J. Mol. Hid. (1989) 207, 99-109

Genetic Analysis of the Lytic Replicon of Bacteriophage Pl

I. Isolation and Partial Characterization

Gerald Cohen192 and Nat Sternberg’

‘Central Research and Development Departm,ent E. I. du Pont de Nemours and Co.,

Wilmington, DE 19898, U.S.A.

2Department of Microbiology, University of Tel Aviv Ramat Aviv, 69978, Israel

(Received 7 July 1988, and in revised form 19 October 1988)

Despite the extensive genetic analysis of bacteriophage Pl, the region of the viral genome that is responsible for its lytic (vegetative) replication has not been identified. In this paper we describe the identification of various fragments of PI DNA that can replicate an otherwise replication-defective A vector when they are cloned into that vector. The fragments share a 2800 base-pair segment of the Pl genome that is located adjacent to the immT region of the phage. Replication mediated by the cloned Pl fragments is abolished by the product, of the PI cl gene, the repressor of phage lytic functions. Since these propert,ies resemble those of the Pl lytic replicon, we suggest that the 2800 base-pair segment identified here contains that replicon.

1. Introduction

Bacteriophage Pl is a temperate virus of Escherichia coli that has lytic (vegetative) and lysogenic growth cycles (for reviews, see Sternberg & Hoess, 1983; Yarmolinsky & Sternberg, 1987). In its lysogenic state, PI exists as an autonomously replicating plasmid with a copy number about equal to that of the chromosome of its bacterial host (Ikeda & Tomizawa, 1969); in its lytic state, Pl multiplies by producing a burst of progeny phage particles. At least two distinct replication systems, or replicons, determine these growth cycles (Austin et al., 1978). The plasmid replicon has been studied intensively and is contained in a 1.5 kbt segment of Pl DNA that encodes an essential replication protein, and regions for initiation and control of replication (Abeles et al., 1984; Chattoraj et al., 1985).

In contrast, little is known about the Pl DNA region(s) that encodes the vegetative or lytic replicon(s). Previous studies on A-P1 miniplasmids

t Abbreviations used: kb, lo3 base-pairs; bp, base- pairs: p.f.u., plaque-forming units; m.o.i., multiplicity of infection: cm. t,he Tn9 transposon carrying the chloramphenicol transacetylase gene.

tentatively assigned a lytic replicon to a region of the Pl map near to that of the plasmid replicon (Sternberg & Austin, 1983). This replicon. termed the L replicon, directs high copy number replication of a replication-deficient 1 phage vector. However, studies of this replicon have been hampered by the instability of the I, replicon plasmid. Moreover, efforts to identify the Pl genes and sites involved in lytic replication have met with unexpected diffi- culty and, to date, no single mutation blocking lytic growth has been reported. Several factors could account for this situation. One is that PI could contain more than one lytic replicaon and that inactivation of all of the replicons is needed to prevent lytic growth. Consequent.ly, Pl mutants that inactivate only one of the replicons might go undetected. Alternatively, host functions might, complement mutations in PI replication genes and, unless one knows what those host) functions are. they would interfere with a PI mutant search. A case in point is the Pl ban gene. The product of this gene is an analog of the bacterial DnaB prot,ein and Pl ban mutants grow normally in the wild-type host (D’Ari et al., 1975). A further possibility is that the Pl genes and sites needed for lytic replication occupy a relatively small regiomof t)he genome, and t’hat the number of mutants screened for replication

100 G. Cohen and N. Sternberg

defects has been too small to ensure that one of the mutants contains a lytic replication defect.

(b) Media and phage growth

Here, we describe the isolation and partial characterization of a Pl lytic replicon. The approach used to identify the replicon is based on the ability of a PI DNA fragment to direct exten- sive replication of an otherwise replication-defective 3, vector. For this purpose, we used a library of Pl DNA fragments of 5 to 10 kb in the vector AD69. To facilitate screening of this library, we chose 18 of the A-P1 hybrid phages whose Pl DNA inserts cover, in an overlapping way, most of the Pl genome (O’Regan et aE., 1987). We show here that one of these phages contains a Pl DNA fragment that directs its replication to a high copy number, and that this replication is regulated by the product of the Pl cl gene, the repressor of phage lytic functions (Scott, 1970). Analysis of the Pl DNA in this phage (Sternberg & Cohen, 1989) indicates that the replicon it contains is different from the L replicon and that it possesses a promoter, P53, that overlaps with a Cl repressor binding site, 0~53. These findings suggest that lytic replication is blocked in the prophage state by Cl-mediated repression of P53.

L broth, L top agar and L agar have been described (Sternberg & Hamilton, 1981). When supplemented with ampicillin, the media contained 100 pg/ml. Small-scale preparations of I-P1 phages were made by transferring a single plaque into 10 ml of L broth containing 10 mM- MgCl, and inoculating with 0.1 ml of an overnight culture of strain MBM7014. Cultures were vigorously shaken for 4 to 45 h at 37”C, 05 ml of CHCI, was added, and shaking was continued for an additional 10 min or until complete cell lysis was detectable. The lysed cultures were centrifuged at 5000 revs/min for 10 min in a Sorvall SS34 rotor and the supernatants stored at 4°C. Large-scale preparations of I-P1 phages were made according to Silhavy et al. (1984). Phage, in 400 ml of a lysate that had been cleared of cell debris by centrifugation in a GSA Sorvall rotor for 15 min at 5000 revs/min, were concentrated by centrifuging the supernatant overnight at 5000 revs/min. The pelleted phage were resuspended by gentle agitation with 2 ml of Tris/Mg buffer (10 mM- Tris.HCl (pH 7.6), 10 mM-MgSG,) for several hours, the volume was then adjusted to 5.8 ml with Tris/Mg buffer, 4.5 g CsCl was added, and the phage were centrifuged for 24 h at 38,000 revs/min in a Ti70.1 rotor at 4°C. The phage band was collected by puncturing the centri- fugation tubes with a 21.gauge needle and the phage were then dialyzed against Tris/Mg buffer at 4°C.

2. Materials and Methods (c) General phage procedures

(a) Bacteria, plasmids and phage strains (i) Spot tests

E. coli K12 strains used in this work were as follows: NS303 is W3102 sup+ gal+ strR; NS1539 is a &mm21 lysogen of this strain; NS3006 and NS3007 were made by transforming these 2 strains, respectively, with plasmid pNS3006, which is a pBR322 plasmid derivative that carries the PI cl repressor gene (N. Sternberg, unpub- lished results). This plasmid contains the segment of PI DNA located between 97 and 995 map units on the standard Pl map (Eliason & Sternberg, 1987). Present in that Pl DNA is the cl structural gene, a region of approximately 200 bp upstream from that gene containing the cl transcription and translation control elements and a region of about 2 kb downstream from the cl gene. The latter contains a gene, without a promoter, that is necessary for cleaving the Pl packaging site (Sternberg & Coulby, 1987). All of the properties attributed to the cl gene in pNS3006 are abolished if the cl.100 mutation (Rosner, 1972) is introduced into the plasmid. NS1813 is a recA derivative of NS1539; MBM7014 supF was used for propagating ID69 hybrid phages (Silhavy et al., 1984). Phage 1D69 is a vector possessing a single BamHI site in the 1 int gene and the immunity of phage 21 (Mizusawa t Ward, 1982); 1D69 Pl hybrid phages were previously constructed by cloning 5 to 10 kb Sau3AI fragments of PlCmc1.100 DNA into the BamHI site of 1D69 (O’Regan et al., 1987). A cof- lection of 54 I-P1 hybrid phages has been physically characterized and a subset of 18 of those phages with Pl DNA fragments that cover, in overlapping fashion, some 98% of the Pl genome was identified (Fig. 1). The &mm21 clear phage B17 contains mutations in the i int, red and c1 genes, and was used in genetic crosses to introduce a c1 mutation into I-P1 hybrid phages. Plasmid ~1007 contains the EamB gene of E. coli (S. Benson, unpublished results) and was used here as a probe to quantify the amount of lamB DNA in various DNA preparations.

Spot tests were made by placing 5 ~1 of A-P1 phage lysates on L plates inoculated with the appropriate indicator bacteria and incubating overnight at 37°C.

(ii) Phage crosses and isolation of i-P1 phages containing a PI lytic replicon

Phage crosses were carried out to introduce a clear mutation into the &mm21 repressor gene of several I-PI hybrid phages suspected of potentially containing a Pl lytic replicon. An overnight culture of NS303 grown in L broth supplemented with 0.20/6 (w/v) maltose was diluted 1 : 100 and grown to A,,, = 0.5 (approx. 1.5x 10s cells/ml), centrifuged, and resuspended in 0.1 vol. L broth containing 10 miw-MgSG,. To 6 x lo7 cells was added 2 x lo* p.f.u. each of I-P1 phages numbers 15, 60, 76, 95, 124 or 139, and 2 x lo* p.f.u. of phage B17, corresponding to an m.o.i. of 3.5 for each phage. The mixtures were incubated for 10 min at 37°C for phage adsorption. They were then diluted 1 : 100 in L broth and shaken for 90 min at 37°C. CHCl, was added to 5oj, (v/v) to complete lysis, and the cultures were shaken for an additional 10 min. Recombinant phage possessing a Pl lytic replicon and a limm2lcI- mutation were identified by their ability to form plaques on a 1imm21 lysogen, but not on a limm21 lysogen containing Pl cl repressor.

(d) General DNA procedures

Restriction digestions and DNA ligations with bacteriophage T4 polynucleotide ligase were performed with enzymes purchased from New England Biolabs or Bethesda Research Laboratories and used under conditions specified by the vendor. Proteinase K was obtained from Boehringer-Mannheim.

Transformation of E. coli with plasmid DNA was according to the method of Dagert & Ehrlich (1979). DNA extraction from purified I-P1 phage stocks was

Isolation of the PI Lytic Replicon 101

carried out as described by Maniatis et al. (1982). Purification of DNA fragments from agarose gels for restriction mapping studies, DNA ligations and labeling of probes was as described by Lin et al. (1984).

(e) Southwn a,salysis of DNA from I-PI-infected cells

Cultures of a limmX1 lysogen (NS1539) and a transformant (NS3007) carrying a plasmid expressing the PI cl repressor gene were grown overnight at 30°C in L broth supplemented with 0.2 y0 maltose. The cultures then were diluted 1: 100 and incubated at 37°C until they reached A 60,,=0.5 (approx. 1.5 x 10s cells/ml). A por- tion (30 ml) of each culture was centrifuged at 7000 revs/min for 10 min in an SS34 rotor and the cell pellet was resuspended in 0.1 vol. L broth containing 10 mM- MgSO,. Samples of 0.2 to 0.4 ml of phage stocks containing 3 x lo9 p.f.u. of IPl-15, ,Icl-Pl-15 and APl-93 were separately added to 1 ml portions of cells to give an m.o.i. of 3 phage/cell, and the mixtures were incubated for 5 min at 37’C for phage adsorption. L broth was added to a final volume of 10 ml, and samples of 3 ml of the mixtures immediately were distributed to each of 3 test-t)ubes and shaken vigorously in a water-bath at 37 “C. At 2. 17 and 32 min, tubes were removed from the bath and chilled prior to DNA extraction. Approximately 3 x lO* infected cells were centrifuged at 5000 revs/min for 10 min in an SS34 rotor and resuspended in 0.70 ml of .50 mw-Tris.HCl (pH %O), lOoi, (w/v) sucrose. Cells were disrupted and the DNA extracted as described by St#ernberg & Coulby (1987). The extracted DNA was dia- lyzed against 4 changes of 1 liter each of 10 mlcl-Tris . HCI (pH 8.(l), 1 mM-Na,EDTA and stored at 4°C. Portions of 220 ng of DNA were digested in a final volume of 300 ~1 with restriction endonuclease HpaI. The reactions were stopped by heating them at 68°C for 10 min: then dried in a Speed Vat, and finally dissolved in 25 ~1 of 10 mM- Tris.HCl (pH &O), 1 mM-Xa,EDTA. Portions of 1, 4 and 16 ~1 from each digestion were analyzed by electro- phoresis in 0.80/, (w/v) agarose gels. Southern transfer to nitrocellulose filt’ers and hybridization with 32P-labeled probes were carried out, as described by Lin et al. (1984). A purified 2 kb 1’1 HpaT fragment that overlaps the Pl rrplicon region was used as a probe to quantify amounts of Pl DNA. ~I007 DNA was used as a probe for chromosomal 1)NA. Probes were labeled with [sr-32P]dCTP (sprc. act. 800Ci/mmol) according to the procedure described by Maniatis et al. (1982). The specific activit,y of labeled probes was greater than 10s cts/min per pg of DNA and approx. lo7 cts/min were used in each hybridization reaction. DNA bands were visualized by autoradiography with Kodak XRP film.

(f) Quantijcation of DNA fragments after Southern transfer

The relative amount of DNA present in DNA bands in Southern transfer experiments was determined by scanning exposed film using a Hoefer GS300 scanning densitometer. To permit more accurate comparison of amounts of a particular DNA fragment in different samples, int,ensities of DNA bands produced by several dilutions of a given sample were compared to bands generated by another undiluted sample containing the lesser amount of that particular fragment. The dilution factor needed to produce the same band intensity in the caomparison was used to calculate the difference in amount of fragment present m the 2 DNA samples. The relative amounts of DNA in a given fragment in the

various samples were corrected for differences in amounts of total DNA on the filters using the ZamR chromosomal gene probe.

3. Results

(a) Isolation of A-PI hybrid phages containing a lytic replicon

To isolate the Pl lytic replicon WC screened a library of 18 I-P1 hybrid phage containing Pl DNA fragments of 5 to 10 kb in size (see Fig. 1) whose Pl DNA inserts comprise an overlapping set of fragments that cover amost) the entire phage genome (O’Regan et al., 1987). We reasoned tha,t if any one of these fragments possessed a functional Pl lytic replicon, then the L-P1 phage with that fragment would not be able to establish efficiently a repressed prophage state because that lytic replicon would not be repressed by &I repressor. Conse- quently, that phage should exhibit a clear plaque phenotype on a A-sensitive host. Preliminary analysis of our I-P1 library by simple spot tests indicated that six of the hybrid phages made clear zones of lysis when lysates of those phage were spotted on a lawn of a I-sensitive host; ID69 (the vector in which the Pl inserts were cloned) and the remaining phages made turbid zones of lysis (Table 1 and Fig. 2(a)). When similarly tested on a l-sensitive host carrying a plasmid expressing the PI cl repressor gene, these six phages now made turbid zones of lysis indistinguishable from t)hose of the other phages and the vect,or (Table 1 and Fig. 2(b)).

To determine which of the six hybrid phages exhibits a clear plaque phenotype because it possesses a Pl replicon, we asked which of the hybrids would generate plaques when 10’ phage are spotted on a lawn of a homoimmune (Limm21) lysogen. The rationale is that a hybrid containing a constitutive replicon (in t,his case. the Pl lvtic replicon) should be able to replicate extensively in a homoimmune host, and should be able to over- titrate the host,‘s &mm21 repressor, provided it) does not make its own limwb21 repressor (Pereira da Silva & Jacob, 1968; Packman & Sly. 1968). Thus; while none of the original hybrid phage forms plaques on a homoimmune host because they possess a wild-type &mm21 repressor, rare imm2lcI- mutants in the lysate should be able t’o form plaques. When phage lysates were spotted on a lawn of a &mm21 lysogen, four of t,he six hybrids that produced clear plaques on a sensitive host (iPl-15, llPl-60, LPl-76, 1Pl-124) also made distinct zones of lysis on the imm21 lysogen (Table 1 and Fig. 2). However, phage that form plaques as efficiently on a homoimmune lysogen as on a sensitive host could be isolated only from the spot generated by the 1Pl-15 phage. Presumably, in the other three cases the zone of lysis on the homoimmune lysogen is not due to the presence of virulent phage in the lysate but refle&s the constitutive expression of lethal genes from the Pl DNA fragment present in these phapes.

102 G. Cohen and N. Sternberg

18

Figure 1. Physical alignment of Pl DNA inserts with respect to the Pl physical map in 18 &Pl hybrid phage DNAs that cover the Pl genome. The locations of all but one of the Pl DNA inserts (O’Regan et al., 1987), shown as open boxes, have been superimposed on the recent restriction and genetic map of Yarmolinsky (1987), to which the reader is referred for gene definitions and relevant references. The remaining Pl DNA insert covers the gap between 1Pl-31 and 1Pl-141. The Pl genome is, by convention, taken to be 100 kb and is sub-divided into 100 map units (mu). Genes are indicated outside the circles by bold-face numbers or acronyms, or bot.h. Bracketed symbols denote genes that have been identified only in the related phage P7 but are assumed to correspond to similarly positioned homologs in Pl. The Cl protein binding sites or operator sequences (Op) are numbered according to their integral position on the map. Bold- face letters and black boxes indicate insertions and substitutions relative to Pl; C designates the invertible segment of Pl. The IS1 element (part of B) is shown as an open bar and an arrow at, map position 96 depicts the site and direction of packaging of Pl DKA.

To substantiate the conclusion that the IPl-15 phage contains a lytic replicon, and that it can grow on a homoimmune Xmm21 lysogen if it contains an imm21cI- mutation, each of the six A-P1 hybrid phages that form clear plaques were crossed with a &mm21 phage carrying a cI- mutation (B17) and phage from the crosses were assayed on strain NS1539. Only the phage from the APl-15 cross gave rise to recombinants that could form a plaques on

that strain. These recombinants arose at a frequency of approximately 1 %, which is about the expected recombination frequency for two markers as far apart as the Pl lytic replicon fragment and the imm21 region. Following plaque purification, we could show that the plating efficiency of the ICI-Pl-15 recombinant phage was the same on &mm21-sensitive and immune hosts, although plaque size was much smaller on the latter. The PI

Isolation of the PI Lytic Replicon 103

Table 1 Spot tests of I-PI hybrids

E.-PI hybrid Indicator bacteria u 1 0. NS303 NS3006 Ml539 PI cl binding sites

t w Op53 t Op99.Op2 t t t t

t w 0~72

t w 0~86 t t t t Opdam t

t op51 ,op53 t -~ t

Spot tests of 1X I-PI hybrid phages. Symbols used: c, appearance of a clear zone of lysis on plates; t, appearance of a turbid zone of lysis: w, the appearance of a weak zone of lysis; a dash, the absence of a zone of lysis. Six of the iP1 hybrid phages LPI-15, -18. -60, -76, -124 and -139 make clear zones of iysis (and clear plaques) on a limm21-sensitive strain, NS303; thrse revert to turbid zones (and plaques) on a limm21-sensitive strain containing a plasmid (pNS3006) with the Pl cl gene (NS3006) (see Fig. 2). None of the I-P1 hybrids forms plaques on a I.imm21 lysogen (NS1539); however, 4 of the 6 1 phages, /:PI-16. -60, -76 and -124 produce weak zones of lysis on this strain (Fig. 2). Known and putative Pl Cl repressor binding sites present in the Pl DNA inserts of I-P1 hybrid phages are listed in t,he last column and are named for their location in integral map units of t,he Pl genome (see Fig. 1 and Yarmolinsky, 1987).

DNA fragment in APl-15 appears, therefore, to csontain a replicon that can substitute for the replicon of LD69. Moreover, since &I-Pl-15 is unable to plaque on a limm21 lysogen cont*aining

the Pl Cl repressor (NS3007), the PI replicon carried on this phage appears to mimic the properties of the Pl lytic replicon. These results are summarized in Table 2.

(b) Analysis qf PI-directed replication, in I-PI phages possessing a lytic replieon

Confirmation that API-15 possesses a lytic replicon comes from direct measurements of Pl replication following phage infection of &mm21 lysogens lacking or containing Pl Cl repressor. The phages used in this experiment were APl-15, ICI-PI-15 and 1Pl-93. The latter (see section (c), below) is a hybrid phage that does not contain a functional PI lytic replicon according to the t,ests described in the previous sect)ion. (iultures of infected cells were lysed at various times after infection, total cellular DNA was extracted, digested with rest,riction endonuclease IlpaI and examined for Pl DNA fragments by Southern analysis using a 2 kb Pl HpaI DNA fragment as probe (upper panels of Fig. 3(a) to (c)). This fragment had been shown in preliminary restriction digestion studies to overlap t,he DNA region contained in the Pl replicon (see section (c), below). The left half of each of the upper panels in Figure 3 describes the results of L-PI infections of homo- immune cells lacking Pl Cl repressor, and the right half of each of the upper pannels describes the results of infections of homoimmune (‘ells wibh that repressor. The lower panels show the same Dh’A samples as in the upper panels aft,er the UpaT probe was washed off the filter and the filter was rehybridized with a chromosomal 1anzR probe.

Inspection of the left half of each of the upper panels in Figure 3(a) and (b) shows that the amount of PI DNA present in total DNA in E,Pl-15 and LcI-Pl-15 infections increases bv some ten- to 15. fold during the 30 minute infection period. This is most clearly seen from the similar band intensities

(a) (b) (cl

Figure 2. Spot tests of I-P1 hybrid phages. Portions (5 ~1) of lysates (10” p.f.u./ml) of each of 18 k-l’1 phages and the vector AD69 were transferred to L plates inoculated with different indicator bacteria. Positions of phage isolates are arranged, starting at the top of the plate and running from left to right. in ascending numerical order of the isolate and finishing with AD69 (see the list in Table 1). Indicator bacteria; plate (a), NS303. a limm21-sensitive strain; plate (b), pljS3006. a t,ransformant of NS303 with the PI cl repressor plasmid, pNS3006: plate (c), NS1539. a limm21 1,vsogen of XS303.

104 G. Cohen and N. Sternberg

Table 2 Eficiency of plating of Aimm21-PI hybrid phages

possessing a PI lytic replicon

R-P 1 Indicator bacteria hybrid NS303 NS1539 NS3007 NSl813

RPl-15 1 2x10-s <lo-’ <lo-’ ICI-Pl-15 1 1 <lo-’ 0.9 API-143 1 2 x 1o-5 <lo-’ <lo-’ A-Pl-143 1 1 <lo-’ 0.9 limm2 ICI - 1 <lo-’ n.d n.d.

Efficiency of plating of A-P1 phages possessing a Pl lytic replicon. Numbers show the relative plating efficiencies of phages on bacterial strains with respect to that on strain NS303. nd., not determined. The indicator bacteria listed are NS303, a I-sensitive strain; NS1539, a kimm21 lysogen of NS303; NS3007, a transformant of NS1539 containing the PI cl plasmid pNS3006; and NS1813 a recA derivative of NS1539. Each of the phages has the immunity of phage 21. Where indicated, they also carry the imm2lcI- mutation of phage U17 (see Materials and Methods). Of several I-P1 hybrid phages tested (see the text) only APl-15 and APL143 were able to form a plaque on a limmfl lysogen after acquiring a imm2lcl mutation. These phages no longer form plaques on a knm21 Iysogen if Pl Cl repressor is present.

in the undiluted DNA samples at two minutes after infection and the 16-fold diluted DNA samples at 32 minutes after infection. Since the multiplicity of infection used in these experiments was three phage per cell, the number of Pl copies present at 32 minutes after infection is 30 to 45 per cell. A similar estimate is obtained by matching band intensities from IPl-l&infected cells with those of the HpaI- cleaved APl-15 DNA standards that are shown in the rightmost three lanes of the upper panels of Figure 3(a) and (b). No significant increase in the level of Pl DNA occurs in the control APl-93 infection (Fig. 3(c)). The right half of each of the upper panels in Figure 3 shows the effect of Pl Cl repressor on Pl DNA synthesis. No stimulation of Pl replication was found under these conditions in any of the A-Pl-infected cultures throughout the course of infection. These results demonstrate that the Pl DNA insert in APl-15 is able to direct its own replication and that this replication is inhibited by the Pl Cl repressor. The data also show that the extent of Pl replication is essentially the same whether the APl-15 phage carries a wild-type or a defective ICI gene.

Tn a parallel study, we measured the amounts of A DNA present in IPI-15, ICI-Pl-15 and IPl-93 infections of Lmm21 lysogens in the absence or presence of Pl Cl repressor. Figure 4 shows Southern analysis of 2 DNA in total cellular DNA employing 1D69 as probe. The DNA examined is that present in the blots used in the experiment described in Figure 3. Most of the larger J DNA HpaI restriction fragments can be seen in each of the DNA samples, even at’ the earliest time-point after infection. The intensities of these fragments increases in IPl-15 and AcIIPl-15 infections during the course of the experiment in the strain without PI repressor (left half of panels). There is ten to 15

times more DNA at 32 minutes after infection than at two minutes after infection. As in the previous experiment, this stimulation of replication is abolished by the Pl Cl repressor (right half of panels). No increase in band intensities was found in the control APl-93 infections either in the absence or presence of Pl Cl repressor. These results and the data presented in Figure 3 provide clear evidence that Z-15 possesses a Pl lyt’ic replicon that directs the i vector to replicate to a high copy number.

(c) Restriction endonucEease digestion analyses of the Pl lytic replicon

The Pl DNA content of the lytic replicon in LPI-15 and several related hybrid phages was initially determined from EcoRI restriction diges- tion of phage DNAs. Table 3 lists the EcoRT fragments present in four hybrid phage DNAs. Like iPI-15, 1Pl-143 possesses a functional Pl lytic rep- licon as judged by its ability to form plaques on a homoimmune lysogen when it carries an imm2lcI mutation (Table 2). The other two hybrid phages,

LPI-15 -Pl repressor +Pl repressor

Time 2 17 32 2 17 32 ------ Dilution 1 4 1 4 161 4161 4 1 4 16 1 4 16 St&

(a) Fig. 3.

Isolation of the Pl Lytic Rep&on 105

hd PI-15 LPI-93 -PI repressor +Pl repressor -PI repressor +Pl repressor

Time 2 17 32 2 17 32 Time 2 17 32 2 17 32 ------ --pm-- Dilution 1 4 1 4 161 4 16 1 4 1 4 16 1 4 16 St& Dilution 1 4 1 4 161 4 16 1 4 1 4 16 1 4 16 St&

Figure 3. Quanitification of Pl-specific DNA following I-P1 infection of cells containing a homoimmune 1 prophage. Southern analysis of total cellular DNA from lysates of L-Pl-infected cells was used to measure PI DNA replication in Pl Cl repressor-free and repressor-containing cells. Cultures of a &mm21 lysogen (NS1539) and a transformant that contains the Pl Cl repressor (NS3007) were infected at a m.o.i. of 3 with (a) LPl-15 or (b) ICI-Pl-15. both of which possess a Pl lytic replicon. or (c) 1Pl-93, which lacks a functional lytic replicon. After phage adsorption, and subsequent incubation of the culture, samples of infected cells were removed at 2, 17 and 32 min and lysed. Total cellular DNA was extracted as described in Materials and Methods. DNA was digested with restriction endonuclease Hpal, concentrated and samples of dilutions electrophoresed in 06% agarose gels and transferred to nitrocellulose filters, Pl DNA was measured by hybridization with a 32P-labeled 2 kb Pl HpaI DNA fragment (Fig. 5) that overlaps the Pl DNA inserts contained in the infecting I-P1 hybrid phages (upper panels). Following autoradiography, the filters were washed to remove the bound radioactive material and rehybridized with a pBR322 1amB (~1007) probe to quantify chromosomal 1amB DRiA (lower panels). The left half of each of the panels contains DNA extracted from phage-infect,ed cells that do not have a Pl cl gene; the right half of each of the panels contains DNA from phage-infected cells that have a Pl cl gene. Several dilutions were analyzed for each DNA sample; 2 min samples were diluted 1 : 1 and 4-fold; 17 and 32 min samples were diluted 1 : 1, 4-fold and 16-fold. Dilutions for each time-point are arranged from left to right in order of increasing dilution. The 3 rightmost lanes in each panel contain HpaI-digested 1Pl-15 DNA samples with 0.5, 2.5 and 10 ng of phage DNA, corresponding to approximately 25, 125 and 500 pg of the HpaI 2 kb Pl fragment. The position of the 2 kb HpaI fragment is indicated by the arrow to the left of each of the upper panels. Note that, since iPl-93 DNA lacks I of the 2 PI HpaI sites that defines the 2 kb Pl HpaI fragment in iPl-15, the HpaI band derived from 1Pl-93 DNA (upper panel of Fig. 3(c)) is not 2 kb. Inspection of the lower 3 panels shows that the amounts of the 1amB chromosomal DNA (indicated by the arrows) present in different DNA samples, at the same dilution, are approximately the same. The single exception is the 32 min API-15 sample. Correcting for this deviation would raise the estimated amount of PI DNA made at this time compared to that at 2 min. A more precise analysis of the data corroborates t,he above conclusions. Other, unrelated, bands seen in the autoradiograms belong to several of the molecular weight DNA markers (lower 3 panels, leftmost lane) and the plasmid containing the Pl cl gene (right half of lower 3 panels), which hybridizes with the pBR322 sequences in the 1amB chromosomal probe. In addition, traces of Pl-labeled fragments that were not entirely removed prior to the 2nd hybridization can be seen in some of the lower panels.

106 G. Cohen and N. Sternberg

-PI repressor +Pl repressor

Time 2 17 32 2 17 32 _----- Dilution 1 4 1 4 161 4 16 1 4 1 4 16 1 4 16 Stds

hd Pl-15

LPI-93

Figure 4. Quantification of I-specific DNA following i-P1 infection of cells containing a homoimmune i prophage. Southern analysis of total cellular DNA from lysates of I-Pl-infected cells was used to measure i DNA replication in cells containing or lacking the Pl Cl repressor. The experiment was carried out using the same Southern blot used to analyze Pl and chromosomal DNA in Fig. 3. Filters were washed to remove label and rehybridized with a labeled AD69 DNA probe. The order of DNA samples is that shown in Fig. 3. Most of the larger HpaI fragments of the 1D69 vector can be seen in each of the DNA samples from the phage-infected cells and each dilution. They are also seen in the 1Pl-15 DNA standards present in the rightmost 3 lanes.

APl-93 and IPl-139, contain Pl DNA from the same region of the Pl genome as the IPl-15 and iPl-143 phages, but were judged not to contain a functional replicon because imm21 CT- derivatives of these phages were unable to form plaques on a &mm21 lysogen (data not shown). Hybrid phages APl-15 and APl-139 are members of the set of 18 recombinant phages whose Pl DNA inserts cover the Pl genome (Fig. l), while LPl-93 and IPl-143 are members of a larger library of 54 characterized hybrids (O’Regan et al., 1987). Comparison of the EcoRI fragments present in these hybrid phage DNAs indicates that they all share only the adjacent EcoRT fragments 25 and 21 (Fig. 5).

To generate a more detailed restriction site map of the PI lytic replicon contained in LPl-15 and llPl-143, we isolated the unique HindIII-Sal1 fragments, containing the entire Pl inserts, from all four of the I-P1 hybrid phages shown in Table 3. The fragments were digested with a variety of restriction enzymes and the resulting products were analyzed by agarose and polyacrylamide gel electrophoresis (data not shown). Figure 5 shows the Pl DNA content and the restriction sites of those A-PI DNA fragments. Comparison of the inserts present in IzPl-15 and ;iPl-143 reveals that they share approximately 2.8 kb of Pl DNA, of which 2 kb is contained within a single HpaI

Isolation of the Pl Lytic Replicon 107

Table 3 PI EcoRl restriction DNA fragments present in related L-PI phages contairGng a

functional or defective PI lytic replicon

Phage

Z-IF, iPI -93 11’1-143 LPI-139

Pl EcoRI restriction fragments Presence of intact 5 15 18 23 17 21 25 14 9 lytic replicon

- + + + + + + - - + - - + + + + + - - - - + + + - + - - - - - + + + + -

1’1 EcoRI restriction fragments present in I-P1 hybrid phages used in this work. Phage DNAs were analyzed as described (O’Regan et al., 1987). The order of EcoRI fragments corresponds to that in thr PI chromosome (see Fig. 5). All of the EcoRI fragments in digests, other than EcoRI-25 were detect)ed by agarose gel electrophoresis. EcoRI-25 was detected by polyacrylamide gel electrophoresis. Symbols used: + denotes t,he presence of an EcoRI fragment and - denotes its absence.

fragment. This fragment includes unique EcoRV, accompanying paper; Sternberg & Cohen, 1989) BglII, KpnI, ClaI and AsuII restriction sites. show that the HpaI 2 kb fragment includes the Comparison of the shared Pl DNA sequence in the entire lytic replicon. two replication-proficient phages S-15 and APl-143 with the Pl DNA sequence in the two replicon-defective phages IPl-93 and A-Pl-139 shows that, iPl-93 lacks a region in EcoRI fragment 4. Discussion

14, and E.Pl-139 lacks a region in EcoRI fragment In this paper, we report the isolation of a Pl lytic 17. that are presumably needed for a functional replicon in a i library of Pl DXA and present’ lytic replicon. Ot,her experiments (described in the information on its size and position on the Pl

P53/Op53 AH EBKCAIH

XPI- I5 rep+

API-93 rep-

XPI - 143 rep+

XPI-139 rep-

Figure 5. Partial restriction site map of the Pl replicon region and A-P1 hybrid phages cont,aining,that region. The open rectangular box at the top of the Figure represents the region of Pl DNA containing the DNA inserts present in these L-P1 phages. The box is subdivided to indicate the relative position and size of Pl EcoRI restriction fragments belonging to this region; other restriction siteas are shown by arrows. The extent and position of the Pl DNA inserts in different l-P1 hybrids is shown by the stippled bars. These were det,ermined by restriction analysis of L&I’1 DXAs. Hroken lines at the left and right ends of the Pl DNA inserts in APl-93 and API-139. respect,ively. indicate that the end-points were not determined precisely. The left boundary of the Pl DNA insert in the replicon-defective phage 1Pl- 93 is localized near the end of Pl EcoRI fragment 15, and the right boundary is localized between the AsuII and HpaI restriction sites in Pl EGoRI fragment 14. Presumably LPl-93 lacks the P53 promoter, which is positioned about 500 bp from the junction of Pl EcoRI fragments 25 and 14 (Sternberg & Cohen, 1989). The right boundary of the Pl DNA insert in IPI-139 is located beyond Pl EcoRI fragment 9 and the left boundary falls within a 125 bp region separating the junction of 1’1 EcoRI fragments 21 and 17 and the BgZII restriction site in Pl EcoRI fragment li. The 2 Pl replicon-containing hybrid phages 1Pl-15 and 1Pl-143 share a common region of 2.8 kb of PI DI\;A. A. ,4~~11: B, Bg/II: E. EcoRV: K. K@; C, CZuI: H. HpaI.

108 G. Cohen and N. Sternberg

physical map. Several phages in our library of 18 & Pl hybrid phages were found to make distinctly clear plaques on a A-sensitive host, whereas other phages as well as the vector make turbid plaques. In each case, when Pl Cl repressor was provided, the clear plaque phenotype disappeared. The Pl DNA inserts in the clear plaque-forming phages all contain one or more Cl repressor binding sites (Table l), which, in so far as their locations are known, map within promoter elements positioned upstream from genes involved in vegetative growth (Velleman et al., 1987; Yarmolinsky & Sternberg, 1987; Eliason & Sternberg, 1987). These include genes involved in lytic replication, DNA methyla- tion and control of repression. We suppose that overexpression of these genes from a A-PI phage interferes with establishment of ,? lysogeny and results in clear plaques. One of the hybrid phages, APl-15, was subsequently shown to contain a Pl lytic replicon through its ability to direct the high copy number replication of this phage under conditions in which the 1 replication system cannot function. Moreover, as this replication is abolished by the Pl Cl repressor, it mimics Pl lytic replication.

Restriction analysis of the Pl DNA region in two &Pl phages containing a functional Pl lytic replicon, IPl-15 and 1Pl-143, reveals that the replicon includes part of EcoRI fragment 14, all of EcoRI fragments 25 and 21, and part of EcoRI fragment 17. The replicon is, therefore, located on the standard Pl map between regions determining the plasmid replicon and the imm1 immunity region (see Fig. 1). Recent studies have shown that EcoRI fragment 14 contains a Cl binding site, designated 0~53, that overlaps a strong promoter, termed P53, which directs transcription clockwise on the Pl map, and that P53 is repressible by the Pl Cl gene product (Sternberg et al., 1986). Data presented in the accompanying paper place this promoter between the HpaI and AsuII sites of EcoRI fragment 14 (Fig. 5). The Pl DNA inserts of both hybrid phages that possess a functional lytic replicon contain this region, whereas the Pl DNA insert of a replicon-defective phage, APl-93, lacks this region, and presumably the promoter element as well. In another replicon-defective phage, APl-139, the P53 promoter region is present but a region of EcoRI fragment 17 found in the two replication-proficient phages is missing. The missing region presumably contains sites and/or genes needed for lytic replication. The juxtaposition of PI control elements (P53 promoter and 0~53 repressor binding sites) within the Pl DNA region containing the replicon strongly suggests the possibility that these elements form an integral part of the replicon. It would appear likely that lytic replication is blocked in the prophage state by Cl-mediated repression of P53. Evidence presented here that supports a role for Cl as a direct repressor of the lytic replicon comes from Southern analysis of cellular DNA isolated following APl- 15 infection of cells containing a homoimmune ;1 prophage.

Evidence that supports the proposal that the site of Cl action is P53 comes from experiments described by Sternberg & Cohen (1989).

The approach we describe in this paper for isolating a Pl lytic replicon led to the identification of a single replicon. It is not clear if this lytic replicon is the only one specified by Pl. In previous studies, two classes of Pl replicons were isolated in a 1 phage vector by the extension in viva of a cloned Pl fragment, EcoRI-5 (see Fig. l), which by itself is not capable of directing replication (Sternberg & Austin, 1983). One of these replicons, called the R replicon, corresponds to that used for maintenance of the plasmid state in Pl lysogens. The other, called the I, replicon, has properties that suggest that it more closely resembles plasmid copy number mutant replicon than a lytic replicon, since it is not, repressible by the Pl Cl gene product and is sensitive to incompatibility by Pl. However, the fact that l--P1 plasmids containing it replicate to a high copy number (15 to 20 copies per cell chromosome) raises the possibility that it may play a role in lytic replication. The L replicon contains Pl EcoRI fragments 5, 15, 18 and 23, and parts of fragments 8 and 17. Thus, it is physically different from the lytic replicon we report here. Compelling evidence that the Pl replicon isolated in this work is, in fact, the primary lytic replicon is based on experiments presented by Sternberg & Cohen (1989).

The question of whether Pl uses more than one replicon in vegetative growth is raised by other studies, which show that lytic replication appears to occur in two stages; an early circular theta phase and a later rolling circle phase (Cohen, 1983). Transition from early to late replication is depen- dent, in an unknown way, on a functional host recombination system, so that Pl replication is reduced by some ten- to 20-fold in recombination- deficient strains (Zabrovitz et al., 1977). Both I-P1 hybrid phages described in this paper that possess a Pl lytic replicon grow equally well on recombina- tion-deficient and wild-type strains (Table 2), implying that the replicon they contain is that used to initiate early Pl replication. This idea is consistent with the fact that Pl Cl repressor largely blocks initiation of replication in the prophage state, and similarly blocks initiation of replication in the Pl replicon we have described here.

We thank Drs Ronald Hoess and Jeffrey Chernak for their careful reading of the manuscript, and Penni Lockhart for her diligent typing of the manuscript.

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Edited by M. Gottesman