dependence on DNA concentration. The effect of HPUra on SPO-1 DNA transfection was tested by the same scheme as reported in Fig. 2 for SPP1 transfection and NAL. Competent cells of strain SB 202 were preincubated for 10 min with 30 jig of HPUra per ml, and then with SPO-1 DNA for 50 min; after deoxyribonuclease action, the infective centers were scored on plates containing the same concentration of drug, using as indicator strain the azp80 mutant. The results, reported in Table 7, demonstrated a pronounced inhibition of SPO-1 transfection. The possibility that this inhibition depends on an effect of the drug on the host DNA replication seems extremely unlikely, the more so considering that the interference of host replication by other means (NAL) affects neither this process (Table 2) nor the host transformation (44). We can summarize our findings by stating that HPUra inhibited transformation and transfection most probably by a direct interference with the polymerizing activity of Pol III. This observation is in agreement with the data obtained in our laboratory with a temperaturesensitive mutant containing low levels of Pol III at a permissive temperature; this mutant is reduced in the ability to sustain transformation and transfection to 10% of that of the wild type, also under permissive conditions (manuscript in preparation). The possibility that the inhibition may be due to the need of cell DNA replication for the achievement of competence or for the expression of transformants is made unlikely by (i) the lack of effect of NAL and (ii) the inhibition of transfection for which a requirement of host DNA replication seems unreasonable. Effect of HPUra on repair of UV damage. Brown (7) showed that HPUra does not appreciably affect the repair replication of B. subtilis after UV irradiation, implying that Pol III is not important in this process. In agreement with this, we observed that the HCR of SPO-1 phage (a process depending, e.g., on the Pol I of its host [30, 53]) was not appreciably affected by the drug, as shown in Fig. 5. This would confine the function of Pol III (other than DNA replication) to the DNA synthesis involved in recombination. A confirmation of this is obtained from the data of Table 8 on the effect of HPUra on the transfection of UV-irradiated DNA. The presence of HPUra, as already seen in Table 7, depressed transfection efficiency, probably by

VOL. 126, 1976

DNA REPLICATION INHIBITORS AND RECOMBINATION

117

TABLE 7. Effect of HPUra on SPO-1 transfection" SPO-1 DNA concn

"

(tg/

Plating 1 (-HPUra) Transfection fre-

ml)

PFU/ml

12.5 25

1.29 x 10 8.45 x 10

quency (A)

3.7 x 10-4 2.41 x 10-1 The titer of competent cells was 3.5 x 108 cell/ml.

0

500 1000 UV dose (erg/mm2 )

FIG. 5. Effect of HPUra on HCR of UV-irradiated SPO-1 phage with SB 202 and PB 1642 strains. The plating efficiencies of irradiated suspensions of SPO1 were determined by the procedures reported in Materials and Methods.

interfering during recombination; the transfection of UV-irradiated DNA was only slightly depressed (down to 68%, versus a reduction to 29% of the unirradiated DNA). Thus, the irradiation of DNA did not cause an important further decrease in transfection ability. If one uses a strain in which the excision repair mechanism is severely damaged, like strain PB 1642 (bearing the polA mutation), the situation is different; the removal of the major repair machinery reveals a further appreciable reduction in survival of UV-irradiated DNA caused by HPUra. The most simple explanation is that this reduction corresponds to the contribution of recombination for the repair of UV damage; only this would be sensitive to HPUra, in agreement with the observations of the previous paragraph.

Plating 2 (+HPUra 30 /Ag/ml) Transfection frePFU/ml quency (B)

8.4 x 103 6.15 x 104

2.4 x 10-1.76 x 10-4

B/A ratio

0.065 0.072

Genetic mapping of nal-3 and azp-80 markers. The genetic location of the mutations conferring NAL resistance to the strain PB 1706 (nal-3) HPUra resistance in the strain PB 1728 (azp-80) was determined by PBS-1 transduction crosses. The results of a three-point cross experiment involving purA, cysA, and nal-3 are reported in Table 9. The data show that the nal-3 mutation is located between the purA and cysA markers. As we have previously described (10), several independent NAL-resistant mutations are located in this area. As it can be seen in Fig. 6, the nal marker is in a chromosomal region that contains several other genes involved in DNA metabolism, namely, recF, recD (39), dnaC, dnaG, dnaH (28), dna-8132 (23), and resistance to novobiocin (novA) (25). In the search of the linkage relationship of azp-80 to known markers on the B. subtilis chromosome, a series of two-factor PBS-1 transduction crosses was performed. The first indication of linkage was obtained with an unmapped marker for the requirement of glycine (strain PB 3292). The data for this linkage are reported in Table 10. This gly marker is different from the one described by Naumov et al. (41), located between recA and argC, on the basis of absence of linkage with these markers (unpublished data) and of differences in growth requirements: the Naumov strain requires either glycine or serine, whereas our mutant has a strict requirement for glycine, which cannot be replaced by serine. The absence of linkage of the gly marker of strain PB 3292 (measured by PBS-1 transduction) with Iys and trpC (unpublished data) suggests that this mutation is also different from the one described by Kelly and Pritchard (29). So we propose to denominate the mutation mapped by an unstable linkage in transformation, between rib and tyr (29) glyA, the Naumov mutation glyB and glyC this new locus for glycine requirement. The glyC and azp-80 mutations are located in the hisA-sacA region. The linkage of these new markers with other ones described in this region is reported in Table 11 (see also Fig. 6).

TABLE 8. Effect of HPUra on transfection with UV-irradiated SPO-1 DNA" Plating 1 (-HPUra)

Plating 2 (+HPUra)

Transfection frequency Transfection frequency Competent Competent cellscells ~~~~B/A Control DNA DNA+V(B) tio Control DNA DNA+UV(D) (A) DN+V()(C) ra-

1 X 10-3 2.29 x 10-4 0.229 SB 202 (paren2.86 X 10-4 tal) PB 1642 (polA42) 1.07 x 10-3 5.0 x 10-5 0.0467 4.67 X 10-4 a Transfection was performed at 10 ,ug of DNA per ml.

1.56

x

10-4

1.63 X 10-;

DIC ra- C/Atiora-

D/B ratio

tio

0.546

0.286

0.68

0.0346

0.436

0.32

TABLE 9. PBS-1 mediated three-factor crosses involving purA, cysA, and nal-3. Transductants

Implied order

Recipient strain

Donor strain

Selection

PB 1706 trpC2 hisB2 tyrA1 aroB2 nal-3

PB3409 purA16 cysA14 leu-8

Ade+

uvr

Ade+ Ade+ Ade+ Ade+

No.

Cys+ Nalr Cys+ NalS Cys- Nalr Cys- NalS

23 7 75 40

purA16 nal-3 cysA14

Cys+

hisA

Classes

Cys+ Ade+ Nalr Cys+ Ade+ NalS Cys+ Ade- Nalr Cys+ Ade- Na1S

19 10 46 59 dna-s132 recF recD dnaC dnaG dnaH purA nov-i nal-3 cysA

i

?C

0

-V

E FIG. 6. Genetic map of B. subtilis drawn according to Harford (24) and Lep6sant-Kejzlarovd et al. (32), in which are indicated several markers regularly scattered along the chromosome. In the enlarged sectors of the map are reported the linkage relationships of nal-3, azp-80, and glyC with other markers of known location. Map distances are expressed as percentages of recombination (see Materials and Methods). 118

VOL. 126, 1976

DNA REPLICATION INHIBITORS AND RECOMBINATION

The orderglyC ctrAl azp-80 was determined by three-factor crosses (Table 11). Concluding remarks. We can sum up our findings as reported in Table 12. This is our interpretation of the table. The NAL target is specific for the synthesis taking place at the bacterial growing point and is not involved in the DNA synthesis necessary for repair or recombination; the HPUra target Pol III is involved in the DNA synthesis necessary for transformation and transfection, and therefore is most likely directly used in the recombination process. It is not important in the repair process, except probably in the fraction of repair that depends on recombination. The Pol III of E. coli is demonstrably involved in repair process, as well as in DNA replication; the idea that the homologous enzyme of B. subtilis is

119

TABLE 12. Summary of inhibitory effects of NAL and HPUraa Process

Effect by NAL

DNA replication Bacillus subtilis SPP1 phage SPO-1 phage

a a a

11 Fig. 1 18

Recombination Transformation Transfection

-

44 Tables 2, 3, 4

-

This work

HCR in wild-type host a

Symbols:

Reference

Effect by HPUra

Reference

a a

7 Fig. 3

Fig. 4 48

-

4 4

Tables 5, 6 Tables 7, 8

-

Fig. 5

(-a) No effect; (4) appreciable reduction.

TABLE 10. Two-factor crosses by PBS-I-mediated transduction involving azp-80 and glyC Donor strain

Recipient strain

Selection

Transductants Classes

No.

% Reconbination

Gly+ Azpr Gly+ AzpS

4

31

69

92

Map units

PB 1728 tyrAl hisB2 trpC2 aroB2 azp-80

PB 3292 thy trpC2 glyC

Gly+

PB 3292 thy trpC2 glyC

CU 479 trpC2 ctrAl

Ctr+

Ctr+ Gly+ Ctr+ Gly-

69 159

70

30

PB 1728 tyrAl hisB2 trpC2 aroB2 azp-80

CU 479 trpC2 ctrAl

Ctr+

Ctr+ Azpr Ctr+ Azps

100 97

51

49

QB 2 sacA321 purA16

PB 3292 thy trpC2 glyC

Gly+

Gly+ Suc+ Gly+ Suc-

125 19

13

87

GSY 1027 trpC2 metB4 uvr-1

PB 3292 thy trpC2 glyC

Gly+

Gly+ Uvr+ Gly+ Uvr-

86 14

14

86

GSY 1027 trpC2 metB4 uvr-1

BD 92 trpC2 hisAl cysB3

His+

His+ Uvr+ His+ Uvr-

32 70

69

31

PB 3292 thy trpC2 glyC

BD 92 trpC2 hisAl cysB3

His+

His+ Gly+ His+ Gly-

250 0

0

>100

TABLE 11. Three-factor crosses by PBS-I-mediated transduction to order glyC and azp-80 markers Transductants Donor strain

Order implied by results

Recipient strain Selection

Classes

No.

Gly+ Azpr Ctr+ Gly+ Azpr CtrGly+ Azps Ctr+ Gly+ Azp5 Ctr-

20 70 0 50

Gly+ Azpr Suc+ Gly+ Azpr SucGly+ Azps SucGly+ Azp-5 Suc-

92

CU 479 trpC2 ctrAI

PB 1731 thy trpC2 glyC azp-80

Gly+

QB 2 sacA321

PB 1731 thy trpC2 glyC

Gly+

purAl6

azp-80

0

glyC ctrAI azp-80

g8glyCazp-80sacA321

28

120 CANOSI ET AL. involved in a process concerning the exchange of sequences between DNA molecules is not unreasonable.

J. BACTERIOL.

20.

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DNA REPLICATION INHIBITORS AND RECOMBINATION

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