Replication and plasmid-bacteriophage recombination I. Marker rescue analysis

VIROLOGY 115,

223-236 (1981)

Replication and Plasmid-Bacteriophage Recombination I . Marker Rescue Analysis

RICHARD D . SMITH' AND ROBERT C . MILLER, JR. 2 Departments of Microbiology and Medical Genetics, University geBritish Columbia, Vancouver, British Columbia V6T IW5, Canada Received February 20, 1981 ; accepted July 1, 1981 Recombinant DNA plasmids of pBR322 and T7 DNA were constructed and tested for the presence of various T7 genes . A plasmid containing part of T7 gene 5 (DNA polymerase) was used to transform various strains of Escherichia coii, including a dna B mutant . [3 HTrhymidine incorporation and a new method of copy number analysis showed that plasmid DNA was not degraded after infection by T7 as is the E coil host DNA . Marker rescue between recombinant T7 plasmids and mutant infecting bacteriophage was quantitated by determining the percentage of wild-type progeny phage produced after infection . Replication of the plasmid DNA and infecting phage DNA was controlled independently by a dna B mutation in the host and by gene 5 mutations in the phage, respectively. Marker rescue frequencies decreased slightly, if either plasmid or phage replication was blocked . However, marker rescue dropped below detectable levels, if neither the plasmid nor the phage could replicate . These results show clearly that replication plays a role in plasmid-phage recombination, and possible roles for replication in this process are discussed . INTRODUCTION

During bacteriophage infection, the processes of DNA replication and recombination are intimately related . For example, several enzymes are known to operate in both replication and recombination processes (Roeder and Sadowski, 1978 ; Lee et al., 1976 ; Broker and Doermann, 1975; Miller, 1975a, b) . Furthermore, the generation of concatemers, normal replicative intermediates, occurs during T4 infection through a process involving replication and recombination (Frankel, 1968 ; Miller et al ., 1970 ; Kozinski and Kosturko, 1976) ; during T7 infection elimination of the exonuclease (gene 6), which is required for normal recombination, leads to the insta' Current address : Department of Cellular Biology, Scripps Clinic and Research Foundation, La Jolla, Calif. 92037 . 2 To whom reprint requests should be addressed .

223

bility of concatemers (Frohlich et al ., 1975 ; Miller et al., 1976) . And last, during crossreactivation experiments markers which are replicated most efficiently are those which are rescued most efficiently (Burck and Miller, 1978; Burck et al ., 1979) . All of the information available indicates that normal levels of recombination during bacteriophage infections depends on at least a limited amount of DNA replication . Although some recombination intermediates can be detected in the absence of replication, these are found in low amounts only after very extended periods of incubation . There are several possible explanations for the above conclusion . The absence of DNA replication could lead to a pool of DNA molecules too small to interact effectively during the recombination process . Alternatively, the conditions used to block DNA replication (DO) might also block recombination. For example, DO conditions are frequently established 0042-6822/81/160223-14$02.00/0 Copyright ®1981 by Academic Prees, Inc . All rights of reproduction in any form reserved .



224

SMITH AND MILLER

by infecting with a particular amber phage; if the enzyme made defective by the amber mutation is required directly for both processes, then of course, both replication and recombination would be eliminated, e.g ., gene product 32 of T4 . Or last, it is possible that some replicative intermediate is used normally to initiate recombination during bacteriophage infection . In order to obtain direct evidence for a role for replication in the recombination process, we sought to develop a system where the replication of the two recombining species of DNA could be controlled independently . Hopefully, such a system would demonstrate a need for replication in the recombination process and would allow us to discriminate between the three possible explanations for such a role outlined above. One appropriate system is a recombination of a genetically marked bacteriophage with a DNA plasmid carrying a wild-type allele. For example, the T7 gene product 5 is a DNA polymerase required absolutely for DNA replication (Studier, 1972 ; Grippo and Richardson, 1971) . Therefore, recombination between a plasmid carrying a fragment of the T7 gene 5 and an infecting gene 5 mutant phage could be monitored by scoring for the production of wild-type phage during the infection process. Under nonpermissive conditions the phage would not replicate until it recombined . The effect of blocking replication for either the plasmid or the phage DNA or both can be tested by establishing the appropriate conditions of infection . The results of such experiments are reported here . Several major conclusions arise from these experiments . First, either the plasmid or the bacteriophage must be able to replicate in order for marker rescue to occur. Second, this result does not reflect the need for a critical amount of intracellular DNA . Third, it does not reflect the absence of an enzyme common to the two processes of replication and recombination . Therefore, the need for replication reflects the involvement of a replicative structure . And last, unlike host DNA the plasmid DNA is not degraded after bacteriophage infection .

MATERIALS AND METHODS

(a) Bacterial and Phage Strains (i) Bacterial strains. E. coli B2$ (sup°) and E. coli 0-11'(sup E) were used as the nonpermissive and permissive hosts for T7 amber phage, respectively (Studier, 1973) . Strains used in marker rescue experiments were all E. coli K12 derivatives and are described in Table 1 . E279 is a derivative of CR34 . Strains containing plasmid molecules were constructed by the transformation procedure of either Wensink et al . (1974) or Cohen et al. (1972) . (ii) Phage stocks . Phage used in this work were described by Studier (1973) . T7 am5-28 and T7 ts5 are both defective in the T7-specific DNA polymerase (gene 5) . High titer phage stocks were prepared according to Studier (1969) . Radioactive phage were prepared according to Benbasat et al . (1978) . (b) Media All bacterial strains were grown in LB containing 10 gg thiamine/ml and 50 gg thymidine/mI; [3Hjthymidine incorporation experiments were performed in TCG phosphate media (Benbasat et al ., 1978). T7 Tris-salt is 0 .01 M Tris-HCI, pH 7 .4,1 .0 M NaCl . (c) Construction of Clones Carrying Recombinant DNA Molecules The cloning vector pBR322 (Bolivar et al ., 1977) was digested with the restriction endonuclease PstI . pBR322 contains a single PstI restriction site in the gene coding for ampicillin resistance. Digestion with PstI gave a population of linear molecules with extended 3'-OH ends to which short tracts of poly(dA) were attached by the enzyme terminal deoxynucleotidyl transferase (Loban and Kaiser, 1973) . T7 wildtype DNA was digested with HpaI, and the resulting fragments were extended with poly(dT) tracts by terminal transferase . The two populations of extended molecules were annealed and the resulting mixture was used to transform CR34, utilizing the



PLASMID-PHAGE RECOMBINATION

225

TABLE 1 BACTERIAL STRAINS Strain

Reference

Genotype

44

CR34 CR34-pBR322 CR34-pRS202

thr, thi, thyA, lea, lacY, supE as CR34 plus pBR322

E279 E279-pBR322 E279-pRS202

as CR34 plus dna B ts279 as E279 plus pBR322 as E279 plus pRS202

HMS174 HMS174-pBR322 HMS174-pRS202

r', mk,, recAl rifn sup

as HMS174 plus pBR322 as HMS174 plus pRS202

This paper This paper

CR34-pRS142 CR34-pRS88

as CR34 plus pRS142 as CR34 plus pRS88

This paper This paper

as CR34 plus pRS202

2

°

Hirota et al . (1970) This paper This paper Wechsler et al . (1973) This paper This paper Campbell et al. (1978)

procedure of Cohen et al . (1972) . Trans- (e) Marker Rescue Experiments formed cells were grown in LB at 30° for (i) Single-step marker rescue experi2 hr at which time 10 jug tetracycline/ml ment. Plasmid containing bacteria were was added and incubation continued for grown from a 1 :50 dilution of a fresh overan additional 60 min . Samples of 0 .1 ml were plated on LB agar containing 10 µg night culture in LB supplemented with 10 tetracycline/ml and grown 12 hr at 30° . µg thiamine/mi and 50 µg thymidine/ml Transformed cell lines were stored in glyc- to a density of 2 X 10 8 cells/ml at 30° . Inerol-LB media at -20° . All recombinant fecting phage were added to aliquots of the DNA procedures were carried out under growing culture at an m .o.i . of 5.0. At 6 .5 A-M containment conditions as specified min postinfection, infective centers and by the Medical Research Council of survivors were plated through T7 antisera on various indicator bacteria . Canada . Also, at 6 .5 min infected cells were diluted through antisera and incubated to (d) Isolation and Purification of Plasmid lysis. Phage were titered under permissive DNAs and nonpermissive conditions . RecombiE. coli strains carrying plasmid mole- nation frequencies were calculated as the number of wild-type phage divided by the cules were grown in LB supplemented with 10 µg tetracycline/ml at 30° to a density total phage yield . In order to compare levof 10 9 bacteria/mi. Chloramphenicol was els of marker rescue that were indepenadded to a final concentration of 200 µg/ dent of burst size, the percentage of cells ml . Cultures then were incubated an ad- yielding wild-type infective centers was ditional 12 hr. Cells were harvested by cen- measured by plating infective centers at trifugation in a Beckman type 15 rotor at 6.5 min under permissive and nonpermis8000 rpm for 20 min . Cells were gently sive conditions . (ii) Marker rescue spot tests . A marker lysed with lysozyme-triton . Cleared lysates were placed in CsCI-ethidium bro- rescue test on plates was developed as a mide gradients and centrifuged for 72 hr fast screening method for detecting the at 33,000 rpm . DNA was removed with a genetic markers carried by recombinant syringe, extracted with n-butanol, and di- molecules . L B agar plates were seeded alyzed against 20 mM Tris-HC1, pH 7.4, with a lawn of nonpermissive bacteria. 20 mM NaCl, 1 mM EDTA . Two microliters of various dilutions of



2 26

SMITH AND MILLER

plasmid carrying bacteria were overspotted with 2 µl of amber phage . The test was considered positive, when plaques arose from spots containing recombinant plasmid strains and no plaques appeared on the control plates which contained cells with pBR322 alone at an equivalent dilution .

tal plasmid or recombinant plasmids not carrying T7' gene 5 fragments did not yield wild-type phage after infection with T7 am 5-28 or T7 ts5 . When pRS202 was labeled with BZP by nick translation, it hybridized only to T7 Hpal restriction fragment D . These two results indicate that pRS202 carries the restriction fragment expected . Restriction maps (McDonnel et al., 1977) of HpaI fragments and nucleotide sequence analysis (John Dunn, personal communication) show that the T7 HpaI restriction fragment D ends near the middle of TV gene 5 . That is, HpaI cuts in T7' gene 5, and a large part of gene 5 is on T7 HpaI fragment I . Therefore, pRS202 carries only part of TV gene 5 and could not be expected to complement a T7 am or ts-infecting phage, Data presented here (Results) and by Campbell et al . (1978) and by Studier and Rosenberg (in press) confirm this conclusion .

(f) Characterization of Clones Carrying T7 Gene 5 Four hundred ampicillin sensitive, tetracycline resistant clones were tested for inserted T7 DNA by affixing the DNA of each clone onto nitrocellulose membranes by the method of Grunstein and Hogness (1975) . These membranes then were hybridized with 'P-labeled TV prepared by nick translation . Out of the 400 clones, 380 hybridized the T7 DNA probe . In order to isolate clones containing T7 gene 5, 2 µg of T7 HpaI fragment D (Fig . 1) was isolated from a 1 .5% agarose gel . Fragment D carries the 5' end of T7' gene 5 (McDonnel et al., 1977 ; Studier and Rosenberg, 1981) . This DNA was labeled in vitro by nick translation and used as a hybridization probe on Grunstein-Hogness type colony filters carrying 200 recombinant clones . Fifteen clones hybridized the fragment D probe, and four were selected for further analysis . Marker rescue tests indicated that at least one clone, CR34-pRS202, contained part of T7 gene 5. T7 am 5-28 and T7 ts5 recombined with pRS202 in CR34 with frequencies of 1 .42 X 10-2 and 2 .1 X 10-2, respectively . Bacteria containing the paren-

A IL5I

(g) Copy Number Analysis (i) Purification of total intracellular DNA . Cells containing plasmid DNA were grown in supplemented LB at 30° to a density of 2 X 10 8/ml . At various times, 10-m1 samples were withdrawn, centrifuged, washed in 20 mM Tris-HCI, pH 7 .4, 50 mM NaCl, 1 mM EDTA, and concentrated 10fold . Cells were lysed with 0 .1% SDS plus 1 mg Pronase/ml at 37° for 12 hr . Lysates were extracted with an equal volume of phenol and extensively ether washed . (ii) Labeling of intracellular DNA . An aliquot of purified intracellular DNA,

.7 I II II2 I 3 I 4 I 5 161 7 18

191 112I13I

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FIG . 1 . Map of Hpal fragments released from TT DNA . (A) Approximate position of T7 genes (Simon and Studier, 1973) . (B) HpaI restriction fragments of T7 DNA (MeDonell et at., 1977) . (C) T7 map units .



Plasmid DNA bound to filter is at 100-

PLASMID-PHAGE RECOMBINATION equivalent to 1 µg of A2N material, was labeled in vitro by nick translation (Maniatis et al., 1975) . Labeled DNA was separated from radioactive nucleotides by passing the reaction mixture over a G-100 Sephadex column and collecting the appropriate material . (iii) Preparation of filters and hybridization conditions . Purified plasmid and chromosomal DNAs were bound to nitrocellulose membranes by the method of Denhardt (1966) . Hybridizations were performed in PM buffer (0 .2 mg/ml Ficoll, 0 .2 mg/ml polyvinylpyrolidone, 0 .2 mg/ml BSA, 0 .01 mg/ml thymidine, 0 .1% SDS in 3X SSC) . Filters were incubated 4 hr at 65° in PM prior to addition of the labeled probe . Labeled DNAs were heat denatured and were added directly to the preincubated filters . Hybridizations proceeded 15 hr at 65° . Filters then were extensively washed in 3X SSC, dried, and the radioactivity bound was determined by scintillation counting. Under our conditions plasmid filters contained 2 .5 µg of bound DNA which was 100 times greater than the amount of input labeled plasmid DNA . During calibration experiments input plasmid DNA hybridized to filters with an efficiency of 35% . Hybridized filters contained at least 50 times the amount of radioactivity compared to background filters ; e .g ., plasmid filters generally contained over 10,000 cpm while background filters contained 100 to 200 cpm . (iv) Copy number determination . Copy number was determined by hybridizing 82P-labeled total intracellular DNA with purified host and plasmid DNA fixed to separate nitrocellulose filters . The ratio of the radioactivity on the plasmid and host genome filters reflected the copy number of the plasmid . In order for the ratio of radioactivity to accurately reflect the copy number it was necessary that plasmid and host DNA molecules be labeled with the same efficiency during nick translation . Purified samples of host genome and plasmid DNA were prepared by banding each in two successive CsCI-ethidium bromide gradients, extracting with n-butanol to remove ethidium bromide, and dialyzing against 20 mM Tris, pH 7 .4, 20 mM NaCl,

227

1 mM EDTA . These samples then were treated with RNase at a final concentration of 20 gg/ml. RNase-treated samples were phenol extracted, ether washed, ethanol precipitated and resuspended in buffer. Equal A2,u amounts of each sample were labeled in vitro with [a-92P]dATP by nick translation . Ten-microliter aliquots of labeled material were extensively washed in cold 5% TCA and 82P incorporation determined by scintillation counting . The incorporation of label into each species was found to be identical . Therefore, in experimental samples where total

10

20

30 40 50 COPY NUMBER FIG. 2. Standard curve for the analysis of hybridization ratios . A standard curve was constructed by mixing known ratios of nP-labeled plasmid and host chromosomal DNA. Ratios were Constructed to represent copy numbers of 10, 20, 30, 40, and 50. These mixtures of DNA were hybridized in vials containing a set of three filters : (1) plasmid DNA, (2) host DNA, (3) no DNA . Two concentrations of input DNA were hybridized in PM at 65° for 12 hr . The amount of input DNA was at least 100 times less than the amount of DNA on each plasmid filter. The above figure represents a typical standard curve . The hybridization ratio can be defined as : epm3EP-labeled plasmid DNA hybridized/epm°2P-labeled host DNA hybridized . Both concentrations of input DNA yielded linear and very similar lines, showing that the DNA on the filter was in adequate excess . (A) Plasmid DNA bound to filter is at 200-fold excess at copy number of 50. (•) fold excess at copy number of 50.



228

SMITH AND MILLER

intracellular DNA was labeled, the ratios obtained could be interpreted directly . Plasmid to chromosome radioactivity ratios after hybridization were converted into copy numbers via a standard curve . Standard curves were constructed by mixing known ratios of labeled plasmid and chromosome molecules, hybridizing these samples with nitrocellulose membranes carrying E. eoli DNA, plasmid DNA, and no DNA at 65° for 15 hr (Fig. 2) . The reconstructed ratios represented copy numbers based on molecular weights of 2 X 10' daltons for the EK coli genome and 3 .85 X 106 daltons for pRS202 DNA . The experimental hybridization ratios obtained were compared to the constructed ratio of molecules ; then these values were used to calculate the copy numbers obtained in experimental samples .

formed at several input DNA concentrations with the filter bound DNA being at least 100-fold excess . After hybridization, filters were washed in 3X SSC, dried, and the bound radioactivity determined . All filters contained at least 1000 cpm, and background hybridization was less than 50 cpm . Bound 8H-labeled material was linear with respect to input . Values of annealed radioactivity were divided by recovered infective centers or recovered 32 Plabeled intracellular DNA . These divisions yielded identical results and calibrated all samples for recovery of intracellular DNA . Copy numbers before infection were determined as outlined above and calculated after infection in relation to the normalized [8H]thymidine-labeled material annealed to filter-bound plasmid DNA . RESULTS

(h) Copy Number Analysis in T7-Irtfected Cells

(a) The Plasmid-Phage Recombination System

E279-pRS202 was grown in LB at 30° to a density of 5 X 10 7 cells/ml . Cells were pelleted by centrifugation, washed in TCG media, centrifuged, and resuspended in TCG containing 250 jig KH 2PO4/ml, 10 µg thiamine/ml, 5 µg thymidine/ml, 100 µg deoxyadenosine/ml, 10 µg 5-fluorodeoxyuridine/ml, and 20 µg uracil/ml . [8H]Thymidine was added to a final concentration of 50 µCi/ml . The culture then was incubated at 30 ° and 42 ° to a density of 3 X 108 cells/ml . At this time, the culture was split and incubated at 30° and 42° for 15 min . Cells were infected with 32 P-labeled T7 am5 phage (specific activity = 2.0 mCi/mg P) at an m .o .i . = 5 .0 . Samples (2.0 ml) were removed at various times, ice chilled, centrifuged, and resuspended in 0 .02 MTris-0 .15 MNaCI, pH 7 .4 . Aliquots of 50 µl were removed and plated for infective centers . Ten-microliter aliquots were removed, and the cold TCA precipitable radioactivity was determined . The remainder of each sample was incubated with 0 .01 M EDTA, 0.1% SDS, and Pronase . After lysis, phenol and other washing, equal volume aliquots were removed and hybridized at 65° in PM buffer (Denhardt, 1966) to filters containing pBR322 DNA . Hybridizations were per-

Plasmid pRS202 recombines with amber or is mutants within gene 5 (Materials and Methods, section f). This plasmid provides a system in which the replication of two interacting DNA species, i .e ., T7 and plasmid, can be manipulated independently during marker rescue experiments . (i) T7 replication. It has been shown previously that amber or is mutations in genes 4 and 5 are defective in DNA synthesis under nonpermissive conditions (Studier, 1972) . The product of gene 5 is the T7-specific DNA polymerase (Grippe and Richardson, 1971 ; Oey et at., 1971) . Density transfer experiments have shown that not even one round of DNA replication occurs in a gene 5 mutant under nonpermissive conditions (R . C. Miller, unpublished observations) . (ii) Plasmid replication. pBR322 is a composite plasmid constructed by Bolivar et al . (1977) from ColEl and pSC101 . The nucleotide sequence for pBR322 is known (Sutcliffe, 1978) . The copy number of pBR322 is amplifiable in the presence of chloramphenicol as is the plasmid ColEl which suggests that their mode of replication may be similar . Several authors have determined the effects of various E. coli DNA synthesis mutants on ColEl rep-



PLASMID-PHAGE RECOMBINATION

lication (Goebel, 1970, 1973 ; Kingsbury and Helinski, 1973) . The effect of several DNA synthesis mutations on pSC101 replication has been documented also (Hasunuma and Sekiguchi, 1977, 1979). The effects of the dna B mutation on pRS202 replication was examined . The E. coil strain E279 was transformed with pRS202 . 3H-Labeled thymidine incorporation was used to follow the total DNA synthesis in these mutants . Figure 3 shows the results of this analysis . DNA synthesis in the dna B mutant with and without the plasmid ceases after shift to 42° . Therefore, plasmid replication ceases at 42° in a dna B mutant. This conclusion is supported by copy number experiments described later in this paper. A close ex-

(e)

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20 40 60 80100120+40 160 190 200 TIME (min.)

FIG. 3 . Total DNA synthesis in E279 and E279pRS202. Cells were grown to 5 x 107 /ml in LB at 30°, centrifuged, washed and resuspended in TCG media containing 10 pg thiamine/ml, 5 pg thymidine/ml, and 100 pg deoxyadenosine/ml. Cultures were incubated at 30° to a density of 1 X 10 8/ml. [3 Hfrhymidine was added to a final concentration of 10 pCi/ml . Samples of 100 pl were withdrawn at various times . The culture then was split and one-half was shifted to 42° (indicated by arrows) and samples withdrawn . The TCA insoluble material was monitored by scintillation counting . (a) E279 at 30° and 42° ; (b) E279 + pRS202 at 30° and 42° .

229

amination of Fig. 3 might lead one to think that a small amount of [3H]thymidine incorporation does occur for up to 15 min at 42° in E. coil carrying this plasmid . Therefore, in subsequent experiments, cells were incubated for 20 min at 42° before infection . (b) Marker Rescue Experiments In the experiments described here, infecting am 5 or is 5 phage rescue the wildtype marker from recombinant plasmids carrying a wild-type T7` gene 5 fragment . The process of recombination between the plasmid and the infecting mutant phage produces wild-type phage . The experiments described below investigate the role of replication in the marker rescue process by comparing levels of marker rescue when one or both of the interacting DNA molecules can not replicate. (i) Marker rescue when both phage and plasmid can replicate. Maximum marker rescue frequencies were established in experiments where both phage and plasmid could replicate . CR34-pRS202, CR34pBR322, E279-pRS202, and E279-pBR322 were grown to 2 X 10 8 cells/ml and infected with either T7 am 5 or T7 is 5 at an m .o.i. of 5 .0. Infective centers and progeny phage were plated under conditions permissive for the parental phage . The frequency of marker rescue was quantitated by two procedures . First, the number of wild-type phage produced was divided by the number of infective centers (under permissive conditions the infective centers were always greater than 90% of the cells) . The second approach was to divide the number of infective centers detected under nonpermissive conditions by the number of cells infected . Interpretations of marker rescue frequencies generated by the two procedures are identical . Measuring marker rescue frequencies by infective centers or progeny phage also yielded identical results during cross-reactivation studies (Burck and Miller, 1978) . (ii) Marker rescue when only bacteriophage DNA can replicate. Cultures of E279-pBR322 and E279-pRS202 were grown to a density of 2 X 10 8 cells/ml. Onehalf of each culture was shifted to 42° and



SMITH AND MILLER

230

TABLE 2 EFFECT OF DNA REPLICATION ON T7 MARKER RESCUE Replication Strain

Phage

Temperature

Phage

Plasmid

Burst

wt b/cell

wt IC/cell

a. b. c. d. e. f. g. h.

CR34-pBR322 CR34-pRS202 E279-pBR322 E279-pRS202 E279-pBR322 E279-pRS202 HMS174-pBR322 HMS174-pRS202

am5 am5 am5 am5 am5 am5 am5 am5

30 30 30 30 42 42 30 30

+ + + + + + -

+ + + + + +

135 120 125 142 48 20 1 .9 4 .4

0 .003 0 .86 <0 .019 0 .55 <0.016 0.19 0.004 0 .166

0.0005 0.628 <0.0008 0.178 0.0003 0.066 0.0007 0.069

i. j. k. 1. m. n. o. p.

CR34-pBR322 CR34-pRS202 CR34-pBR322 CR34-pRS202 E279-pBR322 E279-pRS202 E279-pBR322 E279-pRS202

ts5 ts5 ts5 ts5 ts5 ts5 ts5 ts5

30 30 42 42 30 30 42 42

+ + -

+ + + + + + -

150 195 16 10 95 144 0.07 0.14

+ + -

0.014 0 .383 0 .0065 0.190 0.065 0.83 0.017 0 .007

Note. Strains of E . coli were grown to 2 X 10 8 cells/ml in L broth . Cell numbers were quantitated by plating and Petroff-Hausser counting . The cells were infected with the indicated T7 mutants at an M .O.I . = 5, incubated 7 min at 30° or 42°, and diluted through antisera . The infected cells then were either plated for infective centers or incubated until lysis and plated for phage. Infective centers and progeny phage were titered under permissive and nonpermissive conditions . Phage and plasmid replication were regulated by temperature and the presence of an amber suppressor . Strains are described in Table 1 . incubated for 20 min before infection with T7 am 5-28 phage . This 20 min period was sufficient time for DNA synthesis in the host cells at 42° to stop completely due to the dna B mutation . The analysis of marker rescue experiments showed a decrease in the production of wild-type phage at 42 ° (Table 2, lines d, f) . Therefore, under conditions when only phage DNA can replicate, fewer wild-type phage are produced than when both plasmid and phage are replicating . (iii) Marker rescue when only plasmid DNA can replicate, To create the condition where plasmid DNA was allowed to replicate but T7 DNA was not, the strains CR34-pBR322 and CR34-pRS202 were grown to a density of 2 X 108 cells/ml at which time one-half of each culture was shifted to 42° . The strains were incubated 20 min at 42° prior to infection with T7 is 5 phage . Comparison of the rescue frequencies at 30° and 42° (Table 2, lines j and 1) indicated that the absence of T7

replication did reduce the number of wildtype phage produced; but the amount of marker rescue was still -25 times the background level . A similar result was obtained when the sup° strains HMS174pBR322 and HMS174-pRS202 were infected with amber 5 phage . Under these conditions rescue frequencies are decreased significantly (compare Table 2, lines h and b), but are still more than 40fold background (compare Table 2, lines g and h) . Therefore, if the plasmid can replicate, then the plasmid and phage recombine, a T7+ gene 5 is produced, the T7 DNA polymerase (gene 5) is produced, and a burst of phage occurs . (iv) Marker rescue in the absence of replication . Since it appeared that only one of the participating molecules must replicate in order to recombine DNA, it was possible that the complete lack of replication would stop the marker rescue process altogether. This was tested by analyzing wild-type phage per cell from E279-

PLASMID-PHAGE RECOMBINATION

pBR322 and E279-pRS202 at 42° infected with T7 is 5 phage . Cells were infected 20 min after shift to 42° . Under these conditions all replication was shut off during the time course of infection. Table 2, lines n, p, indicates that no marker rescue is detectable above background when there is no DNA synthesis . There is no burst at all during these infections, because there is no gene 5 product synthesized . Without marker rescue no progeny phage are produced since the gene 5 product is essential for phage growth. If one compares lines g, h, o, and p, one can see that T7' are liberated only when the plasmid carries a fragment of the T7' gene 5 and only when the plasmid can replicate (line h) . If the plasmid does not carry the correct part of T7' gene 5 (g or o) or if the plasmid cannot replicate (p), then no T7' phage are liberated . Line h has a wt s/cell which is about 10 times higher than lines g, o, and p . Sucrose gradient analysis shows that the T7 DNA is present in high-molecular-weight form even after extensive incubation under nonpermissive conditions . The size of T7 is 5 DNA is the same at 42° in dna B ts- or dna B'-infected cells . Consequently, the large difference in rescue frequencies is not due to DNA degradation (sucrose gradient analysis, data not shown) . Therefore, the only significant difference between lines p (Table 2), where there was no rescue, and lines h and 1, where there were high levels of rescue, is that plasmid DNA replication is also inhibited, thus preventing marker rescue which is necessary for phage growth. Similar results are obtained when marker rescue is measured in a dna B mutant carrying plasmid pRS88, which normally rescues T7 ts4-101 ; at 42° there is no rescue (data not shown) . However, the gene 4 mutants can be rescued, if either the phage or the plasmid can replicate, as has been shown above for gene 5 mutants . (c) Analysis of Plasmid DNA after Infec-

tion Infection by bacteriophage T7 is accompanied by the degradation of the host genome . T7 genes 3 and 6 code for an en-

231

donuclease and exonuclease responsible for this breakdown (Kerr and Sadowski, 1972 ; Center et al., 1970). The liberated host nucleotides are utilized in phage replication . In order to establish the integrity of plasmid DNA molecules after infection, HMS174-pRS202 (sup°) was infected with amber 5 phage at 30° and the total intracellular DNA was analyzed on agarose gels at various times after infection. Degradation of host DNA was detectable 20 min after infection . This is demonstrated by the conversion of the high-molecularweight DNA near the origin of the gel to lower-molecular-weight fragments (Fig . 4) . A Southern blot was made from this gel and hybridized with 'P-labeled pBR322 DNA. The autoradiogram (Fig . 5) showed that plasmid DNA escaped the degradative process since the plasmid molecules formed discrete bands even 30 min after infection . Densitometer tracings of the autoradiogram in Fig. 5 indicated that

FIG. 4 . Electrophoretic analysis of plasmid DNAs after infection . Purified total intracellular DNA was prepared from HMS174-pRS202 infected with am 5 phage . Equal aliquots were removed from the infected culture at (A) 0, (B) 5, (C) 10, (D) 20, (E) 30 min postinfection . Samples of equal volume were analyzed on a 0 .5% agarose gel containing 1 µg ethidium bromide/ml . The arrow indicates the origin of the gel .



232

SMITH AND MILLER

bacteriophage T7 is completed by 25 min at 42°, these results indicate that there is no significant decrease in the number of plasmid molecules during the course of marker rescue experiments due to shifting the temperature from 30° to 42° . (e) Effect of Phage Infection on Plasmid DNA

A

B

C

D

E

FIG . 5 . Autoradiogram of Southern blot containing plasmid DNAs electrophoresed on 0.5% agarose gels . The DNAs from the agarose gel in Fig . 4 were transferred by the method of Southern (1975) to nitrocellulose paper . This bound DNA was hybridized with "P-labeled pBR322 DNA for 15 hr at 65° . This method visualizes plasmid DNA molecules from samples removed at (A) 0, (B) 5, (C) 10, (D) 20, (E) 30 min postinfection . The arrow indicates the origin of the gel .

there was no loss of plasmid DNA up to 30 min after infection . One obtains identical results if a dna B is cell is infected with T7 is 5 at 42° . These results were confirmed by the hybridization analysis described below . (d) Hybridization Analysis of Plasmid Copy Numbers An analysis of plasmid copy number in and E279-pRS202 was performed in order to establish the number of interacting plasmid molecules present during the temperature shifts of a marker rescue experiment. The copy number was determined by hybridizing 32P-labeled total intracellular DNA with purified host and plasmid DNA fixed to separate nitrocellulose filters . The ratio of the radioactivity on the plasmid and host genome filters reflected the copy number of the plasmid . Table 3 shows the copy numbers obtained with the analysis of CR34-pRS202 and E279-pRS202 during a temperature shift regime . Since the infection cycle of CR34-pRS202

The fate of plasmid DNA after infection was examined by labeling plasmids continuously with [3H]thymidine and quantitating the radioactive material annealing to plasmid DNA bound to nitrocellulose filters . This procedure determined the total amount of plasmid DNA at any time after infection ; the procedure had the capacity to measure both increases and decreases of plasmid DNA . 32P-Labeled T7 phage were used to infect the plasmid-carrying cells so that an internal standard was available to monitor the recovery of intracellular DNA at each stage of the experiment . This approach eliminated several problems with copy number analysis after infection . Copy number experiments normally measure ratios of plasmid DNA to host DNA ; however, host DNA is degraded by enzymes coded for by T7, so traditional copy numbers are not useful measurements after infection . The determination of [3H]thymidine-labeled DNA annealed to filter-bound plasmid circumvents this problem . Copy numbers before infection can be related to plasmid moleTABLE 3 COPY NUMBER ANALYSIS IN NONINFECTED CELLS

Temperature (°)

Time (min)

CR34pRS202 copy no .

E279pRS202 copy no.

30 30-42 42

-15 0 40

38 38 30

20 20 21

Note. CR34-pRS202 and E279-pRS202 were grown exactly as in marker rescue experiments . Samples were removed at the indicated times relative to the 30 to 42 temperature shift . Samples were prepared as described in text. The copy number is expressed as the number of plasmid molecules per genome .



PLASMID-PHAGE RECOMBINATION TABLE 4 HYRRIOIZATION ANALYSIS OF PLASMID DNA AFTER INFECTION Time after infection (min) 0 o 15 20 0 a 10 15 20

Temperature (°)

Plasmid hybridization" (X10-2 )

Copy No . °

30 30 30 30 42 42 42 42 42

4.21 4.23 4.73 5.34 3.2 3.20 3.20 3.57 3.4

20 21 22 24 20 21 20 22 21

Note. E. coli were grown in TCG containing [3 Hlthymidine and were infected with nP-labeled phage ; 2-ml aliquots were removed from the cultures at various times, the DNA was extracted, and plasmid DNA was annealed to filter-bound DNA . The amount of annealed plasmid DNA was divided by the recovered infective centers or the amount of recovered 3P-labeled DNA . This calculation corrected values of the plasmid DNA as determined by hybridization for recovery of intracellular DNA (described under Materials and Methods) . " Plasmid hybridization is defined as the 811-labeled plasmid radioactivity annealed to plasmid-bound DNA divided by the recovered infective centers or nP-labeled DNA (cpm/cell) . 'The copy number is calculated by multiplying the copy number determined at 0 min by the ratio of the plasmid hybridization at 0 min to the plasmid hybridization at any other time .

cules per cell after infection by correlating the amount of 'H-labeled material annealed to filter-bound plasmid DNA at any particular time . The results (Table 4) show that there is little change in the amount of plasmid DNA per cell after infection and are in agreement with the indicators from the less quantitative approach of hybridization according to the procedure of Southern (1975) . DISCUSSION

The objective of experiments described here was to study the role of replication

233

in recombination between a recombinant DNA plasmid and a mutant infecting bacteriophage . The results show that if either DNA molecule can replicate, then recombination occurs, and a wild-type phage is produced . However, if neither the phage nor the plasmid can replicate, then recombination does not occur at a detectable level . Replication could influence recombination indirectly by several mechanisms . First, the number of DNA molecules in the cell available for recombination could be important in determining recombination frequencies, and this would be affected by replication . However, experiments reported here show that the number of interacting molecules is not the limiting factor . First, marker rescue is high even when phage replication is blocked prior to recombination . If the plasmid can replicate, then marker rescue occurs, and this leads to a burst of phage . Second, there is no marker rescue, and therefore no burst, if neither the plasmid nor the phage can replicate. There is only a small difference in intracellular DNA concentration between these two conditions (24 plasmid molecules versus 20 plasmid molecules and only 1-2 intracellular phage DNA molecules per cell) (Benbasat et aL, 1978), since there is no prior phage replication in either case . Yet, there is a very large difference in marker rescue frequencies (0 .166 versus 0.007, Table 1), depending on plasmid replication . Furthermore, there are much less dramatic drops in the rescue frequency when only phage replication is blocked . The intracellular DNA concentration, however, has been cut enormously from about 400 phage DNA molecules per cell to only 1 to 2. So, one can decrease drastically the amount of intracellular DNA without greatly decreasing marker rescue frequencies ; and alternatively, one can eliminate marker rescue without changing significantly the intracellular DNA . Therefore, there is no strong correlation between intracellular DNA concentration and marker rescue frequencies (Table 5) . These results are consistent with data from T7 phage recombination experiments where the number of intracellular T7 DNA molecules was limited either by naladixic



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SMITH AND MILLER

TABLE 5 INTRACELLULAR T7 AND PLASMID DNA Rescue T7 Plasmid frequency" equivalents equivalents wt o per per cell cell Phage Plasmid per cell Replication

+

+

4

-

+

400° 400 1-2` 1-2

24 20 24 20

0 .860 0 .190 0 .166 0 .007

Note. This table summarizes published data on amounts of intracellular DNA under various conditions and relates those data to rescue frequencies . The table shows that there are only weak correlations between decreased DNA and decreased rescue frequencies . " See Table 2 . 'Kelly and Thomas (1969) . `Benbasat et al. (1978) .

acid or a DNA polymerase mutation (Kerr and Sadowski, 1975 ; Miller et al., 1976) ; neither condition significantly affected recombination until replication was abolished. Hybridization, copy number analysis, agarose gel electrophoresis, and sucrose gradient sedimentation show that neither the plasmid nor the phage DNA are degraded under nonpermissive conditions . A second possibility is that the apparent dependence of this plasmid-phage recombination on replication merely reflects the need for either the dna B protein or the T7 DNA polymerase (gene 5) at some critical stage of the recombination process. In other words, it might be speculated that the dna B protein complements the gene 5 product in a recombination function . Both the T7 am 5-28 and T7 ts5 mutations used map within the gene known to code for the T7-specific DNA polymerase (Studier, 1969; Grippo and Richardson, 1971) which is essential for phage replication (Studier, 1972) . Results presented here indicate that the dna B protein of E. coli is required for the replication of pBR322 and recombinant plasmids where pBR322 serves as a vector . There is no evidence that the dna B protein has any polymerase or nuclease activity . Therefore, it is on-

likely that the dna B protein complements the T7 DNA polymerase in an essential recombination function . Finally, both gene 5 and gene 4 temperature sensitive mutants fail to recombine with plasmids carrying homologous DNA in a dna B host at 42°. Yet both gene 5 and gene 4 mutants are rescued under nonpermissive conditions in wild-type E. coli (see results ; Campbell et aL, 1978) . It is extremely unlikely that the dna B protein would complement both the gene 4 and gene 5 products in an essential recombination function . Therefore, it seems most likely then that under conditions nonpermissive for the dna B protein and the T7 polymerase, or the gene 4 product, recombination is affected, not by the failure of these proteins to perform a common task in recombination per se, but by the absence of DNA replication . The Meselson-Radding model (1975) and the unisex-circle model (Stahl, 1978) describe mechanisms for genetic recombination which include DNA synthesis as an important element of the process leading to the formation of joint molecules . The data presented here confirm that DNA synthesis stimulates recombination . It should be pointed out that DNA synthesis could be essential for the efficient conversion of joint molecules to covalent recombinants ; in other words, it may be necessary for resolving a heteroduplex structure . Our data show merely that some replication is required for efficient recombination . ACKNOWLEDGMENTS This research was supported by an operating grant from the National Research Council of Canada . We would like to thank D . M . Taylor for excellent technical assistance, and R. A . J. Warren and G . Spiegelman for helpful discussions. REFERENCES BENBASAT, J., BURCK, K ., and MILLER, R . C ., JR . (1978) . Superinfection exclusion and lack of conservative transfer of bacteriophage T7 DNA . Virology 87,164-171 . BOLIVAR, F ., RODRIGUEZ, R. L ., GREENE, P . J ., BETLACH, M . C ., HEYNECKER, H. L., CROSSA, J . H ., FALKOW, S ., and BOYER, H . W. (1977) . Gene 2, 95-113 .



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