JOURNAL OF VIROLOGY, Sept. 1977, P. 476-482 Vol. 23, No. 3Copyright © 1977 American Society for Microbiology Printed in U.S.A.

Maintenance of Bacteriophage P1 PlasmidALINE JAFF9-BRACHET' AND RICHARD D'ARI*

Unite de Ggnitique Microbienne, Centre National de la Recherche Scientifique, Institut de BiologieMoleculaire, UniversitW de Paris VII, 75221 Paris Cedex 05, France

Received for publication 30 November 1976

Three mutants of bacteriophage P1 affected in their ability to maintain thelysogenic state stably are described here. These mutants were normal in lyticgrowth, but lysogenic derivatives segregated nonlysogens at abnormally highrates (1 to 30% per division). Cells harboring these mutant prophages wereelongated or filamentous. The mutations responsible for this prophage instabil-ity fell into two classes on the bases of their genetic location, their effect on theability to lysogenize recA bacteria, and their suppressibility by ant mutationseliminating antirepressor activity. The two mutants that were able to formrecA lysogens showed the same prophage instability and partial inhibition of celldivision in recA as in rec+ lysogens. The fact that plasmid-linked mutations cancause prophage instability suggests that P1 codes for at least some of thefunctions determining its own autonomy and segregation.

P1 plasmid consists of covalently closed cir-cular duplex DNA molecules of molecularweight about 60 x 106 (10, 12, 36). It is normallypresent in lysogenic cells in a number of copiesper chromosome that is close to unity (12). Pro-phage loss is a rare event, occurring at frequen-cies of less than 10-4 per division (26). The factthat this element is not frequently lost in grow-ing populations implies the existence of a strictcontrol mechanism governing its replicationand segregation to daughter cells at cell divi-sion.Two classes of models have been proposed for

the control of plasmid replication. In 1963, Ja-cob et al. postulated that the regulation ofrepli-cation is under positive control, dependent on areplicon-specific initiator active only at somespecific time in the cell cycle (13). To explainthe accurate segregation into daughter cells atcell division, they suggested that replicons maybe attached to a site on the cell membrane thatduplicates in such a way that at least one copy,with replicon attached, is passed to each daugh-ter cell. In 1969, Pritchard proposed a negativecontrol of replication under which a replicon-specific inhibitor of replication, synthesized atthe moment of initiation, prevents initiation ofa further cycle of replication until the inhibitorconcentration falls below a critical value due tocell volume increase during growth (24).We have chosen to study the coordination of

replication and segregation of the plasmidphage P1 through the isolation and characteri-

1 Present address: Bact6riologie M6dicale, Institut Pasteur,75015 Paris, France.

zation of mutants that cannot be maintainedstably as prophage. A preliminary characteri-zation of three such mutants is described here.These plasmid stability mutations fall into twoclasses on the bases of their genetic location,their effect on the capacity to lysogenize recAbacteria, and their suppressibility by ant muta-tions; the P1 ant gene, as shown by Chesneyand Scott (3) and in the accompanying article(5), appears to code for an antirepressor.Lysogens carrying these mutant prophage

are unstable and give rise to nonlysogens at anabnormally high frequency. Cultures of theselysogens contain elongated cells, and in somecases considerable filamentation is observed.This partial interference with normal cell divi-sion has been reported for cells harboring main-tenance mutants of other plasmids-ColEl (14),ColVB (17), R1 (9)-and suggests that certainplasmid-linked mutations may also affect somebacterial process related to cell division. Thefact that mutations in P1 can cause lysogeninstability suggests that part of the controlmechanism(s) governing the maintenance andsegregation of this extrachromosomal repliconis coded for by the plasmid itself.

MATERIALS AND METHODSBacterial strains. Table 1 lists the nonlysogenic

Escherichia coli K-12 derivatives used. Sh16 (19) is anonsuppressing streptomycin-resistant strain ofShigella dysenteriae furnished by J. L. Rosner.

Bacteriophage strains. All P1 phages are deriva-tives of Plkc (18). The different mutations used arelisted in Table 2, except for seg-1 and seg-5, whichare described in the text. Recombinants were ob-

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MAINTENANCE OF PHAGE P1 PLASMID 477

TABLz 1. E. coli K-12 strains used

Strain Relevant char- Source or referenceacteristicsC600 Str' supE44 1Q1008 Str' sup+ 6Q1504 dnaB27 6Q1i08 dnaB70 6N99 Strr sup+ gal W3102, parent of N100 (35)N100 Str' sup+ gal Strain 152 (35)

recAQ205 mutD5 galU Pir 7Q206 mutD5gal+ P1' Spontaneousgal+ mutant of

Q205GY2168 lacYl mal/F'2- mal derivative of 200PS/

lac+ F'2-lac (8) from J. George

tained from the progeny of appropriate crosses. P7(34) is the 4)amp+ of H. W. Smith (31).Media and diluents; growth and assay of phages.

For a description of these materials and procedures,see D'Ari et al. (6).Phage mutagenesis. Mutagenized stocks ofPlCm

were prepared by confluent lysis of the P1-sensitivemutD mutator strain Q206. Cultures of Q206 weregrown overnight in glucose-minimal salts mediumat 370C and then diluted 100-fold into LB brothsupplemented with 50 ,ug of thymidine per ml andaerated for 3 h at 370C before use. LMC plates wereseeded with 5 x 108 bacteria and 108 PlCm,incubated at 370C for 7 h and then scraped; thelysates, after chloroform treatment, were centri-fuged. The number of clear plaques on Shl6 wastaken as a measure of the extent of mutagenesis;mutagenized stocks typically contained about 3%clear mutants, compared with less than 0.1% inunmutagenized stocks.Phage crosses. For a description of phage crosses,

see D'Ari (5).Scoring of markers. (i) The seg markers. The seg

markers in a cl+ genetic background were scored bymeasuring the stability ofthe resulting lysogens, asdescribed below. In a c1.100 background, the follow-ing rapid test for seg was devised. C600 cells were

lysogenized at 300C with the progeny from a cross;after 2 nights of incubation, isolated colonies werepicked with sterile toothpicks and inoculated into0.5 ml ofLB broth containing citrate in nylon micro-culture containers, which were incubated overnightat 300C to obtain saturated cultures. The followingday the cultures were diluted about 100-fold withstainless-steel prongs into 0.7 ml of LB broth con-taining citrate, incubated for 90 min at 420C to in-duce the lysogens, and then replicated onto LB-citrate plates, which were incubated overnight at420C. A confluent spot of bacterial growth indicatedPlseg lysogens, which segregate many temperature-resistant nonlysogens; no growth indicated Plseg+lysogens.

(ii) Amber markers. Lysates of the phage to betested, prepared as for the seg test, were replicatedonto LMC plates containing streptomycin andseeded with the sup+ Sti' strain Q1008 and 3 x 107amber tester phage; absence of lysis indicated thepresence of the tester amber allele.

Lysogen stability. Lysogens of PlCm and deriva-tives, identified as chloramphenicol-resistant colo-nies, were restreaked twice on selective media be-fore use. Measurements of lysogen stability weremade on exponential cultures at 300C in LB brothcontaining chloramphenicol (25 pug/ml) and citrate(5 x 10-3 M); the former prevents growth of nonlyso-genic segregants without affecting their viability,and the latter, by blocking Ca2+-dependent P1 ad-sorption (2), prevents relysogenization ofsegregantsby free phage. Cultures were plated on citrate platesand incubated for 2 nights at 300C; about 200 isolatedcolonies were picked and replicated onto plates withand without chloramphenicol and incubated over-night at 3000. For thermoinducible lysogens, theratio of colonies on citrate plates at 420C to coloniesat 300C is also used as a measure oflysogen stability.Immunity tests. Immunity was tested by spotting

Plvire onto an LMC plate seeded with the strain tobe tested. For cultures containing nonlysogenic seg-regants, chloramphenicol was added to the plates.Phage concentrations ranging from 104 to 108 per mlwere used; at the higher concentrations, nonim-mune strains show clear circles of lysis, even recAstrains, on which P1 does not make visible plaques.

RESULTSStability of the Cm insertion. To isolate mu-

tants of P1 affected in their maintenance,PlCm was used; its cat gene (chloramphenicolacetyl transferase), derived from an R factor(15), confers chloramphenicol resistance to lyso-gens and thus serves as a convenient prophagemarker. To check the stability of the Cm inser-tion in lysogens, a culture ofQ1008 (PlCm) wasgrown and plated at 300C without drug selec-tion, and colonies were picked and replicated toplates with or without chloramphenicol. Of1,500 clones tested, only one was chlorampheni-col sensitive; analysis showed it to be a defec-tive lysogen, immune to superinfecting P1 butdeleted for several prophage genes. Thus, theloss of Cm from a lysogen is seen to be a rareevent. This is in striking contrast to the insta-

TABLz 2. P1 genetic markers

Name Description Referencecat(Cml) Chloramphenicol acety- 15

lease insertionci. 100 Temperature-sensitive 26

repressorc5.482 Double mutant (see text) 28virs Virulent 27dan-1 Suppressor of c.i100 5sud-2 Suppressor of dan-i 5ant-i Antirepressor negative 5bac-i ban-i ban negative 6am3.6 27am8.13 L 27am34.62 a Lethal embers, sup- 33am56.32 ) premed bysupE 29, 33amM 33

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478 JAFF9-BRACHET AND D'ARI

bility of the Cm insertion during lytic growth(15); our PlCm stocks generally contain 10 to20% phage that have lost Cm.

Isolation of P1 seg-1. We tried to isolate athermosensitive segregation mutant from astock of PlCm mutagenized by growth in amutD strain (7) as described in Materials andMethods. C600 was lysogenized at 300C with themutagenized stock, and isolated colonies werepicked and replicated to plates with and with-out chloramphenicol and incubated at 30 and420C. Of 2,000 clones tested, none showed com-plete growth inhibition at 420C in the presenceof the drug, but one grew better in the absenceof chloramphenicol than in its presence at bothtemperatures. Phage isolated from this clonewere purified and reintroduced into C600; theresulting lysogens segregate nonlysogens at anabnormally high frequency at 300C. The mu-tant phage was designated PlCm seg-1; itmakes turbid plaques on Shi6 at 420C. To facili-tate the scoring of the seg marker, we intro-duced the cl.100 mutation into PlCm seg-1 bycrossing it against PlCm am3.6 cl.100; theprobability per division of prophage loss wasmeasured as described in Materials and Meth-ods.

Isolation of Plseg-5. Another P1 mutant,Plc5.482, isolated and described by Scott (28),contains a mutation similar to seg-1. Plc5.482makes clear plaques on Shi6 at 420C and turbidplaques at low temperature. It lysogenizes E.coli at 300C; the lysogens are not induced at420C, but at both 30 and 420C lysogenic culturescontain nonlysogenic segregants. The clear mu-tation was found to lie between am3.6 andam7.14 (see Fig. 1). As is shown below, thisphage carries at least two mutations; we pro-pose the name seg-5 for the mutation causinglysogen instability and the name c5 for theother mutation, which presumably confers atemperature-sensitive clear phenotype on Shi6.The Cm marker was introduced into the orig-

inal Pic5 seg-5 phage to estimate the frequencyof segregation of nonlysogens in the presence ofchloramphenicol, thus avoiding a selective ac-cumulation of the faster-growing nonlysogeniccells. Since amM lies near c5 on the geneticmap, P1c5 seg-5 was crossed against PlCmamM (multiplicities of 0.5 and 5, respectively);a Cm am+ recombinant producing clear plaqueson Shi6 at 42°C was isolated, and an E. colilysogenic derivative of this recombinant wasshown to segregate nonlysogens. This phage isthus of genotype Cm c5 seg-5.To have a rapid test for the seg character, the

temperature-sensitive mutation cl.100 was in-troduced into PlCm c5 seg-5 by crossing the

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latter phage against PlCm am56.32 cl.100; anam+ phage that segregates nonlysogens at highfrequency was recovered. This recombinant isshown below to have the genotype Cm c5 seg-5cl.100 (direct detection of the c5 marker is ob-scured by the cl.100 mutation). Lysogens forPlCm c5 seg-5 cl.100 are thermoinducible;when plated at 42°C on citrate plates, survivalis about 10-2 and the survivors are mainly non-lysogens; PlCm cl.100 lysogens give rise toabout 10-5 survival. This permits simple detec-tion ofthe seg marker (see Materials and Meth-ods). Both lysogens give survival frequencies ofabout 10-5 when plated on chloramphenicol-citrate plates at 420C.The clear mutation of Pic5 seg-5 lies in the

same region as the ban-i mutation, which islocated between am3.6 and am56.32 (6). The P1ban protein, analogous to the bacterial dnaB, isable to replicate bacterial and phage DNA (23,6); ban mutants, unlike wild-type P1, dependon functional dnaB product to replicate theirDNA and cannot grow lyrically in dnaB (ts)hosts at nonpermissive temperatures. To seewhether ban function is normal in P1c5 seg-5,lytic growth was measured in dnaB70 (Q1508)and dnaB27 (Q1504) hosts. Scott's original P1c5seg-5 and the recombinants PlCm c5 seg-5 andPlCm c5 seg-5 cl.100 were all ban (burst sizes<1 at 420C and 80 to 160 at 300C); in mixedinfection with PlCm bac-1 ban-i, no comple-mentation was observed. It should be noted,however, that PlCm bac-1 ban-i makes turbidplaques on Shl6 at 420C and is perfectly stableas a prophage (6; unpublished observations).To determine whether the Ban and Seg phe-

notypes were due to the same mutation, PlCmc5 seg-5 cl.100 was crossed against PlCmam34.62 am3.6 am8.13 cl.100. Several seg-5 re-combinants carrying different amber mutationswere purified and tested for their ban charac-ter; three were ban+, thus dissociating ban andseg. One of these ban+ phage, PlCm am8.13seg-5 ci.100, was taken as our reference seg-5stock; all phage strains carrying the 5+ seg-5alleles derive ultimately from this phage. In across against Plvirs am56.32, two PlCm seg-5(am+ cl+ vir+) recombinants were isolated. Ly-sogens for these phage segregated nonlysogensat high frequency, but the phage themselvesmade turbid plaques on Shi6 at 420C, thus dis-sociating the clear and Seg phenotypes. Theclear and Ban phenotypes have not been sepa-rated; for simplicity, we assume that they aredue to a single mutation c5, but phage of geno-type c5 seg+ have not been isolated.

Isolation of P1Cm sud-2 cl.100 and PlCmsud-2 c1.100 dan-i. The dan-i mutation, de-

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MAINTENANCE OF PHAGE P1 PLASMID 479

scribed in the accompanying article (5), wasisolated as a suppressor of cl.100; P1 cl.100dan-i produces an altered cl product capable ofmaintaining lysogeny at 420C. This phage isunable to make plaques at 320C; sud-2 (5) wasisolated as a suppressor of dan-i, permittinglytic growth at 320C. The sud-2 mutation ap-

pears to cause a low constitutive expression ofthe ant (antirepressor) gene. Lysogens forPlCm sud-2 cl.100 dan-i and for PlCm sud-2cl.100 (constructed by recombination) growslowly, form filaments, and segregate nonlyso-gens at high frequencies; cultures of these lyso-gens risk accumulating faster-growing mu-tants.

Effects of the seg-i seg-5 and sud-2 muta-tions. PlCm seg-1 cl.100, PlCm seg-5 cl.100,PlCm sud-2 cl.100, and PlCm sud-2 cl.100dan-1 all show normal lytic growth; they formnormal size plaques from 30 to 420C.

Lysogenization frequencies are also normalin rec+ strains (about 20%). Lysogens harboringa sud-2 prophage grow more slowly than seg-1or seg-5 lysogens. Exponential cultures of theselysogens were observed under the microscope.Control PlCm cl. 100 lysogens show normal cellsize; the cells of seg-i and seg-5 lysogens areelongated, and those of the two sud-2 lysogensinclude many long filaments.

In the recA strain N100, PlCm seg-1 cl.100and PlCm seg-5 cl.100 lysogenize as efficientlyas PlCm cl.100; cells of the resulting seg lyso-gens are elongated. PlCm sud-2 cl.100 andPlCm sud-2 cl.100 dan-i, on the other hand,

are unable to form lysogens in NIOO or in recBbacteria (data not shown).

All of these lysogens, rec+ and recA (wherepossible), segregate nonlysogens at abnormallyhigh frequencies. These segregants generallygrow faster than the lysogens. To measure theprobability per division of prophage loss, thecultures were grown in the presence of chlor-amphenicol and citrate. The bacteriostatic anti-biotic prevents growth of chloramphenicol-sen-sitive (nonlysogenic) segregants without affect-ing their viability, and the citrate, by blockingP1 adsorption, prevents relysogenization ofsegregants by free phage. Under these condi-tions, the frequency of segregants in a cultureis a direct measure of the probability per divi-sion of forming a nonlysogen: each faulty divi-sion produces a new segregant; each correctdivision produces a new lysogen. Table 3 givesthese frequencies in rec+ (N99) and, for seg-1and seg-5, recA (N100) lysogens at 300C. Thesegregants in the mutant cultures were de-tected by their chloramphenicol sensitivity;several from each culture were purified andshown to be nonimmune and temperature re-sistant, and their genotype was checked (galsup+ Strr prototroph, and UVs for N100 segre-gants). The segregants from the seg-1 and seg-5lysogens were relysogenized with PlCm cl.100and shown not to affect the rate of prophageloss. By all these criteria, the chloramphenicol-sensitive segregants were indistinguishablefrom the original nonlysogens N99 or N100.

Genetic mapping of seg-1, seg-5, and sud-2.

TABLE 3. Segregation frequencies ofP1 lysogens'rec+ (N99) recA (N100)

ProphageSurvival at 420C" Cm' at 30°Cc Survival at 42oCb CmW at 300Cc

PlCm cl.100 2 x 10-5 0/203PlCmam8.13cl.100 8 x 10-5 8 x 10-5PlCm am3.6 cl.100 2 x 10-5 3 x 10-4PlCm cl.100 dan-i 0/204PlCm am8.13 seg-i cl.100 2 x 10-2 24/204 2 x 10-2 6/191PlCm am3.6 seg-5 ci.100 2 x 10-3 2/178 2 x 10-2 3/204PlCm sud-2 cl.100 2/201 _ d _ dPlCm sud-2 cl.100 dan-i 4/200 _ d _ dPlCm sud-2 ant-i cl.i00 dan-1 1 x 106 0/204PlCm sud-2 ant-i am8.13 1 X 10-2

seg-i cl.100PlCm sud-2 ant-i am8.13 1 x 10-3

seg-5 ci.100a Exponential cultures grown at 300C in the presence ofchloramphenicol and citrate were plated at 30 and

420C on citrate plates.b Ratio of colonies at 4200 to colonies at 300C.e About 200 isolated colonies from the 30TC plates were picked and replicated onto plates with and without

chloramphenicol and incubated at 3000 overnight.d Phage unable to lysogenize recA.

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To locate the seg-i and seg-5 mutations on theP1 genetic map, genetic crosses were performedas described in Materials and Methods; thecl.100 mutation was present on all phage, per-mitting easy scoring of the seg allele. The de-tection ofthe Seg phenotype in cultures ofther-moinducible lysogens is based on their highsurvival frequencies at 420C in the absence ofchloramphenicol due to the presence of temper-ature-resistant nonlysogenic segregants. Table4 shows typical data of a three-factor cross in-volving seg-i; unselected progeny phage arescored for all markers. Assuming the minoritygenotype to be that of the double recombinantclass, this cross places seg-i between am3.6 andam56.32 (Fig. 1). Similar conclusions werereached from crosses involving am3.6 andam8.13, with the seg-1 mutation associatedwith either amber mutation. Although seg-1has not been shown to be a single mutation, noanomalous mapping results were obtained.To locate the seg-5 mutation with respect to

seg-1, PlCm am3.6 seg-1 cl.100 was crossedagainst PlCm am8.13 seg-5 c 1.100, and the fre-quency of seg+ recombinants was measured(seg-1 and seg-5 being indistinguishable fromone another). The unselected progeny con-tained 10% am+ recombinants but only 3 of 400seg+ recombinants (genotypes am8.13, am3.6,and am+), showing that seg-i and seg-5 areclosely linked but not identical. No seg+ revert-ants were found among the more than 400 lyso-gens of each parent phage.The phenotype of the seg-1 seg-5 double mu-

tant is unknown; if the two mutations on lyso-gen stability affect independent maintenancemechanisms, the double mutant might be to-

TABLE 4. Mapping of seg-1Cross: PlCm am 56.32 c 1.100 x P1Cm am 3.6 seg-1 c 1.100

Genotypeam3.6 seg-1 am56.32 No.+ + - 229- - + 192_ + - 5+ _ + 16+ + + 17_ _ - 6

+ - - 2_ + + 1

tally incapable of maintaining itself as pro-phage. From the above cross, unselectedplaques were picked and tested for their abilityto lysogenize; the presence ofCm was tested byability to transduce the lysogen Q1008(P7) tochloramphenicol resistance (even non-lysogen-izing phage show a positive response). Of 400Cm phage tested, all were able to lysogenize.Lysogens for PlCm sud-2 cl.100 and PlCm

sud-2 cl.100 dan-i show variable survivalfrequencies at 420C, making it impossible touse the rapid test developed for seg-i and seg-5. Advantage was taken of the ability of sud-2to suppress dan-i. Crosses in which both par-ents carry the cl.100 dan-i mutations are de-scribed in the accompanying article (5); theyshow that sud-2 is tightly linked or allelic tovirs (<4 x 10-4 recombination).

Effect of an ant mutation on segregation.As shown in the accompanying article, the sud-2 mutation creates a low constitutive expres-sion of the ant protein, which in turn inacti-vates the cl repressor. The properties of thesud-2 mutation depend on this expression ofant; sud-2 ant-i phage are distinguished fromsud+ ant+ phage solely by their inability toplate on P7 lysogens (5). In particular, the seg-regation of nonlysogens observed in cultures ofPlCm sud-2 cl.100 dan-i lysogens is sup-pressed by the ant-i mutation (Table 3). To seewhether seg-i- and seg-5-induced segregationdepends on the ant protein, PlCm sud-2 ant-icl.100 was crossed against PlCm am8.13 seg-icl.100 and PlCm am8.13 seg-5 cl.100; in bothcases, sud-2 ant-i seg recombinants were re-covered. They are unable to grow on P7 lyso-gens, and N99 lysogenic derivatives of theserecombinants show the same segregation fre-quencies as the sud+ ant+ seg lysogens (Table3). Thus, the segregation observed in seg-i andseg-5 lysogens differs from that caused by sud-2in that it does not depend on the presence oftheant protein.

Segregation in an F'-lac strain. Kingsburyand Helinski (14), in their study of tempera-ture-sensitive replication mutants of ColEl,found that for 5 out of 35 such mutants F'-laccould complement the ColEl mutation and per-mit replication at the nonpermissive tempera-ture. We performed similar tests in the F'-lacstrain GY2168 lysogenized with phage bearing

seg-1seg-5

sud-2 c5Iyg amM am7.14 dan-1

am34.62 cat am3.6 vir bac-1 ban-1 am56.32 am8.13 c1.100FIG. 1. Genetic map ofP1 (not drawn to scale). Mapping data are taken from references 5, 6, 28, 30, 33,

and this work; am3.6, formerly reported to lie to the right of virs, has recently been mapped between cat andvirs (D. H. Walker, Jr., and J. T. Walker, personal communication).

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MAINTENANCE OF PHAGE P1 PLASMID 481

a seg-1, seg-5, or sud-2 mutation. These F'-laclysogens continue to segregate nonlysogens athigh frequencies. In addition, the seg-1 and seg-5 prophages do not affect the frequency of segre-gation of lac- cells.

DISCUSSIONThe temperate phage P1, because of its ca-

pacity to grow lyrically, offers a considerableadvantage over other plasmids for genetic andphysiological studies of plasmid maintenance.The existence of P1 mutants affecting prophagesegregation suggests that at least part of themechanisms) controlling plasmid maintenanceis coded for by P1. Similar observations havebeen made in other systems. Temperature-sen-sitive maintenance mutations, both chromo-somal and plasmid linked, have been reportedfor F (13), ColEl (14), ColVB (16), and for theStaphylococcus aureus penicillinase plasmid(22); plasmid-linked mutations ofR1 causing anaccumulation of cured cells have also been re-ported (32).The P1 mutations seg-1, seg-5 (recovered

from Scott's P1c5.482 [28]) and sud-2, as de-scribed in this work, all affect plasmid mainte-nance. In addition, Scott described a turbidplaque-forming phage, Pllyg, that is unable toform stable lysogens. The lyg mutation wasmapped near virs (30); its relationship to sud-2is unknown. Certain defective Pldlac deletion-substitution mutants are also unstable as pro-phages in both rec+ (19, 25) and recA (11)strains. Walker and Walker have measured theextent of several of these deletions by markerrescue tests (33); in at least two cases the sud-2,seg-1, and seg-5 regions were apparently intact,and the prophages were reported to be main-tained as plasmids (M. E. Rae, Ph.D. thesis;quoted in Walker and Walker [33]).Experiments are currently in progress, in

collaboration with M. van Montagu, to deter-mine the physical state ofthe prophage DNA inlysogens carrying a seg-1, seg-5, or sud-2 pro-phage. Preliminary results indicate that PlCmseg-1 and PlCm c5 seg-5 prophages, like PlCm,are in the form of covalently closed circularDNA molecules.

Wild-type P1 prophage can be destabilizedafter superinfection by wild-type P1. This su-perinfection curing was reported for rec' lyso-gens by Mise and Arber (20) and has beenobserved in recA lysogens by Meurs and D'Ari(manuscript in preparation). It is possible thatthe diverse mutations or treatments that re-sult in prophage loss do so in different ways,with varying effects, direct or indirect, on thepostulated maintenance control mechanismss.

It is noteworthy that plasmid-linked muta-

tions resulting in plasmid destabilization oftenhave an effect on host cell division, causingfilamentation. This correlation has been ob-served in mutants of ColEl (14), ColVB (17), R1(9), and P1 (this work). Such perturbationscaused by R1 mutants have been attributed byNordstrom et al. (21) to an unbalance in macro-molecular biosynthesis caused by competitionbetween the R factor and the chromosome foran essential replication product. Koyama andYura, on the other hand, have suggested (17)that plasmids, although unnecessary for bacte-rial growth, may, when present, constitute partof the host regulatory system governing celldivision.At present, it is difficult to determine

whether the primary effect of a given mutationis on replication of the plasmid or on its segre-gation into daughter cells. Uhlin and Nord-strom have approached this problem throughthe isolation and characterization of R1 mu-tants having an increased number of plasmidDNA molecules per bacterial chromosome(copy mutants) (32). These mutants presum-ably have an abnormality in their replicationcontrol; those studied also show altered incom-patibility properties (32). Filamentation of bac-teria carrying R1 copy mutants was also ob-served (9) and, in some cases, segregation ofcured cells at an abnormally high frequency(32). There thus seems to exist a relationshipbetween the copy number, the expression ofincompatibility, cell division, and plasmid seg-regation. This pleiotropy is not always ob-served. For example, chromosomal mutationsin E. coli (pcn) isolated and characterized byCress and Kline caused a threefold increase ofP1 copy number but no detectable prophagedestabilization (4). Nevertheless, these correla-tions encourage one to consider models inwhich control of cell division and plasmid repli-cation, incompatibility, and segregation are allaffected by some common element.

ACKNOWLEDGMENTSWe are grateful to Michael Yarmolinsky for suggesting

P1 as a model system for studying the maintenance andsegregation problem. We thank Martine Chemana andFrangoise Gire for secretarial assistance and Solange Dorsi-mont, Yvette Mallet, Ren6e Marquise, and Frangois Coues-surel for services so cheerfully rendered.

During the course of this work, R.D. benefited from afellowship from the European Molecular Biology Organiza-tion. The research was funded by the Delegation Generale ala Recherche Scientifique et Technique.

LITERATURE CITED1. Bachmann, B. J. 1972. Pedigrees of some mutant

strains of Escherichia coli K-12. Bacteriol. Rev.36:525-557.

2. Bertani, G. 1951. Studies on lysogenesis. I. The mode ofphage liberation by lysogenic Escherichia coli. J. Bac-teriol. 62:293-300.

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3. Chesney, R. H., and J. R. Scott. 1976. Superinfectionimmunity and prophage repression in phage P1. II.Mapping of the immunity-difference and ampicillin-resistance loci of P1 and Oamp. Virology 67:375-384.

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