Vol. 153, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1983, p. 800-8120021-9193/83/020800-13$02.00/0Copyright © 1983, American Society for Microbiology

Isolation and Characterization of P1 Minireplicons, X-P1:5Rand X-P1:5L

NAT STERNBERG* AND STUART AUSTINBasic Research Program-LBI, Frederick Cancer Research Facility, Frederick, Maryland 21701

Received 22 October 1982/Accepted 19 November 1982

We have isolated two new classes of Pl miniplasmids, called X-P1:5R and X-P1 :5L, by the in vivo extension of a cloned Pl fragment, EcoRI-5, which by itselfis not capable of plasmid replication. The X-P1:5R plasmids contain EcoRI-5 plusa variable portion of the adjacent P1 EcoRI fragment 8. They have a copy numberlike that of Pl (about 1 per host chromosome), are faithfully segregated at celldivision, and are subject to incompatibility exerted by either a single copy of Pl ora single copy ofEcoRI-5. In contrast, the X-P1 :5L plasmids contain EcoRI-5 and aportion of adjacent Pl DNA that includes at least Pl EcoRI fragments 15, 18, and23 and a part offragment 17. These plasmids have a copy number of about 15 percell chromosome. Despite this they are segregated to daughter cells somewhatless faithfully than are X-P1:5R plasmids. They are sensitive to incompatibilityexerted by a single copy of P1, but not to incompatibility exerted by a single copyof EcoRI-5. X-P1:SL plasmids are, however, sensitive to incompatibility exertedby multiple copies of EcoRI-5. These results show that the relative copy numbersof exerting and responding elements are important for the incompatibility pheno-type and strongly suggest that X-P1:5L plasmids lack a repressor of replicationthat can be supplied in trans from P1 but not from EcoRI fragment 5. We suggestthat P1 normally uses the SR replicon and that the 5L replicon may be a backupsystem that ensures plasmid maintenance should the primary replication event failto initiate.

Bacteriophage P1 is a temperate virus that hasboth a lytic and a lysogenic phase in its lifecycle. In its lysogenic phase, Pt is an autono-mously replicating plasmid with a copy numberabout equal to the chromosome number of itshost bacterium, Escherichia coli (3, 19, 27). Thefaithful inheritance of plasmid DNA is assuredby a partitioning process that produces plasmid-free cells as rarely as once in every 105 celldivision events (4, 28). To learn how these pro-cesses of controlled plasmid replication andequipartition are achieved, we and others havechosen to dissect the large 95-kilobase (kb) PtDNA molecule into smaller segments that stillretain some or all of the plasmid maintenancefunctions (2, 3, 26). Initially we were interestedin two properties of the plasmid state: incom-patibility and replication. Incompatibility is theinability of differentially marked isogenic plas-mids to be maintained stably in the same cell.We were able to localize at least one elementthat exerts P1 incompatibility to a 7.0-kb frag-ment of P1 DNA, EcdRI-5 (36). Thus, cells thatcontain an integrated copy of that fragment intheir chromosome could not maintain an autono-mously replicating P1 plasmid. More recently,two incompatibility elements (incA and incB)

have been localized to separate parts ofEcoRI-5(2).Our initial strategy for defining the plasmid

replication region was based on a false premise,namely, that the region of overlap between twoP1-derived miniplasmids, pIH1972 (30) andPlAN19 (3) (Fig. 1), must contain all the neces-sary information for plasmid replication. In fact,the overlap region, a 4.5-kb segment of P1EcoRI-5, is not itself capable of autonomousreplication (36). This paradox has been resolvedby the studies described here. We have isolatedand characterized new P1 miniplasmids. Theirproperties show that two separate but adjacentregions of P1 DNA can replicate autonomously.One contains a segment of P1 DNA present onpIH1972, and the other contains a segment of P1DNA present in P1AN19.

MATERIALS AND METHODSBacterial and phage strains. The bacterial strains

used in this paper are listed in Table 1. X-P1 hybridphages containing EcoRI fragments of P1 DNA insert-ed into the EcoRl site of the X D-srIX3 vector aredescribed in Stemnberg et al. (37) and in Stemnberg (33).The X-P1:BglII-4 phage was constructed by cloning theBglII-4 fragment of Pl DNA into the BamHI site of X

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CHARACTERIZATION OF Pl MINIREPLICONS 801

P1 map

R R8

FTb c-t d| e tK K H H

-Hidll-3-

PIH1972

P1AN19K K b538

X-P1:5 36 39

R R R R -ARR25

R5 g 15 1 18 23J 1;1 i j 14 j

I t Sl t h f|tBg K K Bg

*0 BgIII -4

R

65H R

R

65R4X-P1 :5R-1

X-P1 :5R-2

X-P1:5R-3

39K K K H R R

4 f-k 7 + j iFmwwvvv v

39K K K H R R

39

B Bg H H Bg nin5 H

+ + ++ + V i71 73 76 77 70 91~V"

IO kblo

R B4 +

71R R R+ + 15 +

71R R R R

+ 15 + 18 +29H

76R R R R R R R

15 4 18 W23+ 17 i i

Bg/B Bg+

FIG. 1. Partial restriction map of P1 and P1 miniplasmid DNAs. Pl DNA is represented by a straight line, andDNA is represented by a wavy line. The location ofEcoRI (R), HindlIl (H), and BglII (Bg) sites on the Pl DNA

is from Bachi and Arber (5). The location of KpnI (K) sites in EcoRI-8 and EcoRI-5 is based on the restrictionenzyme analyses shown in Fig. 3. The letters a through i designate particular P1 restriction fragments referred toin Fig. 3, 4, and 5. The boundaries of P1 DNA present in miniplasmids pIH1972 and P1lAN19 are from Shaffermanet al. (30) and R. Mural (personal communication), respectively. The dashed line represents the extent ofuncertainty in these measurements. The construction of X-P1:5 DNA is described in Stemnberg (33) with thecoordinates of restriction sites on X DNA from Daniels et al. (11). b538 and nin5 are A DNA deletions locatedbetween k map coordinates 44 and 60 and 83 and 89, respectively (15). The maps of X-P1:5R and X-P1:5L DNAswere generated from the restriction analysis shown in Fig. 4 and 5. We have drawn the map of X-P1:5L-1 toinclude a portion of P1 EcoRI-15 because genetic rescue experiments indicate that this phage contains the wild-type alleles of two P1 amber mutations known to be on fragment 15, am136 and ami80 (26, 33).

D-srIX3 at A map coordinate 71.3 (37). All of these X-P1phages retain the original cIts857 repressor mutationand b538 deletion of the vector. The latter removes theA integrative recombination system. immA clearphages (W30 and W248, henceforth called WW) wereused to select X immX lysogens, and imm434 clearphages (G9 and G217, henceforth called GG) wereused to select A imm434 lysogens (34). A-Pl:5R and K-

P1:5L phages are described in this paper. PlasmidpALA8 is pBR325 with P1 EcoRl fragment 5 cloned atthe EcoRI site (2). The P1 phage used here is the PlCmphage described by Kondo and Mitsuhashi (21).Media and buffers. Li broth was used as a liquid

medium (33). Li agar is LI broth with 15 g of agar perliter. EMBO agar (18) and TMG buffer and TASEbuffer (33) have been described elsewhere. TE bufferis 10 mM Tris-hydrochloride (pH 8.0)-i mM EDTA.

Standard phage methods. Preparation of K lysatesand the purification of phages and phage DNA havebeen described by Stemnberg et al. (37). Measurementof the frequency of lysogenization and the perform-ance of phage crosses have been described by Stern-berg and Hamilton (34). A immA cIts857 Oam29-P1:5Rand A immX cIts857 Pam8O-P1:5L were constructed bycrossing A immA cIts857-P1:5R with imm434 Oam29and immk cIts857-P1:5L with imm434 Pam8O. Thephages from the cross were plaqued on strain YMCand incubated overnight at 38°C. Phages containingthe immk cIts857 marker (clear plaques) were trans-ferred with toothpicks to plates containing either strainW3350 or YMC to screen for recombinants containingamber mutations.

Construction of A-P1:5R and A-P1:5L by the exten-sion of P1 EcoRI-5. The strategy for construction of K-

X-P1 :5L-1

X-P1 :5L-2

R4

X-P1 :5L-3R4

X-Pl :5L-4

X-P1 :Bgill-4

R

R Bg/B+ +

Bg

4vwvv73

I I Ivvwvvwvv

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802 STERNBERG AND AUSTIN

TABLE 1. Bacterial strains

Strain Relevant genotype Reference

YMC supF Dennert and Hen-ning (12)

W3350 sup+ Campbell andBalbinder (7)

YMC(P2) supF, P2 lysogen Stemnberg et al.(35)

N205 recA3 sup+ Stemnberg andWeisberg (38)

RW842 sup', cryptic K in Enquist and Weis-galT berg (14)

N1200 Hfr KL16 recA56C600 supE Appleyard (1)NS616 C600 supE recA56 This reportNS1026 C600 supE recA56 This report

groPA15NS1249 N205 (K imm434- Stemnberg et al.

P1:5) (36)aNS1526 N205 loxB::PlCm Stemnberg et al.

(35)b

a The K imm434-Pl:5 prophage in this lysogen isintegrated at attB (17) in the host chromosome.

b The PlCm prophage in this lysogen is integrated atloxB (35) in the host chromosome as described previ-ously (36).

P1:5R and A-P1:5L is illustrated in Fig. 2a. StrainRW842 thyA-(PlCm) was infected with phage A-P1:5,and K lysogens were selected on EMBO agar platescontaining chloramphenicol (25 ,ug/ml) and WW selec-tor phages. The frequency of lysogen formation was 5X 10-4 per infected cell. All of these lysogens containcomposite plasmids consisting of the A-P1:5 DNAintegrated into the P1 plasmid DNA as judged both bygenetic criteria (36) and by electrophoretic analysis ofplasmid DNA on agarose gels (Fig. 2b). The RW842thyA-(P1Cm::X-P1:5) lysogens were converted torecA by mating them with strain N1200, selectingthyA+ Cmr colonies, and screening these colonies forsensitivity to UV radiation. About 50%o of the thy'Cmr colonies were sensitive. The recA lysogens werepurified and grown in Li broth to 2 x 108 cells per mlat 32°C; the K prophages were then induced by shiftingthe temperature of the culture to 42°C for 90 min. Theyield of plaque-forming K phages under these condi-tions is about 0.1 per induced cell. This low yield canbe accounted for by the need for K DNA to be excisedfrom the composite plasmid before it can be packagedinto a virion. In the recA host, excision by homologousrecombination between the fragment 5 sequencesbracketing the K DNA is inefficient; consequently, theresulting phages are enriched for packaged K se-quences that have been excised from the composite byillegitimate recombination (Fig. 2a). The resultingphage lysate was used to lysogenize strain N205, and afrequency of 10-4 per infected cell was observed. Thisvalue is 100 times higher than the lysogenizationfrequency of the original K-P1:5 phage. Three N205lysogens were purified, and their K prophages wereinduced to produce the three X-P1:5R phages studiedin this paper.About 1 of 104 phages produced from the recA

composite lysogen was able to form plaques on strainYMC(P2), an indication that these phages are defec-

tive for K red and gam gene functions (the Spi-phenotype (40]). Four of these phages were purifiedand are the A-P1:5L phages studied here. All of theseA-Pl:5R and K-P1:5L phages were isolated from inde-pendently derived composite lysogens.

Restriction endonuclease digestion and gel electropho-resis. Digestion of K, A-Pt, and Pt DNA with theenzymes EcoRI, KpnI, HindIII, BgIII, and BamHIwas carried out under the conditions recommended bythe vendor (Bethesda Research Laboratories). Agar-ose gel electrophoresis of restriction fragments wasperformed as described by Stemnberg and Hamilton(34), and polyacrylamide gel electrophoresis was per-formed as described by Stemnberg et al. (35).Measurement of rates of plasmid loss. The natural

rate of loss of a A-Pt plasmid prophage was measuredas described in Austin et al. (2). Briefly, coloniescontaining aX immX-P1 miniplasmid prophage growingon agar plates containing WW selector phages werepicked and added to TMG, freed of the selector phagesby centrifugation, and then grown for eight genera-tions in Li broth with 10 mM sodium citrate. Sampleswere then spread on Li citrate agar and incubated at32°C overnight. Colonies were picked with toothpicksand stabbed into Li agar plates and Li plates spreadwith WW selector phages to test for plasmid retention.Plasmid loss due to incompatibility was measured asfollows. Strains containing pBR325 or pALA8 wereconstructed by transformation of N205 (X-P1:5R) or(X-Pl:5L) lysogens with the plasmid DNA (9) andselection of Ampr colonies on Llamp plates with WWselector phages. The colonies were purified once onthese same plates, freed of selector phages by centrifu-gation and washing, and then grown for 12 generationswithout selection in Li broth. The cells were thenspread on Li plates, and after overnight incubation,colonies were picked with toothpicks to agar plateswith selector phages or ampicillin to measure the lossof either immK or Ampr markers.

Detection of plasmid DNA by gel electrophoresis.Plasmid DNA wah detected by a slight modification(36) of the agarose gel electrophoresis method ofEckhardt (13). Either the plasmid was labeled with[3H]thymidine and detected by autoradiography or itwas detected in gels by ethidium bromide staining andquantitated by hybridization as follows. The gel wasfirst soaked for 30 min in 250 ml of 0.25 N HCI, thenfor 30 min in 250 ml of 1.5 M NaCI-0.5 N NaOH, andfinally for 2 h in 1 M ammonium acetate-0.02 NNaOH. The DNA was then transferred from the gel tonitrocellulose filters by the method of Southern (32) in1 M ammonium acetate-0.02 N NaOH for 12 h. TheDNA was dried under vacuum for 3 h at 80°C and washybridized with either K or Pt DNA labeled with[32P]dCTP and [32P]dTTP to a specific activity of 5 x108 cpm per ,ug of DNA by the nick translationprocedure of Maniatis et al. (24). Hybridization wascarried out at 68°C for 12 h with 10' cpm in 10 ml ofhybridization solution consisting of 4x SSC (1 x SSCis 0.15 M sodium chloride-0.015 M sodium citrate),0.02% Ficoll, 0.02% polyvinylpyrollidon, 0.02% bo-vine serum albumin, 40 mg of sonicated calf thymusDNA per ml, and 10%o dextran sulfate. The filters werewashed with 0.1 x SSC-0.1% sodium dodecyl sulfateat 68°C. Kodak XR5 X-ray film was exposed to thefilter for various times. The plasmid bands were quan-titated by scanning the exposed films with a Quick

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(a)

Pi

x - -

recA +

CHARACTERIZATION OF P1 MINIREPLICONS

c1857

recA

420

recA420

c1857 c1857

b. c. d.

"I,. i

FIG. 2. The isolation of X-P1:5R and X-P1:5L phages. (a) P1 DNA is represented by a straight line and A DNAby a wavy line. P1::X-P1:5 composites were constructed in a recA+ strain, the strain was then made recA, and theX prophage was induced. Excision of the X-P1 DNA from the composite by an illegitimate recombination eventgenerates either X-P1:5R or X-P1:5L phages. (b) Gel electrophoresis of whole-cell lysates of strain RW842containing (a) no plasmid, (b) X-P1:5R-3, (c) PlCm, and (d) composite plasmid formed between PlCm and X-P1:5.

Scan scanning densitometer at 525 nm. The bandintensity chosen for scanning was always within thelinear range of the exposure response.

Liquid hybridization determination of A-P1 plasmidcopy number. X-P1 plasmid copy numbers were deter-mined by the liquid reassociation kinetics methoddescribed previously (3), with the folowing modifica-tions. 3H-labeled X cI857S7 DNA was used as the soleDNA probe. No internal standard for total DNAconcentration was used. The total DNA concentrationof the test samples was determined by measuring theoptical density of the samples at 260 nm and applying asmall correction for residual protein based on the 260nm/280 nm ratio of optical densities. The reassociationrate of denatured probe DNA into double-strandedDNA was measured by using a fixed concentration ofDNA extracted from a control strain (N205 with asingle copy of A integrated at attX in the bacterialchromosome) and by using the same concentration of

DNA extracted from N205 lysogenized with X-P1:5Lor X-P1:5R phages. The ratio of reassociation rates oftest and control strains determined by averaging threeseparate assays was taken as the copy number of Xsequences in the test strain. The values obtained wereconsistent with the approximate values for relativeplasmid copy number obtained by gel electrophoresis.

Isolation of A-P1:SR and A-P1:SL piasmid DNA.Plasmid DNA was isolated from exponentially grow-ing cultures (3 x 108 cells per ml in Li broth at 32°C) ofstrains N205 (X-P1:5R) or N205 (X-P1:5L) by the lysismethod of Clewell and Helinski (8). The isolatedplasmid DNA was precipitated with ethanol by centrif-ugation at 15,000 rpm in an SS34 rotor and resus-pended in 6 ml ofTE buffer. CsCl was added to 5.86 g,ethidium bromide was added to 500 ,ug/ml, and thesolution was spun in a 50 Ti rotor at 40,000 rpm for 72h. The supercoiled DNA band was collected with asyringe, the ethidium bromide was extracted four

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804 STERNBERG AND AUSTIN

times with CsCI-saturated isopropanol, and the DNAsolution was extensively dialyzed against TE.

RESULTSConstrucon ofnew P1 minIplasmids. When Pl

EcoRI fragment 5 is cloned in a phage X vector,the resulting chimera cannot be maintained as anautonomously replicating plasmid prophage(36). However, EcoRI-5 does contain severalelements involved in P1 plasmid maintenance.These include the region of Pl DNA shared byP1 minireplicons pIH1972 and PlAN19 (3, 30,36) (Fig. 1), P1 incompatibility (36), and a Plgene whose product is essential for plasmidsegregation (31). Therefore, we reasoned that ifPl plasmid maintenance functions are clustered,as they appear to be in other plasmid replicons(23, 25), then additional elements necessary forPl replication might be encoded by segments ofP1 DNA flanking EcoRI-5. By extending EcoRI-5 to include those segments; we hoped to isolatenew minireplicons. The extension protocol isillustrated in Fig. 2a and is described in detailabove. First, a A phage containing EcoRI-5 (X-P1:5) was used to construct a Pl::X-Pl:5 com-posite plasmid by recombination between ho-mologous Pt sequences present in the X-P1 andP1 DNAs. The presence of a composite plasmidin the resulting strain was confirmed by gelelectrophoresis (Fig. 2b). The strain was maderecA, and the A prophage was induced. Phage Xproduction under these circumstances requiresthe excision of the X-P1 DNA from the compos-ite. In a recA host, excision is likely to occur bya recombination event that does not recognizeDNA homology, increasing the chances that theexcised X-P1 DNA will include additional PtDNA to the left or right ofEcoRI-5 in its positionon the conventional P1 map (5). Phages withrightward and leftward extensions are designat-ed X-P1:5R and X-P1:5L, respectively. Becauseof the orientation of EcoRI-5 in the X-P1 :5 phageused here, the map cannot be drawn with boththe A and Pt sequences in their conventionalorientations. We have elected to draw Fig. 1with the X map as it is normally drawn (11), andtherefore the Pt map is reversed (39). Accord-ingly, Pt EcoRI-15 is to the right of EcoRI-5 inFig. 1 and P1 EcoRI-8 is to its left. Three phageswith extensions of EcoRI-5 into EcoRI-8 (X-P1:SR phages) and four phages with extensionsinto and through EcoRI-15 (X-P1:5L phages)were isolated (Fig. 1). In the former case thephages were selected from the total excisedpopulation by virtue of their ability to lysogenizestrain N205. Since the observed frequency oflysogeny of that population was 10-4 (see above)and the lysogenization frequency of X-P1:5R was0.10 to 0.15 (see below), we estimated that about1 of 1,000 phages in the excised phage popula-

tion was a X-P1:5R phage. The selection of A-P1:5L phages was based on the loss of the X redand gam fenes (the Spi- phenotype [40]). About1 of 10- phages in the phage population wasSpi- (see above), and about 50% of these (2 of 4,see below) were X-P1:5L phages.

Partial restriction maps of P1 EcoRI-5, P1EcoRI-8, A-P1:5R, and X-P1:5L DNA. The P1restriction map of Bachi and Arber (5) containsrelatively few restriction sites in either EcoRI-5or EcoRI-8 (Fig. 1). We decided to expand therepertoire of mapped restriction sites in thesefragments before embarking on an analysis of X-P1 :5R and X-P1 :5L DNA. The enzyme we foundmost useful was KpnI, which cleaves EcoRI-5three times and EcoRI-8 twice. The location ofthese KpnI sites was determined by agarose gelelectrophoresis of restriction digests (Fig. 3) andis depicted in Fig. 1.

Restriction maps of the P1 DNA present in X-P1 :5R and X-P1 :SL phages are also shown in Fig.1. Some of the restriction enzyme analyses on

-.P1:8

kb

3.8--- *.4-

c-1

-P1.5 pALA8

4 . 6 7 8 9 1011

-5_5- g-h-i

__--e-f

- h

* 4'a I

FIG. 3. Agarose gel electrophoresis of restricted X-P1:8, X-P1:5, and pALA8 DNA. Lanes 1 through 3 areX-P1:8 DNA restricted with EcoRl, EcoRI plus KpnI,and EcoRI plus KpnI plus HindIII, respectively. Notethe appearance of KpnI fragment b and EcoRI-KpnIfragnent a in both lanes 2 and 3 and the conversion ofKpnI-EcoRI fragment c-d in lane 2 to two new frag-ments, c and d, in lane 3. Lanes 4 through 7 are X-P1:5DNA restricted with EcoRI, EcoRI plus BgIII, EcoRIplus BgllI plus KpnI, and EcoRI plus KpnI, respec-tively. Lanes 8 through 11 are pALA8 (pBR325-EcoRI-5) DNA cleaved with EcoRl, EcoRI plus BgIII,EcoRI plus BglII plus KpnI, and EcoRI plus KpnI,respectively. Note that EcoRI-BgllI fragment e-f(lanes 5 and 9) is not cleaved by KpnI (lanes 6 and 10),but that BgIII-EcoRI fragment g-h-i is cleaved by KpnIinto three fragments, g, h, and i. The size standards (inkb) shown at the left of the figure are from an EcoRIdigest of P1 DNA electrophoresed in a parallel laneand correspond to fragments 5, 9, 14, and 19 (5).

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CHARACTERIZATION OF P1 MINIREPLICONS 805

Kpnl

(a) EcoRI HindM (b)

KpnI-EcoRI

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 B 9 10

kb7.0 -

3.8-

1.9-

-a

- c-d

_,,h\b

-HdM-31.0-

FIG. 4. Agarose gel electrophoresis of restricted X-P1:5R DNAs. (a) Lanes 1 through 5 are X-P1:5, X-P1:5R-1,X-P1:5R-2, A-P1:5R-3, and P1 DNAs, respectively, cleaved with EcoRI. Lanes 6 through 10 are A-P1:5, X-P1:5R-1, A-P1:5R-2, A-Pl:5R-3, and Pt DNAs, respectively, cleaved with HindIII. (b) Lanes 1 through 5 are A-P1:5R-1,A-P1:5R-2, X-P1-5R-3, X-P1:8, and A-P1:5 DNAs, respectively, cleaved with KpnI. The arrows in lanes 1 through3 indicate the location of novel junction fragments j, k, and 1. (Fig. 1). Note that KpnI fragment b in lane 4migrates slightly faster than KpnI fragment h in lane 5 and that the DNAs in lanes 1 through 3 contain fragment h,but not fragment b. This has been confirmed by Southern hybridization of this gel (data not shown). Lanes 6through 10 are K-Pl:5R-1, K-Pl:5R-2, X-Pl:5R-3, A-P1:8, and A-P1:5 DNA, respectively, cleaved with KpnI plusEcoRI. Note the presence of KpnI-EcoRI fragment c-d in all these lanes except in lane 10.

which these maps are based are shown in Fig. 4and 5. The three A-P1:5R phages contain EcoRI-5 plus a 2- to 4-kb portion of EcoRI-8, includingits HindIII site and its EcoRI-5-proximal KpnIsite. None of the three phages contains theEcoRI-5-distal KpnI site offragment 8. The fourK-P1:5L phages contain EcoRI-5 and extensionsof variable length into and through Pt EcoRI-15(Fig. 1). The boundaries of these extensions forK-Pt :5L-1, A-P1 :5L-2, K-Pt :5L-3, and K-Pt :5L-4are, respectively, in Pt EcoRI fragments 15, 18,17, and 14. Since K-Pt:5L-3 does not contain acomplete Pt BglII-4 fragment (Fig. Sc), its K-PtDNA boundary must lie in the portion ofEcoRI-17 flanked by the BglII site and by EcoRI-23.The extent of the K DNA deleted in K-P1:5R

and K-P1:5L phages is also shown in Fig. 1. ForK-P1:5R phages, the junction between K PtDNAs on the K map created by these deletionscould not be precisely determined by restrictionanalysis because the endpoint of the Pl DNAwithin the KpnI fragment of EcoRI-8 is notprecisely known. However, based on the size ofnovel K-Pt KpnI junction fragments (Fig. 4b),we can conclude that no more than 3.6 kb of K

DNA to the right of the KpnI site at K mapcoordinate 39 is retained in any of the three K-

P1:5R phages. We were able to determine thelocation of the novel K-P1 function in K-P1:5L

phages more precisely because of the availabil-ity of appropriate restriction sites in that regionof the K map (Fig. 1). We know that the Pt DNAin A-P1:5L-1 and K-P1:5L-2 phages ends withinan EcoRI-BamHI fragment of X DNA locatedbetween A map coordinates 65 and 71 becauseboth phages retain the BamHI-BglII K fragmentat map coordinates 71 to 73 (Fig. 1 and 5c). Incontrast, the Pt DNA in X-P1:5L-4 and K-Pl:5L-3 ends within BamHI-BglHI and BglII-HindIIIfragments of K DNA at map coordinates 71 to 73and 73 to 76, respectively (Fig. 1 and 5b and c).Note that the retention of the N gene is notrequired for growth of K-P1:5L phages becausethey contain the ninS deletion (15) and thatall four K-P1:5L phages retain the entire K cIgene, whose C-terminal end is located within anHindIII fragment (31) retained by all four K-P1:5L phages (Fig. 5b).A-Pl:5R and A-P1:5L prophages are plasmids.

(i) A-P1:5R. All three of the K-Pt:5R phagesisolated lysogenize strain N205 about 10 timesmore efficiently than does the K-P1:5 phage(Table 2). Since the vector used in the construc-tion of these phages is defective for K integrativerecombination, the K-Pt:5R prophages are prob-ably stably maintained in these lysogens byvirtue of their ability to replicate as plasmids.The presence of a plasmid of the expected size

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806 STERNBERG AND AUSTIN

EcoRi34

2f 3 4 5

Bg34l2 3 4 s 6

1 2 3 4 5 It,

,.....Xw

-A 76-77

-A 71-73

7 8 9 A:C

FIG. 5. Agarose and acrylamide gel electrophoresis of restricted X-P1:5L DNAs. Panels a and c are 1%agarose gels, and panel b is a 5% acrylamide gel. (a) Lanes 1 through 6 are A-PI:5L-1, X-P1:5L-2, X-P1:5L-4, X-

P1:5L-3, XD-srIX3, and P1 DNAs, respectively, cleaved with EcoRI. The markers on the left indicate thepositions of P1 EcoRI fragments, and the markers on the right indicate the sizes (in kb) of selected fragments. (b)Lane 1 is pBR322 DNA digested with the enzyme Hinfl. Lanes 2 through 6 are A-Pl :5L-2, X-P1:5L-4, X-P1:5L-3,X-P1:5L-1, and AD-srIX3 DNAs, respectively, digested with HindIlI. The markers on the left are size standards(in kb), and the marker on the right indicates the location of the K HindIll fragment at coordinates 76 and 77. Thisfragment is clearly present in lanes 2 through 6. (c) Lanes 1 through 6 are Pl, K-P1:5L-2, K-Pl:5L-4, K-PI:5L-3, K-

P1:5L-1, and AD-srIK3 DNAs, respectively, cleaved with BgIII. The upper marker on the left indicates theposition of P1 BglII fragment 4 which is present in lanes 1 and 3. The lower marker on the left indicates theposition of K BgIII fragment at map coordinates 73 to 79 which is present in all lanes except lane 4. Lanes 7through 11 are K-P1:5L-2, K-Pl:5L-4, K-Pl:5L-3, K-P1:5L-1, and KD-srIK3 DNAs, respectively, cleaved withBamHI plus BgIII. The marker on the right indicates the location of BamHI-BgII fragment at map coordinates71 to 73 which is absent in lanes 8 and 9.

(a) (b)6

kb kb 0-7.0 1.6-

-3.80 52-..,

0.39--1.9

Ban)Hi- Bq/iP

S-

15-1 7

21-23-

(c)

4I

4-7

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CHARACTERIZATION OF P1 MINIREPLICONS 807

TABLE 2. Lysogenization frequency of X-P1 hybrid phagesLysogenization

Phage Bacterial strain (lysogens perinfected cell)

X-P1:5 N205 (recA sup') 2 X 10-7X-Pl:5R-1 N205 (recA sup') 1.6 x 10-1X-P1:5R-2 N205 (recA sup') 1.0 x 10-1X-P1:5R-3 N205 (recA sup') 1.2 x 10-1A Oam29-P1:5R-3 N205 (recA sup') 6 X 10-2X-P1:5L-1 N205 (recA sup') 2 X 10-7X-P1:5L-2 N205 (recA sup') 1 X 10-7X-P1:5L-3 N205 (recA sup') 2 X 10-5X-P1:5L-4 N205 (recA sup') 5 X 10-6A Pam8O-Pl:5L-3 N205 (recA sup') 1.2 x 10-5X-P1:5R-3 NS616 2 X 10-2X-P1:5R-3 NS616 groP 1.4 x 10-2X-P1:5L-1 NS616 2 X 10-7X-P1:5L-3 NS616 4 X 10-5X-P1:5L-3 NS616 groP 1.3 x 10-5X-P1:5R-3 N205(Ximm2l-P1:5)attB 2 x 10-6X-P1:5R-3 N205 loxB::PlCm 4 x 10-5bX-P1:5L-3 N205(Aimm2l-Pl:5) 3 X 10-5X-P1:5L-3 N205 loxB::PlCm 3 X 10-7

a The frequency of A immA lysogeny was measured on EMBO plates with WW selector phages as described inStemnberg and Hamilton (34).

b The apparent 20-fold difference in the frequency of X-P1:5R lysogeny when the strain contained a P1prophage at loxB as compared with when it contained a X-P1:5 prophage at attB is probably not meaningfulbecause about 0.1 to 0.5% of the cells in a population containing a prophage at loxB have lost the PlCm byexcision (35). Although these cells can still exert incompatibility against an entering X-Pl :5R prophage becausethey still contain an extrachromosomal Pl plasmid, the incompatibility-exerting element is now itself subject toincompatibility exerted by the entering X-P1:5R DNA. Loss of the P1 plasmid permits the stable establishment ofthe X-P1:5R plasmid. Consistent with this interpretation is the observation that all of the X-P1:5R lysogensisolated using strain N205 loxB::PlCm no longer contain the Pl prophage as judged by their loss of P1 immunityand sensitivity to chloramphenicol. This effect is not significant in the strain with A imm434-Pl:5 at attB becausethe spontaneous rate of excision from attB is very low.

of X in cells containing a X-P1:5R prophage, butnot in cells lacking that prophage, was demon-strated by agarose gel electrophoresis (Fig. 6).To show that this plasmid DNA was X-P1:5R, itwas purified by CsCl-ethidium bromide bandingand analyzed by restriction enzyme digestion.The restriction pattern of this DNA is identicalto that of X-P1:5R phage DNA, except for theexpected replacement of the two X cohesive-endfragments by a single larger plasmid fragment(Fig. 7). Based on the intensity in gels of X-P1 :5Rand larger Pl plasmid DNA bands, the twoplasmids appear to have approximately the samecopy number (Fig. 6a). This conclusion is con-firmed by DNA reassociation kinetics (Table 3).The copy number of both plasmids in cells with a40-min generation time is about one per hostchromosome, the same value as was determinedfor P1 by Prentki et al. (27).We studied one of the X-P1:5R phages, X-

P1:5R-3, in detail (2). The locations of severalplasmid maintenance functions were determinedby mapping deletion mutations by the DNAheteroduplex method (2). We note that, accord-

ing to the restriction-fragment mapping shownhere, the left X-P1 junction of X-P1 :5R-3 appearsto be further to the right than originally suggest-ed (2). The deletion analyses showed that, inaddition to containing information necessary forreplication, X-P1:5R DNA also contains essen-tial elements for plasmid equipartition. Thus,despite its low copy number, the X-P1:5R plas-mid is lost only about once in every 500 celldivision events in strain N205 (Table 4). Al-though the fidelity of this segregation event isquite high, it is still 2 orders of magnitude lowerthan the fidelity of Pl plasmid segregation,which is lost only once in every 105 cell divisionevents (4, 28). The possible reasons for thisdifference are discussed below.

(ii) A-P1:5L. X-P1:5L-3 and X-P1:5L-4 lysoge-nize strain N205 about 100 times more efficientlythan do X-P1:5, X-Pl:5L-1, and X-P1:5L-2 (Table2). Like the X-P1:5R prophage, the X-P1:5L-3and the X-P1:5L-4 prophages are plasmids (Fig.6). The rare X-Pl:5L-1 and X-P1:5L-2 lysogensdo not contain plasmid DNA (data not shown).The restriction enzyme pattern of X-P1 :5L-3

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(b).,:; -:A;@. .. it; * 3 ;1 3 4 6 7

p1__

5R.5L-

FIG. 6. Agarose gel electrophoresis of whole cell lysates. (a) Lanes 1 through 6 contain 10 il of cell lysate(equivalent to about 2 x 10' cells) and lanes 7 through 12 contain 20 ,ul of cell lysate. The lysates are from strainsN205(X-P1:5L-3) (lanes 1 and 7), N205(X-P1:5L-4) (lanes 2 and 8), N205(X-P1:5R-1) (lanes 3 and 9), N205(A-P1:5R-2) (lanes 4 and 10), N205(X-P1:5R-3) (lanes 5 and 11), N205(P1Cm) (Lanes 6 and 12), and N205 (lane 13).The material visible at the bottom of the gel consists of small fragments of the host chromosome (13). Thepositions of the 40- to 45-kb SR and 5L plasmids and the 95-kb P1 plasmid are marked at the left of the gel. Thetritium-labeled DNA was visualized by autoradiography. (b) A gel similar to that shown in (a) was transferred tonitroceliulose paper as described in the text, probed with 32P-labeled A DNA, and then exposed to X-ray film for45 min (upper panel), 450 min (middle panel), and 900 min (lower panel). The exposed films were scanned with aQuick Scan scanning densitometer at 525 nm, and the intensity of bands in bands 1 through 3 at the 45-minexposure was similar to the intensity of bands in lanes 4 through 6 at the 900-min exposure. The bands scannedshow a linear response with exposure time. Lanes 1 through 7 contain 10 Il of cell lysate from N205(X-P1:5L-3)isolate 1, N205(X-P1:5L-3) isolate 2, N205(X-P1:5L-4), N205(X-P1:5R-1), N205(X-P1:5R-2), N205(X-P1:5R-3), andN205, respectively.

plasmid DNA is identical to that of the phageDNA, except for the replacement of the twocohesive-end fragments of the phage DNA by asingle larger plasmid fragment (Fig. 7). In con-trast to X-P1 :5R and P1 plasmids, the copynumber of X-P1-5L plasmids, as judged by theintensity of stained plasmid DNA in gels, is high(Fig. 6). This copy number difference was quanti-tated both by Southern transfer hybridization(Fig. 6, Table 3) and by DNA reassociationkinetics (Table 3). The results of both proce-dures indicate that the copy number of the X-P1:5L plasmids is about 15 to 20 times higherthan that of the X-P1:5R plasmids. Despite thishigher copy number, X-P1:5L plasmids are main-tained somewhat less faithfully than are X-P1:5Rplasmids (Table 4).X-Pl:BglI4 cannot be maintained as a plasmid

prophage. P1 BgIIl fragment 4 (Fig. 1) was

cloned into the X D-srIX3 vector (see above), andthe resulting X-P1 phage was used to lysogenizestrain N205. The frequency of lysogeny is 4 x10-7 lysogens per infected cell, not significantlyhigher than it is for X-P1:5 (Table 1). The rare X-P1:BgII4 lysogens lack any plasmid DNA asjudged by gel electrophoresis. We conclude thatBglII-4 does not contain sufficient informationfor plasmid maintenance.A-PI:5R and A-P1:5L plas are sensitive to

Pl incompatiblIty. The A-P1:5R plasmid is in-compatible with Pl. This shows that X-P1:5Rand its P1 parent make use ofcommon pathwaysfor plasmid maintenance. We also demonstratedthat X-P1:5R is sensitive to the incompatibilitydetermined by the same fragment of P1 DNA(EcoRI-5) that exerts incompatibility against Pl.Incompatibility was determined by measuringthe ability of X-P1 :5R to lysogenize recA strains

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CHARACTERIZATION OF Pl MINIREPLICONS 809

5R

1 2

5L

3 4 5 6

_~~~~~~~~~9 99.9--

5.6 - _ 5.64.3- -4.3

2.5 --

FIG. 7. Agarose gel electrophoresis of restricted A-P1:5L and A-P1:5R plasmid DNA. Lane 1 contains k-

P1:5R-3 phage DNA, and lane 2 contains X-P1:5R-3plasmid DNA. Lanes 3 and 5 contain A-Pl:5L-3 phageDNA, and lanes 2 and 4 contain A-P1:5L-3 plasmidDNA. The DNA in lanes 1, 2, 5, and 6 was digestedwith HindIll plus BamHI, and the DNA in lanes 3 and4 was digested with EcoRI. The only difference be-tween the phage and the plasmid DNAs is the absencein the plasmid digests of the two phage cohesive-endfragments and their appearance as a new, larger fusionfragment. The appropriate fragments are designatedby arrows in each of the lanes. Size standards (in kb)are shown at the left and right.

with or without chromosomally integrated PI orEcoRI-5. The integrated element exerts incom-patibility against a X-P1:5R prophage as shownby the fact that it greatly reduced the ability ofA-P1:5R to form stable lysogens (Table 2). Re-ciprocal experiments lead to the same conclu-sions. In this case strain N205 (A-P1:5R) wasinfected with phage PlCm, and Cmr lysogenswere selected. The frequency of lysogeny wasabout 20%, and all of the 100 Pl lysogens testedhad lost the X-P1:5R prophage. In addition, theintroduction of the plasmid pALA8 (pBR325with EcoRI-5) into strain N205 (X-P1:5R) dra-matically destabilized the K-Pl:5R plasmid (Ta-ble 4).The incompatibility tests performed with K-

P1:5R were also performed with A-P1:5L-3 andA-P1:5L-4. However, in this case, only one ofthe two chromosomally integrated elements (Pt,but not EcoRI-5) was able to exert incompatibil-ity (Table 2). P1 also was able to efficientlydisplace a K-P1:5L plasmid prophage after infec-tion of strain N205 (K-Pl:5L). The failure of a

TABLE 3. Copy number of X-P1:5R and X-P1:5Lplasmids

Method of PlasmidPlasmid copy no. copy no. per

deterniination' hostchromosome

Pi RK 1X-P1:5R-3 RK 1X-P1:5R-1 SH 1.3X-P1:5R-2 SH 0.8X-P1:5R-3 SH 1X-P1:5L-3 RK 15X-P1:5L-3 SH 20X-P1:5L-4 SH 17

a RK, Reassociation kinetics; SH, Southern hybrid-ization. Values determined by the Southern hybridiza-tion method (Fig. 6) were standardized to the copynumber of X-P1:5R-3, which based on the reassocia-tion kinetics method is taken to be 1 per host chromo-some (see the text).

single copy of EcoRI-5 to exert incompatibilityagainst the high-copy-number K-Pt:SL plasmidwas overcome by increasing the gene dosage ofthe exerting element; a high-copy-number plas-mid containing EcoRI-5 (pALA8) did exert in-compatibility against X-P1:5L plasmids (Table4).Phage k replication functions are not necessary

for either X-P1:5R or X-P1:5L plasmid replica-tion. Although susceptibility to Pt incompatibil-ity indicates that at least some of the elementsresponsible for A-Pt:5R and A-P1:5L plasmidmaintenance are Pt encoded, others may be K

encoded. For example, the 5R or 5L plasmidmight be partitioned by Pt functions, but repli-

TABLE 4. Maintenance of X-P1:5R and X-P1:5Lplasmids in recA bacteria

% Plasmid lossaafter 12

Bacterialstraingenerations ofBacterial strain growth without

selectionpressure

N205(X-P1:5R-1).2.5 (5/200)N205(A-P1:5R-3).5.5 (11/200)N205(X-P1:5L-3).6.0 (12/200)N205(X-P1:5L-4).5.5 (11/200)N205(X-P1:5R-3) (pBR325).<3.0 (0/32)N205(X-P1:5R-3) (pALA8).>80.0 (27/32)N205(X-P1:5L-3) (pBR325).3.5 (1/28)N205(X-P1:5L-3) (pALA8).>80.0 (27/32)N205(X-P1:5L-4) (pBR325).7.0 (2/28)N205(X-P1:5L-4) (pALA8) >70.0 (23/32)

a The rate of X-P1 plasmid loss was measured asdescribed in the text. The numbers within parenthesesare the ratios of Imms colonies per total coloniespicked. All of the colonies tested were Ampr.

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cated by functions not fully repressed in theprophage state. To assess this possibility wemeasured the ability of either X-P1:5R or X-

P1 :5L to lysogenize E. coli under conditions thatblock replication. In one case, we introducedamber mutations into the essential replicationgenes, gene 0 in the case of X-P1:5R and gene Pin the case of X-P1 :5L-3, and in another case weused an E. coli strain containing a defective groPgene, the product of which is needed for X

replication (16). In neither case did we observeany significant effect of the mutations on lysoge-ny (Table 2). Three of the X-P1 :5R lysogens andthree of the X-Pl:5L lysogens were isolated ineach of the two experiments, and their DNAswere analyzed by gel electrophoresis for plas-mids. In all 12 cases, the plasmids were presentat the same copy number as the controls (datanot shown). We conclude that X replicationfunctions are not necessary for either X-P1 :5R orX-P1:5L plasmid replication. Additional supportfor this conclusion comes from the recent isola-tion of deletion mutations mapping in the P1portion of X-P1 :5R DNA that destroy the abilityof X-P1:5R to replicate (2) and from the con-struction of miniplasmids derived from X-P1:5Rthat contain no X sequences (A. Abeles, person-al communication).

DISCUSSIONWe have shown that in the prophage state,

both A-P1 :5R and X-P1 :5L exist as plasmids. Thefollowing observations show that the mecha-nisms exploited by both these plasmid types fortheir maintenance are derived entirely from thePl parent and suggest that the mechanismsinvolved have a functional role in the mainte-nance of the parent plasmid: (i) replication of X-

P1:5R and X-P1:5L as plasmids was not affectedeither by X or by host mutations that block X

replication; (ii) deletion mutations that map inthe Pl sequences of X-P1:5R (2) or X-P1:5L(unpublished results) blocked plasmid replica-tion; (iii) subcloning of X-P1:5R yielded unit-copy miniplasmids that contain no X DNA(A. Abeles, unpublished results); and (iv) bothclasses of new miniplasmids were subject to Pl-exerted incompatibility.The existence of both X-Pl:5R and X-P1:5L

replicons provides an explanation for some ofthe conflicting data regarding the previouslystudied Pt minireplicons, P1AN19 and pIH1972.The P1 DNA present in these two repliconswould suggest that P1AN19 is a 5R replicon andpIH1972 is a 5L replicon (Fig. 1). The X-P1:5phage, although it contains a region of Pt DNAthat overlaps both of these replicons, cannotreplicate as a plasmid because it is missing atleast one element needed by each. Recent dataindicate that an essential element of the 5R

replicon spans the junction between EcoRI frag-ments 5 and 8 (Fig. 1) (2). Both pIH1972 (29) andX-P1:5L are missing this region. Whether thesequences essential for 5R maintenance overlapthe sequences essential for pIH1972 or X-P1:5Lmaintenance remains to be determined. Howev-er, since X-Pl:BglII4 is not a SL replicon, atleast a portion of EcoRI-5 missing from thisphage (Fig. 1) is necessary for 5L replication ormaintenance (or for both).Although pIH1972 appears to be a 5L-type

plasmid, its copy number, like that of P1,PlAN19, and X-P1:5R, is low (1 to 2 copies percell chromosome; A. Shafferman, personal com-munication), whereas the copy number of the X-P1:5L plasmids is high (15 to 20 copies per hostchromosome). The simplest explanation for thisdifference is that the X-P1:SL-3 and X-P1:5L-4plasmids are missing P1 sequences that encode arepressor of 5L replication present in pIH1972.The absence of a replication repressor from theX-P1 :5L plasmids may explain their unusualincompatibility properties. The X-P1:5L plas-mid, unlike P1 or X-P1:5R, does not respond toincompatibility exerted by a single copy of PlEcoRI fragment 5, but is sensitive to incompati-bility exerted by a single copy of Pl DNA. Inthis respect the X-P1:5L replicon resembles aclass of P1 copy mutant plasmids that we havepreviously studied (36). The X-P1:5L plasmidsare susceptible to incompatibility exerted byEcoRI fragment 5 when present in the high-copy-number plasmid vector pBR325. We favorthe following explanation for these phenomena.The incompatibility exerted by a single copy ofEcoRI-5 can be overcome by an elevated plas-mid copy number because an approximate paritybetween the numbers of exerting and respondingelements is necessary for susceptibility. Presum-ably, Pl, but not EcoRI-5, encodes a repressorof 5L replication, thereby reducing the copynumber of the 5L replicon to a point where thefragment 5-encoded incompatibility determi-nants of the Pl can exert incompatibility againstthe remaining copies of the 5L replicon. We arepresently trying to localize the P1 gene(s) thatencodes the putative repressor of5L replication.

Besides the difference in plasmid copy num-ber, X-P1:5R and X-P1:5L phages differ in theirability to lysogenize bacterial strain N205. Thelysogenization frequency with X-P1 :5R plasmidsis approximately 0.1, and that with X-P1 :5Lplasmids is in the range of 10-5. The two mostlikely contributing factors to this difference areas follows: (i) X-P1:5L is deleted for the A clIIgene, whereas X-P1:5R is not, and X clIl-phages lysogenize E. coli strains 10 to 100 timesless efficiently than do A cIII+ phages (6); and(ii) the establishment of 5L lysogens requiresreplication to a high copy number. This elevated

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CHARACTERIZATION OF P1 MINIREPLICONS 811

replication potential might tend to direct the Kinfection into a lytic rather than a lysogenicresponse. Despite the low frequency of lysogensobtained after K-P1:5L infection, the resultingprophage is not a mutant capable of more effi-cient lysogeny as shown by the fact that phagereisolated from K-P1:5L lysogens are indistin-guishable from the original K-P1:5L phage. Toobtain these phages we found it necessary to invitro package (37) A-P1:5L plasmid DNA be-cause, for reasons that we do not yet fullyunderstand, it has not been possible to obtain Kphages by induction ofN205 (K-Pl :5L) lysogens.At least part of this induction defect must be dueto the red gam defect of 5L phages whichinhibits their vegetative growth in recA hosts(40).Another poorly understood property of the X-

P1 :5L plasmid prophage is its instability inrecA+ strains. Thus, although A-P1:5L lysoge-nizes recA+ strains as efficiently as it doesisogenic recA strains, the prophage is much lessstable in the former case, in which it is lost at arate that is about 5 to 10% per generation (datanot shown). Since this instability can be ob-served after the introduction of a recA+ alleleinto strain N205 (K-Pt:5L) by mating (data notshown), we believe it to be a maintenance ratherthan establishment problem.The existence of multiple replication elements

on plasmids is not unique to P1. Several plas-mids have secondary origins of replication thatare revealed after the primary origin is deleted(20, 23). In fact, the plasmids F and Rl have twononoverlapping segments of DNA capable ofplasmid replication (10, 22). In the case of Pl wesuggest that the main Pl replicon is the SRreplicon, and that the 5L replicon acts as abackup system. The 5R replicon has a copynumber and a plasmid-partitioning system thatare similar to those of Pl. Despite these similar-ities, the A-P1:5R plasmid is not nearly as stableas P1. A large part of this difference is due to theproblem of rec-promoted recombination (4), buteven in the recA host N205, K-P1:5R is lost at arate of 0.1 to 0.2% per cell generation, whereasPI is lost at a rate of 0.001 to 0.01% per cellgeneration. A possible explanation for this dif-ference is that Pt has a backup replicationsystem that is turned on in the rare instancewhen the SR replicon fails to fire, thereby al-ways providing enough plasmid copies fordaughter cells at division. The 5L replicationmode is an obvious candidate for such a system.Alternatively, the 5L replicon may not representa normal plasmid replication mode at all, butmay represent an unusual manifestation of alytic or plasmid establishment pathway. Furthergenetic and physical analysis of the 5L repliconshould help to distinguish between these possi-

bilities and should shed light on some of theunusual properties of the 5L plasmid state.

ACKNOWLEDGMENTSThis research was sponsored by Public Health Service

contract NO1-CO-23909 with Litton Bionetics, Inc., from theNational Cancer Institute.

LITERATURE CITED1. Appleyard, R. K. 1954. Segregation of new lysogenic

types during growth of a doubly lysogenic strain derivedfrom E. coli K12. Genetics 39:40-452.

2. Austin, S., F. Hart, A. Abeles, and N. Sternberg. 1982.Genetic and physical map of a P1 miniplasmid. J. Bacte-riol. 152:63-71.

3. Austin, S., N. Sternberg, and M. Yarmolinsky. 1978.Miniplasmids of bacteriophage P1. I. Stringent plasmidreplication does not require elements that regulate thelytic cycle. J. Mol. Biol. 120:297-309.

4. Austin, S., M. Ziese, and N. Sternberg. 1981. A novel rolefor site-specific recombination in maintenance of bacterialreplicons. Cell 25:729-736.

5. BAchi, B., and W. Arber. 1977. Physical mapping of BglII,BamHI, EcoRI, HindIII, and PstI restriction fragments ofbacteriophage P1 DNA. Mol. Gen. Genet. 153:311-324.

6. Belfort, M., and D. Wulf. 1971. A mutant of Escherichiacoli that is lysogenized with high frequency, p. 739-742. InA. D. Hershey (ed.), The bacteriophage lambda. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

7. Campbeil, A., and E. Balbinder. 1958. Properties of trans-ducing phage, p. 386-389. Carnegie Institute WashingtonYearbook.

8. Clewell, D. B., and D. R. Helinskl. 1969. Supercoiledcircular DNA-protein complex in Escherichia coli: purifi-cation and induced conversion to an operon circular DNAform. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166.

9. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubationin calcium chloride improves the competence of Esche-richia coli cells. Gene 6:23-28.

10. Danbara, H., J. K. Timmis, R. Lurz, and K. N. Timis.1981. Plasmid replication functions: two distinct segmentsof plasmid RI, repA and repD, express incompatibilityand are capable of autonomous replication. J. Bacteriol.144:1126-1138.

11. Daniels, D. L., J. R. deWet, and F. R. Blattner. 1980. Anew map of bacteriophage lambda DNA. J. Virol. 33:390-400.

12. Dennert, G., and V. Henning. 1968. Tyrosine-incorporat-ing amber suppressor in Escherichia coli. J. Mol. Biol.33:322-329.

13. Eckhardt, T. 1978. A rapid method for identification ofplasmid deoxyribonucleic acid in bacteria. Plasmid 1:584-588.

14. Enquist, L., and R. A. WeLsberg. 1976. The red plaquetest: a rapid method for the identification of excision-defective variants of bacteriophage lambda. Virology72:147-155.

15. Flandt, M., Z. Hradecna, H. A. Lozeron, and W. Szy-balskli. 1971. Electron micrographic mapping of deletions,insertions, inversions and homologies in the DNAs ofcoliphages Lambda and Phi8O, p. 329-354. In A. D.Hershey (ed.), The bacteriophage lambda. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

16. Georgopoulos, C. P., and I. Herskowltz. 1971. Escherichiacoli mutants blocked in lambda DNA synthesis, p. 553-564. In A. D. Hershey (ed.), The bacteriophage lambda.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

17. Gottesman, M. E., and R. A. Weisberg. 1971. Phageinsertion and excision, p. 113-138. In A. D. Hershey(ed.), The bacteriophage lambda. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

18. Gotesman, M. E., and M. B. Yarmolinsky. 1968. Integra-

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tion negative mutants of bacteriophage lambda. J. Mol.Biol. 31:487-505.

19. Ikeda, H., and J. I. Tomlzawa. 1968. Prophage P1, anextrachromosomal element. Cold Spring Harbor Symp.Quant. Biol. 33:791-798.

20. Kolter R. 1981. Replication properties of plasmid R6K.Plasmid 5:2-4.

21. Kondo, E., and S. Mtuhh. 1964. Drug resistance ofenteric bacteria. V. Active transducing bacteriophageP1CM produced by the combination of R Factor withbacteriophage P1. J. Bacteriol. 88:1266-1276.

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