Requirement of E. coli DNA synthesis functions for the lytic replication of bacteriophage P1

VIROLOGY

141. 193-206 (1983)

Requirement of E. co/i DNA Synthesis Functions for the Lytic Replication of Bacteriophage Pl NISSIM

HAY AND GERALD

COHEN1

Departmen? of Microbiology, Thx George S. Wise Faculty qf Life S&TUZS, Uniuersity of Tel Aviv, Tel Aviv, Israel Received April 28, 1983;accepted July 25, 1983 Pl lytic growth was examined in a number of different temperature sensitive mutants Growth was analyzed by measurements of E. coli that affect chromosomal replication. of phage burst sizes and specific DNA synthesis. Efficient Pl growth required each of the bacterial elongation functions dnaE (pdC), dnaZ (sub units of E. cdi polymerase III holoenzyme), and dn& (primase) but was not dependent on the elongation function dnaB (mobile promoter). Of two initiation functions tested the dnuA function was found to be dispensable for normal growth whereas the dnnC function was essential. Temperature component of shift experiments with different dnaC mutants showed that the initiation the dnaC function was needed continuously throughout at least the first half of the lytic cycle, while the dmC elongation activity was probably required during the entire cycle for normal phage yields. In two respects the dependence of Pl lytic growth on E. coli DNA synthesis functions was significantly different from that reported for Pl plasmid replication (Scott and Vapnek, 1930). Thus, lytic replication was far more dependent on a functional poK gene product than was plasmid replication and did not require the bacterial dnoB product.

INTRODUCTION

Bacteriophage P1 is a complex temperature phage that multiplies in the bacterium Escherichia coli Like other temperate coliphages, such as X and P2, its genome can be propagated by lytic growth, with the production of infectious progeny particles, or maintained as a stably inherited prophage. In h and P2 the prophage is integrated at a specific site in the bacterial chromosome and replicates in situ with it. In contrast, Pl prophage exists as an autonomously replicating, low copy number, plasmid that can be isolated as a covalently closed circular DNA molecule (Ikeda and Tomizawa, 1968). Pl prophage replication is, therefore, circular in nature and presumably similar to that of other bacterial replicons. We have recently shown that Pl lytic growth also proceeds, in part, by a circular ’ Author addressed.

to whom requests

for reprints

should be

mode of replication (Segev et uZ., 1980; Cohen, manuscript, in press). Circular DNA molecules are made soon after lytic infection through a Pl promoted, site-specific, recombination event at the terminally redundant ends of the linear phage genome (Hochman et al, manuscript, in press). At early times in infection most Pl replication is circular, or theta-like, whereas at later times in infection a transition to a rolling circle mode of replication probably occurs which depends on a funtional host recombination system. Because of the circular nature of prophage and lytic replication it was of interest to examine the structural and functional requirements of these two processes. At present little is known about the origin and direction of Pl DNA synthesis or of the involvement of phage specific functions in these replication systems. Some information on the role of host functions comes from a study of Nainen and Vapnek who analyzed prophage replication in a number of E. wZi temperature sensitive mutants that affect chromosomal DNA

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synthesis. Their conclusions, cited in the review of Scott and Vapnek (1980), were that each of the bacterial gene products, dnuB (mobile promoter), dnaC (initiation), and dn.aG (primase) were necessary for plasmid replication. Surprisingly, plasmid DNA synthesis occurred extensively in poZC!(dnaE) b mutants at the restrictive temperature, although the Pl DNA made in these conditions could not be recovered in a covalently closed form. In the present study we report on the dependence of Pl lytic growth on E. coli initiation and elongation replication functions. Pl growth was analyzed by measuring phage yields and DNA synthesis in different temperature sensitive mutants that affect chromosomal replication. Our results show that efficient Pl growth required each of the bacterial DNA elongation functions poZC, dnaZ (subunits of polymerase III holoenzyme), and dmG. The dnaB gene product, however, was not required for growth due to the fact that Pl expresses an analogous protein of its own in lytic infection (D’Ari et a+!,1975; Ogawa, 1975). Of two host initiation functions that were tested, the dmC function was found to be essential for Pl replication while the dnaA function could be dispensed with. Similar conclusions concerning the requirement for initiation functions for Pl lytic growth were reported TABLE

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by Hooper and Eagan (1981) based on measurements of phage bursts. Temperature shift experiments with two dnaC mutants showed that the initiation component of the dnaC function was required continuously throughout at least the first half of the growth cycle, whereas the elongation component was probably needed during the entire lytic cycle for normal phage production. MATERIALS

AND

METHODS

Bacteria and phuge Bacteria used in this study were all derivatives of E. coli K12 and are listed in Table 1. Pltir is a virulent strain of phage Pl (Scott, 1968). PlCm cl.100 is a thermoinducible mutant of the derivative PlCm (Kondo and Mitsuhashi, 1964) that forms clear plaques at 37” and turbid plaques at 32” (Rosner, 1972). Media LB broth supplemented with 5 r&f CaCl, (LBC medium) was used for preparation of phage stocks (Miller, 1972). Top and bottom agar for routine assay of phage have been described elsewhere (Rosner, 1972). The standard medium for measuring DNA synthesis was TD, a Trisglucose-Casamino Acid medium (Scott and Shuster, 1973), that contained per liter 0.1 MTris-HCl (pH 7.5), 5.8 g NaCl, 3.7 g KCl, 1

BA(XERIAL STRAINS Strain CR.34 El779 E107” E486” BT.399’ HMT” N167” DG76 PCl” PC2” AX727” AX’i%‘R o Obtained *Obtained ‘Obtained

Genotype thpl la-6 thi-1 thyA dexx-1 lnc Yl strA67 tonA supE44 CR34 dnaA171 CR34 druzBlO’7 CR34 dnaE486 CR34 dnuG399 HfrH thy met HMT dnaA16’7 la-6 thyA deoc3 &Al53 DG76 dnaC1 DG76 dmC2 hc thi str dmZ2016 dnuZ ts revertant from B. Bachman from J. Wechsler. from M. Abe.

from the E. cnli Genetic Stock Center.

Reference Wechsler

and Gross (1971)

Wcchsler and Gross Wechsler and Gross Wechsler and Gross Gross (1972) Abe and Tomizawa Abe and Tomizawa Carl (1970) Carl (1970) Carl (1970) Filip et d, (1974) This work

(1971) (19’71) (1971) (19’71) (1971)

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1.1 g NH&l, 10 g Casamino Acids, 2 g glucose, 2 mg thymidine, and 1 mg vitamin B1. For phage infections TD medium was supplemented with 10 mM MgSOl and 5 m&Z CaClz (TDC medium). Preparation of phage stocha High titer phage stocks were prepared by liquid infection as previously described (Segev et &, 1980). Phage was purified from crude lysates by differential sedimentation and CsCl density gradient centrifugation. Typical phage yields from a 0.4-liter culture were 10” PFU. Low titer stocks were made by the confluent plate lysis method. Measurement of burst size. Cultures of temperature sensitive and wild type parental strains were grown at 30” in TDC medium to an ODSw of 0.3-0.4 (approximately 1 X lo* cells/ml). When burst sizes were determined by infection at the restrictive temperature, samples of cultures were transferred to 40” or 42” (depending on the strain, see section on results) and held at this temperature for 15 min in the

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case of elongation defective mutants or 4045 min for initiation defective and &UJZ mutants (see below). For burst size measurements made at the permissive temperature, 30”, this step was omitted. Plvir was added at a multiplicity at 0.02-0.05 PFU/cell and the mixture incubated for 10 min to allow for phage adsorption. Infection was started by diluting the infected cells lOOO-fold into medium held at the appropriate temperature and vigorously shaking the growth tubes. Unadsorbed phage was assayed immediately. Aliquots from the growth tubes were assayed for phage production at successive intervals of lo-20 min after infection. When burst sizes were determined by thermal induction of lysogens, PlCm cl.100 lysogens were grown in TD medium at 30” to 1 X 108 cells/ml, transferred to the restrictive temperature and then incubated with shaking for 90 min and assayed as described above. Temperature shif experimerzts. Cultures of &UC mutants (PC1 and PC2) and pa-

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FIG. 1. Rates of DNA synthesis in different E. coli temperature sensitive replication defective mutants at the restrictive temperature. Bacteria were grown at 30’ in TDC medium to an ODW of 0.3, or approximately 1 X 10s cells/ml, and then shifted to the restrictive temperature 40° (for dnuC mutants) or 42” (all other mutants). Samples were immediately distributed into prewarmed growth tubes and pulse labeled at selected times with pH]tbymidine for 2 min as described in Materials and Methods. DNA synthesis rates are given by the tritium label present in acid-insoluble material in each 2-min pulse. Symbols: A-dnaE (MC), A-dnaB, @-&WA, 6-dnuG, q -dnaZ, and O-wild type parent strain; II-dnaC1, n -dnaC2, and O-wild type parent strain.

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rental(DG76) cells were grown at 30’ in LBC medium to an ODSw of 0.3-0.4 (approximately 1 X 10’ cells/ml) and infected with Plvir at a multiplicity of infection of 0.05-0.1 PFU/cell. After 10 min, to allow for phage adsorption, samples were diluted lOOO-fold into fresh medium prewarmed at 30” and incubated with vigorous shaking. Immediately, and at successive intervals of every 20 min, samples were transferred to growth tubes kept at 40’ and shaking continued. After a total incubation time of 160 min (sum of times at 30” and 40”) phage titers were assayed. Bursts were calculated after taking into account unadsorbed phage.

Measurement of rates of DNA synthesis. Rates of DNA synthesis in infected and noninfected cells were determined from the incorporation of 13H]thymidine into acidinsoluble material in a 2-min pulse. Bacteria used in this study were, with the exception of the dnaZ mutant and its revertant, all thg- derivatives. Cultures of cells were grown at 30” in TDC medium to approximately 1 X lo8 cells/ml, divided into two equal parts and transferred to the restrictive temperature, 40” or 42” for 15-45 min (see below). Plzrir was added to one part at a multiplicity of infection of 5-10 PFU/cell and incubated for 10 min for adsorption of phage. Samples of 0.5 ml from infected and uninfected cultures were then rapidly distributed into prewarmed growth tubes kept at the restrictive temperature and vigorously aerated. At selected times rH]thymidine (sp act 50 Ci/mmol, Israel Atomic Energy Commission) was added to each sample to a final level of 10 &i/ml and after a 2-min pulse the incorporation of label was stopped by addition of an equal volume of cold 10% trichloroacetic acid containing 0.1% thymidine. Precipitates were collected on Whatman GF/C filters, washed with cold 5% trichloroacetic acid and ethanol and then counted in a liquid scintillation medium with a Packard Tricarb spectrometer. The rate of incorporation of [‘Hjthymidine into acid-insoluble material is a valid measure of the rate of DNA synthesis provided uptake of label and entry into the pool are not rate limiting. The rate of uptake of label in cultures was the

COHEN

same for pulses ranging from 0.5 to 2 min, i.e., incorporation was linear within this range. Also at cell concentrations below 2 X 10’ cells/ml proportionality was found between incorporation rate and cell density. All data were corrected for nonspecific adsorption of label to cells, determined by adding label after acid precipitation. Figure 1 depicts rates of DNA synthesis at the restrictive temperature in each of the temperature sensitive mutants and their parent strains used in this study. The data are presented in this form since we chose to follow DNA synthesis by pulse labeling experiments rather than the customary practice of monitoring accumulation of label. Based on the profiles shown in Fig. 1, appropriate incubation periods for cultures at the restrictive temperature, prior to phage infection, were chosen (15 min for elongation defective mutants, dnaB, dnuG, and polC, and 40-45 min for initiation defective mutants dnuA and dnaC and the elongation mutant, dnaZ) such that bacterial DNA synthesis had dropped to a low level while not affecting significantly the viability of cells. TABLE

2

Pl BURST SIZES w E wli TEMPERATURE SEKSITIVE MUTANTS DEFECTIVE IN DNA SYNTHESIS Burst size” Bacterial Initiation N167 HMT El77 CR34 PC1 PC2 DG76 Elongation El07 E486 BT399 CFt34 AX727 AX727R

strain

Relevant genotype

42”

30”

dnaA ts dnaA ’ dnaA ts dnaA+ dnaC ts dnuC is dnuC+

62 65 65 67
83 68 92

dnaB dnaE dnuG dna+ dnaZ dnaZ

62 5
75 47 48 65 43 47

mutants

mutants t.s ts (pelC) ts ts ts3

DPermissive and restrictive temperatures were 30’ and 42” with the exception fer dnaG (26* and 38’) and druK mutants (30° and 40”), respectively. Average of two or more experiments.

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sensitive mutations in each of these genes. Cultures of cells grown at the permissive temperature, 30”, were shifted to the restrictive temperature, 40”-42”, for up to 45 min to terminate chromosomal DNA synthesis and then infected with phage Pl. A virulent strain, Plvir, was chosen for these studies to prevent formation in infected cells of stable lysogens. In some experiments Pl replication was analyzed following thermal induction of lysogens. At appropriate times Pl growth was monitored by measuring rates of DNA synthesis and phage production.

RESULTS

A variety of temperature sensitive mutations that affect chromosomal DNA synthesis in E. coli have been characterized (Wechsler and Gross, 19’71; Wechsler, 1978). Mutations in genes dnaA and dnaC usually prevent initiation of chromosomal DNA synthesis at the restrictive temperature, but allow completion of a round of replication. Mutations in genes dnaB, dn& (poZC),dnuG, and dnuZ almost exclusively affect chain elongation. In the experiments described below Pl lytic growth was analyzed in bacteria containing temperature

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FIG. 2. Rates of DNA synthesis and growth profiles in Pl infected dnuA temperature sensitive and wild type hosts at the restrictive temperature. Bacteria were grown at 30” in TDC medium to an ODW of 0.3, or about 1 X 108 cells/ml, transferred to 42’ (restrictive temperature for the dnaA mutant) and incubated for 40 min. To one portion of the culture Plwir was added at a multiplicity of infection of 5-10 PFU/cell and incubated for 10 min for phage adsorption. Samples from each portion were then distributed into prewarmed growth tubes and pulse labeled immediately (zero time) and at selected times with rH]thymidine for 2 min as described in Materials and Methods. In a parallel experiment rates of DNA synthesis were measured in noninfected cells. Rates of DNA synthesis are given by the tritium label present in acid-insoluble material in each 2-min pulse. Growth of infected and noninfected cultures was monitored by optical density. Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols: O-infected and O-uninfected dnuA177 host; ~-infected and Cl-uninfected wild type (CR34) host, respectively.

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Lytic Gmwth Mutants

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dnaA. Pl growth was examined in two different dnaA mutants. Data for phage yields and rates of DNA synthesis for one strain are presented in Table 2 and Fig. 2, respectively. As judged by (i) the almost equal phage burst sizes and (ii) the similar growth (optical density) and DNA synthesis profiles in infected normal and mutant hosts at the restrictive temperature, it is evident that Pl lytic growth does not require the product of the dnaA gene. Measurements of rates of DNA synthesis were determined in these and other experiments after chromosomal DNA synthesis had been greatly reduced and we assume, therefore, that the DNA synthesis detected in phage infected cells reflects essentially only phage-specific DNA synthesis. Although we did not carry out hybridization experiments to verify this point, the characteristic bell shape pattern of DNA synthesis seen in Fig. 2 matches that of previous studies where hybridization was used to follow Pl specific DNA synthesis in lytic infection (Zabrovitz et aL, 1977). Moreover, at times later than 15 min after infection more then 90% of the newly synthesized DNA is Pl specific even in a wild type host (Segev et al., 1980). dnaC. Table 2 contains results for burst sizes of Pl in two dnaC mutants, one of which, dnaC2, affects initiation of chromosomal DNA synthesis and the other, dnuC1, affects both initiation and elongation (Wechsler, 1975). Data are given for measurements made at permissive and restrictive temperatures in mutant and wild type hosts. Phage yields were more than 200-fold lower at 40” in both dnaC mutants compared to the parental strain. In fact, as Fig. 3 shows, there appears to be no detectable burst in either mutant at the restrictive temperature. The lo-30%fold reduction in burst size in the mutant strains compared to the wild type strain at 30” suggests that the defect in the dnaC gene product is not fully repaired at this temperature. To determine whether the negligible burst size of Pl in dnuC mutants is due to a block in replication, DNA synthesis rates

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FIG. 3. Kinetics of phage production in dnoC temperature sensitive and wild type hosts at the restrictive temperature. Bacteria were grown at 30” in TDC medium to an ODm of 0.3, or approximately 1 X 10s cells/ml, transferred to 40” (restrictive temperature for the dnaC mutants) and incubated for 40 min. Pltir was added at a multiplicity of infection of 0.02-0.05 PFU/cell and the mixture incubated for 10 min for phage adsorption. Infected cells were then diluted into prewarmed growth tubea and assayed periodically for phage production as described in Materials and Methods. Symbols: +dnaCl, A-dnaC2, and s-wild type hosts, respectively.

were measured in infected and noninfected mutants at the restrictive temperature, 40”. Figure 4 shows that rates of DNA synthesis in infected dnuC1 cells were actually lower than those in uninfected cells, while in infected dmC2 cells rates were marginally higher than in the uninfected cells (lower panels). Rates of DNA synthesis in the wild type strain show the characteristic bell shape profile. By integrating the rates of DNA synthesis with time over the period of O-50 min, we find that Pl-specific DNA synthesis is at least 200-fold less in the dnaC2 mutant and is undetectable in the dnaC1 mutant as compared to the estimated Pl-specific DNA synthesis in the

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FIG. 4. Rates of DNA synthesis and growth profiles in Pl infected dnuC temperature sensitive and wild type hosts at the restrictive temperature. Protocol was the same as that described in the legend to Fig. 2 with the exception that the restrictive temperature used for dnnC mutants was 40” (Wechsler, 1975). Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols: O-infected and O-uninfected dmC1 host; r-infected and A-uninfected dnaC2 host: *infected and U-uninfected wild type (DG76) host, respectively.

parent strain at the restrictive temperature. The latter takes into account residual bacterial DNA synthesis (Segev et al, 1980). The small differences found in phage yields and DNA synthesis between the two dnaC mutants at the restrictive temperature could be explained if one of the mutants, dnaC2, was leaky. Clearly then, the failure of Pl to produce a burst in the two mutant strains is a consequence of its strict requirement for the dnuC gene product for replication. The fact that the infected mutant cultures showed no sign of undergoing lysis at the restrictive temperature (upper panels) emphasizes the point that Pl development must be severely restricted in these conditions. Pl lytic growth can also be initiated by thermal induction of a lysogen. To determine whether the dnaC function is required when growth starts from a plasmid prophage, Pl lysogens of the dnaC1 mutant and the parent strain were constructed. A

thermoinducible mutant of Pl was used so that after transferring cells to 40” induction (destruction of repressor) and inactivation of the dnaC product occurred together. Figure 5 shows rates of DNA synthesis and growth profiles of induced cultures of mutant and wild type lysogens. The patterns are similar to those shown in Fig. 4 and indicate that the dnaC product is necessary for normal growth after induction. Because we have previously shown that the dnuC product is not required for circularization of Pl DNA early in lytic infection (Segev and Cohen, 1981), the above results are consistent with the view that the dnaC function is required for Pl replication at a stage in the lytic cycle later than that of circle formation. Temperature shift experiments were carried out in an attempt to pinpoint at which stage in the lytic cycle the dmC gene product was needed. Since the dnuC gene product is involved in both initiation and

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FIG. 5. Rates of DNA synthesis and growth profiles of thermally induced PlCm cl.100 lysogens of temperature sensitive &&I and wild type hosts. Cultures of mutant and wild type cells containing the thermoindueihle prophage PtCm cl.100 were grown at 30” in TD medium to an ODsW of 0.3, approximately 1 X ld cells/ml, and then transferred to 40” (restrictive temperature for the &z&l mutant and inactivation of the thermosensitive cl repressor). At selected times samples of cells were pulse labeled and rates of DNA synthesis and growth profiles determined as described in the legend to Fig. 2. Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols: l heat induced o!nnCl lysogen and 0--dnaCl nonlysogen; m-heat induced wild type lysogen and Clwild type nonlysogcn, respectively.

elongation of chromosomal DNA synthesis, the results presented in the preceeding sections for the replication dependence of Pl on the dmC gene product could reflect a requirement for one or both functions. Temperature shift experiments were, therefore, carried out using the dmC2 mutant strain which is defective only in initiation and the dnuC1 mutant strain which is defective in both initiation and elongation functions. Cultures of cells grown at 30” were infected with Pl and at selected times shifted to the restrictive temperature for sufficient time to allow for a burst of phage. In each experiment the total time of incubation at 30” and 40” was 160 min, although phage production usually occured after 120-140 min. Figure 6 summarizes the data for two typical shift experiments

and shows the kinetics of burst (insert) at the permissive temperature in the two mutants. With the initiation defective druzC2 mutant no phage burst was obtained (that is a burst size of less than one) if cultures were shifted to the restrictive temperature at times within 25 min after infection. At later times the burst size increased rapidly so that shifts made at about 80 min (at the end of the latent period, see insert Fig. 6) resulted in a full burst size. Since the burst sizes of the parent strain at both permissive and restrictive temperatures were about the same as the mutant at 30”, the simplest interpretation of these results is that the dmC2 initiation function is needed continuously throughout at least the first half of the Pl lytic growth cycle. In similar

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FIG. 6. Influence of temperature shift on growth of Pl in dnaC temperature sensitive and wild type hosts Cultures of cells were grown in LBC medium at 30” to an ODW of 0.3, about 1 X 108 cells/ml, and infected with Pltir at a multiplicity of 0.05-0.1 PFWcell. After 10 min incubation for adsorption, samples were transferred every 20 min to prewarmed growth tubes and shaken at 40’ (restrictive temperature for dnnC host) for a total time of infection of 160 min (sum of time of infection at 30” and 407. Burst sizes were determined as described in Materials and Methods and are given as the ratio of phage titers for every sample at the end of 160 min divided by the titer determined immediately after adsorption, after correcting for unadsorbed phage. Symbols: l -dnaC1, A--dnaC2 and *wild type hosts, respectively. Insert shows kinetics of phage production at the permissive temperature, 30” (same symbols).

experiments with the initiation and elongation defective dnaC1 mutant the earliest time at which phage was produced was at 70-75 min after infection. Even after 100 min the burst size was just 10% that of the final value and only after a shift at 120 min was a normal burst size found. These results, taken together with those for the dnaC2 mutant, indicate that the dnuC elongation function is probably necessary for Pl growth throughout the major part of the lytic cycle. We cannot, however, rule out the possibility that the differences observed between the dnaC mutants in these experiments may be due to differences in

the rates of inactivation of the ts proteins following the temperature shift.

Growth in Ebngatim Mutants

Pl Lytic

Defective

dnuB. Figure ‘7 shows growth profiles and rates of DNA synthesis in Pl infected dnaB and wild type strains at the restrictive temperature. Burst sizes are reported in Table 2. Both sets of data show that Pl grows almost as well in the mutant host as in the parental strain. Pl codes for a dnaB-like function, ban, that is expressed during lytic infection (D’Ari et aL, 1975;

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FIG. ‘7. Rates of DNA synthesis and growth profiles in Pl infected dnuB temperature sensitive and wild type hosts at the restrictive temperature. Protocol as described in the legend to Fig. 2 with the exception that incubation at the restrictive temperature, 42”. was limited to 15 min prior to phage adsorption. Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols; O-infected and O-uninfected dnuB10’7 host; *infected and Cl-uninfected wild type (CR.34) host, respectively.

Ogawa, 1975). Early Pl DNA synthesis begins at lo-15 min after infection in the mutant strain but is immediate in the wild type host (Fig. 7). The delay in Pl DNA synthesis in the dnaB mutant may, therefore, be due to the time needed for the Pl ban protein to be made. dnuE. Burst sizes of Pl in a dnuE (poZC) mutant and the parental strain at the restrictive temperature are given in Table 2. Values in the pole host ranged between 8 and 15% that in the wild type strain. Reproducibly, the latent period in the poZC strain was considerably longer than that in the wild type strain (data not shown), and partial lysis occured at later times (see below). Figure 8 shows rates of DNA synthesis and growth profiles in infected mutant and normal hosts at the restrictive temperature. Assuming that the slightly higher rates of DNA synthesis in the infected culture as compared to the nonin-

fected culture represents phage-specific DNA synthesis, then Pl DNA synthesis in the mutant strain at the restrictive temperature was about 510% that in the parental strain. This estimate was obtained by integrating DNA synthesis rates with time from 0 to 50 min and adjusting the data in the wild type strain for residual bacterial DNA synthesis. The reduced amount of Pl DNA made in the plC host correlates well with the phage burst size in this host. Efficient Pl lytic growth requires, therefore, the polC component of E. coli polymerase III. dnaZ. Table 2 contains burst sizes of Pl in a dn.aZ mutant and a temperature insensitive revertant. We did not test whether the temperature insensitive phenotype of the latter was the result of true reversion or due to an extragenic suppressor (Walker et aL, 1982). Burst sizes in the dnaZ mutant were 15-20% that in

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FIG. 8. Rates of DNA synthesis and growth profiles in Pl infected dnoE (MC) and wild type hosts at the restrictive temperature. Protocol as described in the legend to Fig. 2 with the exception that incubation at the restrictive temperature, 42”. was limited to 15 min prior to phage adsorption. With longer preincubations at 42” the latent period in the dnoE strain was moreextendedthan that after 15 min (50 and 2.5 min in the mutant and parent strains, respectively) and infected mutant cultures were not detectably lysed. Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols: O-infected and O-uninfected dnuE486 host; -infected and Cl-uninfected wild type (CR&t) host, respectively.

the revertant. Compared to other parent strains used in this study the burst of Pl in the revertant strain was somewhat low. Measurements of Pl DNA synthesis and growth profiles in the mutant and revertant host at the restrictive temperature are shown in Fig. 9. Amounts of DNA synthesized in the Pl infected (and uninfected) dnaZ revertant strain appear to be normal compared with other strains used in this work. On the other hand, Pl DNA synthesis in the mutant host was much reduced at the restrictive temperature and was estimated as being no more than 10% that in the revertant strain at this temperature. The dnaZ gene product appears, therefore, to be necessary for normal Pl replication. drtuG. Burst sizes for Pl in a dnaG mutant and its parent strain at the restrictive and permissive temperature are reported

in Table 2. Since the dnaG strain was Pl resistant, we isolated a sensitive variant by selecting for a chloramphenicol resistant derivative with phage PlCrn. The data show that, as with the dnaE and dnuZ elongation functions, the dnaG function is also required for efficient Pl lytic growth. DISCUSSION

Table 3 compares results obtained in this work for the dependence of Pl lytic replication on E. coli DNA synthesis functions with those of a similar study by Nainen and Vapnek on Pl plasmid replication (see Scott and Vapnek, 1930). Both replication processes exhibit an identical requirement for bacterial initiation functions but differ in several respects in their requirement for host elongation functions.

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FIG. 9. Rates of DNA synthesis and growth profiles in Pl infected dnaZ and wild type hosts at the restrictive temperature. Protocol aa described in the legend to Fig. 2. Upper panels growth profiles, lower panels rates of DNA synthesis. Symbols: e-infected and O-uninfected dnaZ2016 host; m-infected and C-uninfected dnaZ temperature insensitive revertant, respectively.

Normal Pl lytic growth did not require the bacterial dnaA initiation function (Lanka and Shuster, 1970). Similarly, this function was dispensabIe for plasmid DNA TABLE DEPENDENCE

OF Pl F’LASMID

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ONE. CO& DNA SYKIWESIS FUNCTIONS Initiation functions Replication mode

dnaA

dnaC

-

+ +

Elongation functions dnaB

dnaG

dnaZ

+” * + ,;; * + sign denotes function is r&uired ior &cient replication; - sign denotes function is not required F.x efiieient replication; f denotes partial replication in the absence of the function. bBased on measurement of pha@ prdduction only. ‘Data of Nainen and Vapnek as reported by Scott and Vapnek (1999).

Lytic” Plasmid’

-.f

polC

,+

synthesis since Pl DNA continued to be synthesized in a drLaA temperature sensitive mutant at the restrictive temperature (Abe, 1974; Scott and Vapnek, 3980). These results suggest that either Pl codes for a dnaA-like protein that substitutes for the bacterial protein or, alternatively, that initiation of Pl DNA synthesis does not require such a protein. In contrast, a functional dnuC gene product was essential for Pl lytic growth (Hooper and Eagen, 1981). Plasmid replication was also dependent on this function since plasmid DNA synthesis was found to decrease in parallel with chromosomal DNA synthesis at the restrictive temperature (see Scott and Vapnek, 1980). The temperature shift experiments described in this work show that both dnaC initiation and elongation functions are needed continuously throughout the major part of the lytic cycle, if not the entire cycle, for efficient Pl growth.

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REPLICATION

OF BACTERIOPHAGE

In previous studies we showed that neither of the d-n.& initiation and elongation functions were needed for the circularization of parental Pl DNA (Segev and Cohen, 198I). Circularization of phage DNA is probably an important early event in I’1 lytic growth since early PI DNA synthesis is characterized, in part, by a circular mode of replication (Sevev et ah, 1980; Cohen, manuscript, in press). Furthermore, plasmid replication is also circular in nature. In view of these findings, as well as the fact that the dnaC protein is necessary for both plasmid and lytic replication, it seems likely that one role for this protein in I’1 growth is, in conjunction with the dnaB protein (Wickner and Hurwitz, 1975; Sclafani and Wechsler, 1981), to promote initiation of replication from circular Pl DNA molecules. This hypothesis would adequately explain the strict requirement for the dnaC function in induction of Pl lysogens which is initiated from the circular plasmid Pl form. Whether the origin of Pl replication at which the dnuC product acts is the same in plasmid and lytic replication, and whether this product can initiate other modes of replication is at present unknown. Plasmid and vegetative replication differed in two noticeable ways with respect to their dependence on the host chain elongation functions tested (Table 3). The products of genes polC and dn.uZ are subunits of E. coli pal III holoenzyme, the dnaG gene product, which codes for primase, and the dnaB protein are involved in priming chromosomal DNA synthesis at the origin of replication and subsequently in directing priming of discontinuous DNA synthesis (Wickner, 1978). Amounts of Pl DNA synthesized in lytic infection in the poLC mutant were 5-10’70 that in the corresponding wild type strain at the restrictive temperature. Plasmid replication, on the other hand, was extensive in the same polC strain, although covalently closed circular DNA could not be recovered (see Scott and Vapnek, 1980). A second significant difference between plasmid and vegetative replication was the lack of requirement for the dnaB function in lytic growth (Beyersman and Shuster, 19’71). This difference is attributed to the fact that Pl ex-

Pl

205

presses a dnuB-like protein during lytic infection, but not in the normal prophage, which can efficiently substitute for the bacterial dnaB protein in vi’~‘o (D’Ari et al., 1975; Lanka et al., 1.978; Touati-Schwartz, 1979). Several other temperate coliphages exhibit a similar dependence as Pl for host elongation functions polC, dnaG, and dmZ for lytic growth but differ in their requirement for host initiation functions. Thus, phage X can dispense with the bacterial dnaC function and replace it with proteins of its own (A gene products 0 and P) which interact with the dnuB protein (Skalka, 1978). Phage 186 on the other hand requires both host dnaA and dnaC replication functions whereas phage P2 requires neither (Bowden et al., 1975; Hooper and Eagen, 1981). At present little is known about the need of Pl functions for lytic replication. The fact that a significant if low level of DNA synthesis occurs in both polC and dnaZ thermosensitive mutants at the rest,rictive temperature may be due to analogous I’1 encoded functions. Residual DNA synthesis could, however, be the result of incomplete inactivation of the thermosensitive proteins at the restrictive temperature, or, perhaps less plausibly, due to other bacterial functions that inefficiently replace the above DNA synthesis functions. Alternatively, this low level of DNA synthesis could be due to repair synthesis rather than replication. Certainly, in the absence of de T~OL’O synthesis of Pl proteins after iafection no phage-specific DNA synthesis occurs (Zabrovitz et aZ., 1977). It is, therefore, surprising that as yet no phage mutants have been reported with defects in vegetative replication (Razza et ai, 1980). We examined 36 independently isolated :temperature sensitive Pl mutants for defects in DNA synthesis. In 34 mutants the amounts of DNA made were normal, in 2 mutants the amounts were approximately half that of wild type (unpublished results). Further attempts are presently underway to isolate Pl r?iutants defective’ in lytic replication. ACKNOWLEDGMENTS We wish to thank Drs. B. Bachman, J. Wechsler. and M. Abe for providing us with bacterial strains,

206

HAY

AND

Dr. D. Vapnek for permitting us to quote from his unpublished data, and Y. Sonyin for excellent technical assistance.

REFERENCES ABE. M., and TOMIZAWA, J. (1971). Chromosome replication in E. coli affected in the process of DNA initiation. Gen&ics 69, 1-15. ABE, M. (1974). The replication of prophage Pl DNA. Molec Ga Gend 132, 63-72. BEYERSMAN, D., and SHUSTER, H. (1971). DNA synthesis in Pl infected E. coli mutants temperature sensitive in DNA replication. MoL Gen Gene% 114, 173-176. BOWDEN, D. W., TWERSKY, R. S., and CALENDAR, R. (1975). Escti~ia coli deoxyribonucleic acid synthesis mutants: Their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyrobonucleic acid synthesis. J. B&L 124. 167-175. CARL, P. L. (1970). Escherichia c&i mutants with temperature sensitive synthesis of DNA. M&X. Gen. Gem%. 109,107-144. D’ARI, R., JAFFE-BRACHET, A., TOUATI-SCHWARTZ, D., and YARMOLINSKY, M. B. (1975). A dnaB analog specified by bacteriophage Pl. J. Mel Bid 94,341366. FILIP, C. C., ALLEN, J. S., GIJSTAFSON, R. A., ALLEN, R. G., and WALKER, J. R. (1974). Bacterial cell division regulation: Characterization of the dnaH locus of Eschaichia coli J. B&e&L 119,44.%449. GROWS,J. D. (1972). DNA replication in bacteria. Curr. Tqmkx Micr&i~L ImmurwL 57,39-74. HOOPER, I., and EAGEN, J. B. (1981). Coliphage 186 infection requires host initiation functions dnnA and dnaC. J. ViroL 40,599-601. IKF,DA, H., and TOMIZAWA, J. (1968). Prophage Pl, an extrachromosomal replication unit. Cou Sprint Harbor Symp Quant. BioL 33,791-798. KONDO, E., and MITSUHASHI, S. (1964). Drug resistance of enteric bacteria. IV. Active transducing bacteriophage PlCm produced by the combination of R factor with bacteriophage Pl. J. B&L 88,12661276. LANKA, E. M., and SHUSTER, H. (1970). Replication of bacteriophages in Eschet-ichiu coli mutants thermosensitive in DNA synthesis. Mokc Gen. Genet 106,274-285. LANKA, E. M., MIKOLAJCZYK, M., SCHIXHT, M., and SCIIUSTF.R, H. (1978). Association of the prophage Pl ban protein with the dnuB protein of Escherichiu di. J. Bid Ch.em. 253,4746-4753. MILLER, J. (1972). Experiments in molecular genetics. Cold Spring Harbor Laboratory, &Id Spring Harbor, New York. NAINEN, O., and VAPNEK, D. (1980). Unpublished observations, cited by Scott, J. R. and Vapnek, D. in Regulation of replication of the Pl plasmid pro-

COHEN

phage. In “Mechanistic Studies of DNA Replication and Genetic Recombination.” Academic Press, New York. OC.AWA, T. (1975). Analysis of the dnuB function of E. wli K12 and the dnaB-like function of Pl prophage. J. Molec Bill 94.327-340. RAZZA, J. B., WATKINS, C. A., AND SCOTT, J. R. (1980). Phage Pl temperature-sensitive mutants with defects in the lytic pathway. virdvyy 105, 52-59. ROSNER, J. L. (1972). Formation, induction and curing of bacteriophage Pl lysogens. virob 48,679-689. SCLAFANI, R. A., and WECHSLER, J. A. (1981). Suppression of dnaC alleles by the dnaB analog (ban) protein of bacteriophage Pl. J. BucterkL 146, 321-324. SCOIT, J. R. (1968). Genetic studies of bacteriophage Pl. Virology 36, 564-574. SCOTT, J. R., and VAPNEK, D. (1980). Regulation of replication of the Pl plasmid prophage. In “Mechanistic Studies of DNA Replication and Genetic Recombination.” Academic Press, New York. Scorr, J. R., and SI~STER, R. C. (1973). DNA synthesis in cells infected with phage PI& Vi-, 53,484486. SEGEV, N., LAUB, A., and COHEN, G. (1980). A circular form of bacteriophage Pl DNA made in lytically infected cells of Eschwichia c&i Virology 101,261271. SEGEV, N., and COHEN, G. (1981). Control of circularization of bacteriophage Pl DNA in Esche-ichiu coli Virology 114, 333-342. SKALKA, A. M. (1978). DNA replication-bacteriophage lambda. CWT. Top. Micro6ioL ImmwwL 78.201-237. TOUATI-SCHWARTZ, D. (1979). A dnaB analog ban specified by bacteriophage Pl: Genetic and physiological evidence for functional analogy between the two products. Molec Gen Genet 174,173-188. WALKER, J. R., RAMSEY, J. A., and HALDEWANG, W. G. (1982). Interaction of the Escherichiu coli dnnA initiation protein with the dnaZ polymerization protein in tivo. Proc Nat Acua! Sci USA 79, 3340-3344. WECHSLER, J. A., and GROSS,J. D. (1971). Es&e&hia c&i mutants temperature sensitive for DNA synthesis. M&c Gen. Gaet 113, 273-284. WECHSLER, J. A. (1975). Genetic and phenotypic characterization of dnaC mutations. J. BacteioL 121, 594-599. WECHSLER, J. A. (1978). In “DNA Synthesis” (I. Molineux and M. Kohiyami, eds.), pp. 49-71. Plenum, New York/London. WICKNER, S., and HURW~, J. (1975). Interaction of E. coli dnaB and dnaC(D) gene products in w&o. Prcxt Nat. Acad Sci USA 72.921-925. WICKNER, S. (1978). DNA replication proteins of Escherichiawti Annu Rev. Biochem 47,116.!-1192. ZABROVITZ, S., SWGEV,N. and COHEN, G. (1977). Growth of bacteriophage Pl in recombination-deficient hosts of Eschxrichia c&i. Virobgy 80.233-248.