Proc. Nati Acad. Sci. USAVol. 79, pp. 515-519, January 1982Genetics

Propagation of satellite phage P4 as a plasmid(cloning vector/colicin/"phasmid"/regulatory element/transcriptional antitermination factor)

RICHARD GOLDSTEIN*, JOHN SEDIVY*, AND ELIZABETH LJUNGQUISTt*Department of Microbiology and Molecular Genetics, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115; and tDepartment of Microbiology,University of Uppsala, Biomedical Center, S-75123, Uppsala, Sweden

Communicated by Bernard D. Davis, August 5, 1981

ABSTRACT Satellite phage P4 has two known options forpropagation. In its lytic cycle, its regulatory functions can act intrans to alter the actions ofa helper virus (P2), which then providesnecessary gene products, including capsid proteins. P4 also canbe propagated in the absence of a helper as a prophage, with dis-tinct sites for integration within the Escherichia coli chromosome.We determined that a single spontaneous mutation (virl) of phageP4 allows a third mode of propagation: as a plasmid (along withcontinued integration into the host chromosome). Hence, the P4regulatory element is capable of (i) temperate; (ii) lytic, helper-dependent; and (iii) plasmid modes of development. These find-ings emphasize the close relationship between defective virusesand plasmids.

Wild-type satellite phage P4 was isolated from a strain of Esch-erichia coli lysogenic for P2-related temperate bacteriophage(1). Its genome is about one-third the size of the P2 genome(i.e., =11,000 base pairs), and the two are not significantly ho-mologous other than having identical 19-base single-strandedcohesive ends (2). P4 was considered to be a defective virusbecause its lytic development was found to require a helpervirus such as P2 (1, 3), which provides at least 18 essential mor-phogenic and cellular lysis functions (4, 5).

Phage P4 acts as a "regulatory element" that can act in transon either an integrated or a coinfecting helper, altering itsexpression in a way that supports the lytic cycle of P4. At leastthree trans regulatory functions can be recognized. First,though heteroimmune, phage P4 can derepress a P2 prophageand, thus, obtain its required morphogenic gene products (6,7). This pathway seems to involve destruction of specific im-munity and concomitant expression of the early genes ofphageP2. Second, though a P2 helper with a lesion in early gene Aor B cannot enter into the assembly steps in lytic development(8), it can do so in the presence of P4. Hence, the P4 regulatoryelement transactivates morphogenic operons of the helperthrough a novel control mechanism (9). Third, the helper nor-mally assembles a capsid large enough to package its own ge-nome, and P4 redirects this process to produce capsids one-third as large, which can completely package only the smallergenome of P4 (10, 11). Geisselsoder et al (12) suggest that themechanism of capsid size determination involves use of a P4-coded antitermination factor to modulate termination of tran-scription of the morphogenic operons of the helper. P4 alsocodes for at least three other functions acting only on itself: ly-sogenization (1, 13), prophage maintenance by a repressor (1,13), and DNA replication (7, 14, 15). None of these three func-tions plays a known role in the satellite-helper relationship.

This paper describes a mutant that reveals the capacity ofphage P4 to be maintained not only as a lytic virus or a prophage,but also as a plasmid. The mutant, P4 virl, isolated as a spon-

taneous mutant of wild-type P4, was able to plate on strains ly-sogenic for both P4 wt and P2 wt (1). Evidently, the virl mu-tation reduces responsiveness of the operator binding site(s) tothe P4 repressor (13). Therefore, P4 virl, unlike P4 wt, shouldnot be able to repress its own replicase; and because it also couldnot cause cell lysis in the absence of a helper, we reasoned thatit should be able to maintain itself as a plasmid.

Several lines of indirect evidence supported this hypothesis.Lindqvist and Six (16) had mentioned that cells infected withphage P4 virl, unlike those infected with P4 wt, can be inducedwith high efficiency by superinfection with helper to produceviable P4 progeny. Similarly, we found that after infection withP4 virl sidl carrying tetracycline- and kanamycin-resistancegenes in transposons TnlO or Tn5, cells maintain drug resistancefor many generations in the absence of any selection (ref. 10;unpublished results). Finally, NaDodSO4polyacrylamide gelelectrophoretic analysis showed that cells infected with P4 virlwere found to indefinitely produce in a coordinated manner allknown phage P4 gene products (ref. 7; unpublished results).

In this paper we directly demonstrate that P4 virl can bemaintained as a stable plasmid. We established a reliable"patch-test" assay to screen potential clones for plasmid main-tenance and, in lysates from positive clones, demonstrated thatthe supercoiled form of DNA molecules are identical with theDNA present in linear form in P4 virions. Moreover, both epi-somal and chromosomally integrated forms of the regulatoryelement are present in the same population of cells. The abilityof P4 DNA to be maintained either as a phage or as a plasmid(pP4) is discussed with respect to the evolutionary relationshipbetween these two classes of replicons.

MATERIALS AND METHODSBacterial Strains. C-la is an F- prototroph ofE. coli (17); C-

la(P4 wt) is lysogenic for P4 wt (13); C-la(pP4 virl)-l carries P4virl as a plasmid (this report); polyauxotrophic C-1792(P2 Ig cc)is a P4 indicator (18); polyauxotrophic C-1055 is a P2 indicator(19).

Bacteriophage Strains. Phage P4 wt was isolated from strainK-235 (which also released helper bacteriophage) (1); P4 virlis an immunity-insensitive derivative of P4 wt (1, 13); P2 virlis an immunity-sensitive clear plaque derivativet of P2 wt (20);and, P2 Ig cc has a mutation correcting for a defect in particleassembly, thereby allowing for a larger burst size (21).

Growth and Purification of Phage Stocks. Phage stocks wereprepared according to Shore et al. (10) and Geisselsoder et al.(7).

Abbreviations: pP4, plasmid P4; kb, kilobase.* P2 virl and P2 Ig cc are clear and turbid plaque immunity-sensitivederivatives of P2 wt, respectively (20, 21). Hence, neither phage willform plaques on the lysogenic indicator lawn strain C-1792(P2 Ig cc).Not used in this assay is the immunity-insensitive derivative, P2 vir3,which forms clear plaques on such a lawn (20).

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Isolation and Purification of Putative Clones CarryingPhage P4 virl as a Plasmid. Logarithmic-phase C-la (ca. 2 X108 cells/ml) was infected with phage P4 virl at a multiplicityof infection of 10. After adsorption for 10 min at room temper-ature, the cells were plated for single colonies. Twelve individ-ual colonies were streak-purified, and clones from each weretested for the presence ofP4 virl as follows. Logarithmic-phasecultures were superinfected with P2 virl at a multiplicity of in-fection of 10 and allowed 10 min at room temperature for ad-sorption. Each culture was then plated for P4 infectious centerson C-1792(P2 ig cc). Plaques appearing were confirmed to beP4 virl, rather than P2 virl, by their failure to plate on C-1055.

For two reasons, only phage P4 virl plaques, and not phageP2 plaques, were expected to arise on these plates. First, be-cause C-1792(P2 ig cc) is lysogenic for P2, it will not plate theimmunity-sensitive P2 virl phage used to superinfect the cul-tures of C-la, but its integrated P2 helper will allow lytic de-velopment of any satellite P4 virl plaque-forming units liber-ated from the superinfected cultures. Second, P4 drastically"interferes" with lytic development of P2 (11). Hence, anyplaques arising on the C-1792(P2 Ig cc) indicator lawn would bedue to P4 virl plasmids that had been induced into lytic de-velopment upon superinfection with P2 virl helper phage.Eight ofthe 12 clones so tested revealed the presence ofP4 virlby their failure to plate on P2 indicator C-1055. One of theseclones, designated C-la(pP4 virl)-1, was used for all furthercharacterization.

Blot-Hybridization Experiments To Detect Plasmid Inte-gration. Cell lysis and phenol extraction of DNA were as de-scribed (22). Where called for, the DNA was digested with re-striction enzyme EcoRI (23). The restriction fragments or totalDNA was resolved by electrophoresis in a 1% agarose gel, trans-ferred to nitrocellulose (24), and hybridized with 32P-labeleddenatured P4 probe DNA (25, 26).

RESULTS

Rationale for Isolating Clones Carrying P4 virl as a Plas-mid. We have shown that satellite phage P4 interferes with lyticgrowth of its phage P2 helper by "directing" small capsid pro-duction (11). With both phages at a multiplicity of infection of10, the degree of interference varies with the temporal orderof infection. When P2 and P4 infect at the same time, the burstof P2 helper is reduced by a factor of 10. When P4 is given a20-min head start, the burst of P2 can be reduced by a factorof 500, whereas the P4 burst increases. This result suggestedthat ifa clone carried P4 as a plasmid, superinfection with helperP2 would give rise to a large burst of P4 and little or no P2. Wereasoned that by selecting for such morphogenic "interference,"we might be able to isolate a stable clone carrying P4 as a plas-mid. P4 virl seemed more favorable than P4 wt because its im-paired lysogenization might increase its tendency to multiplyas a plasmid.

"Patch-Test" for the Stability of Clones Carrying Phage P4virl as a Plasmid. Colonies to be tested were stabbed("patched") into plates prepared as described in the legend toFig. 1. As each stab of clones putatively carrying plasmid P4(pP4) virl grew into a visible patch of cells, it was infected atits outer perimeter by helper phage located in the uppermosttop agar layer. Such superinfection of the perimeter cells of thepatch then induced their pP4 into lytic development. The re-sulting burst of P4 virl phage infected surrounding C-1792(P2Ig cc) indicator lawn cells, giving rise to visible lysis around theperimeter of the patch. Hence, a "halo" was formed in the in-

dicator lawn around any patches of cells harboring pP4 virl.This lysis could not be due to infection by P2 virl phaget fromthe uppermost agar layer nor by P2 Ig cc spontaneously releasedfrom the indicator lawn cells because these phages were sen-sitive to immunity to P2 expressed by indicator lawn cells. Lysisalso would not be due directly to helper P2 because lytic de-velopment of P4 blocks assembly of the helper (11).

Fig. 1 shows a photograph of a patch-test plate. All 70 indi-vidual colonies of C-la(pP4 virl)-l stabbed onto a lawn of theP2 Ig cc lysogenic indicator strain C-1792 show a distinct halosurrounding each patch. In the center of the plate is a row offive patches of strain C-la containing neither phages P2 nor P4.No halo of clearing surrounds these patches. The P4 lysogenicstrain C-la(P4 wt) gave the same result (data not shown).

Kahn and Helinski (27) suggested that clones of pP4 virlwere unstable and not maintained at 42°C. Therefore, we testedthe putative pP4-maintaining strain C-la(pP4 virl)-l by grow-ing single colonies in L broth at 30°C, 37°C, and 42°C until theyreached stationary phase (-2 x 109 cells per ml). Each culturewas then plated to yield single colonies at the temperature atwhich it had been grown in liquid culture. Colonies (500-1000)from plates incubated at each of the three temperatures werethen picked and tested for maintenance of P4 virl as a plasmidby using the patch test at the temperature at which the colonieshad been grown. All patches showed distinct halos of clearingafter incubation for 12-20 hr§. Controls consisting of patchesof C-la and C-la(P4 wt) were negative.

Isolation and Characterization of pP4 virl. Experimentsdescribed in the previous two sections demonstrate that strainC-la(pP4 virl)-l stably maintains pP4 virl through numerouscell divisions at all temperatures tested. Of these cells, 100%produce a large burst ofphage P4 virl when superinfected withP2 helper phage (=35 phage per cell; unpublished results). Incontrast, as expected, no lysogen for P4 wt exhibited nearly thislevel of rescue (ref. 28; unpublished results). These results in-dicate that P4 virl is maintained either as a plasmid or as anunstable, easily rescued prophage.

Direct proof for plasmid maintenance of P4 virl would bethe isolation of supercoiled plasmid forms of P4 DNA. There-fore, DNA from a lysate of strain C-la(pP4 virl)-l was isolatedas described (29). An aliquot was then concentrated and purifiedby ethidium bromide/CsCl equilibrium gradient centrifuga-tion, and the remainder was stored at 4°C. As expected, a bandof supercoiled DNA was visualized in the gradient under UVlight.

Electrophoresis through an agarose gel showed that the sizeof this DNA was about 11 kilobase (kb) (Fig. 2, lane 7). More-over, the unpurified lysate of the same strain (lane 6) showeda band at the same location, in addition to expected host DNAand relaxed plasmid DNA bands. Strain C-la and strain C-la(P4wt) showed no band of supercoiled plasmid DNA molecules,

§ No data were presented by Kahn and Helinski (27). We now under-stand that their observations on the instability of clones carryingplamid P4 virl were most likely based on the method by which theypicked such clones. Unpublished results by one of us show that twodistinct species ofclones carrying pP4 virl were generated after initialinfection of E. coli strains with P4 virl. Such an infection gave riseto large and small size colonies in a ratio of 6:1, respectively. The smallcolonies did not appear until 6-12 hr after large colonies were seen.They were composed offilamentous cells. Of the small colonies, 100%contained pP4 virl, whereas only 50% of the earlier-appearing largecolonies contained pP4 virl, and less than 3% of these continue tomaintain the plasmid. In contrast, the small colonies initially foundto maintain pP4 virl continue to do so regularly. They also give riseto large colonies (at a large/small frequency of 2:1), and these maintainpP4 virl at a frequency of 100%. Strain C-la(pP4 virl)-l describedin this paper is of this last category.

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1 2 3 4 5 6 i

C-la(pP4virl?-1

FIG. 1. Typical agar plate displaying patch tests for clones car-rying pP4 virl. No halo of clearing is seen around five control patchesof C-la cells in the center row. In contrast, to left and right of the con-trols are rows of patches of strain C-la(pP4 virl)-1 showing distincthalos of clearing. Patch-test assay plates to test for clones maintainingP4 virl as a plasmid were set up as follows. Logarithmic phase C-1792(P2 ig cc) (0.2 to 0.4 ml) in top agar was poured onto an agar plate.After solidification, 4109 P2 virl phage in 0.1 ml of P-buffer (7) werespread over the nascent lawn. These plates could be stored at 40C forup to 3 mo before use in the patch test. Alternatively, such patch-testplates can be prepared without the P2 virl overlay. However, on suchplates the halo of clearing surrounding a patch of cells containing plas-mid P4 virl is not as distinct.

either in the ultracentrifuge gradient or on agarose gel electro-phoresis (data not shown).

Restriction digest patterns were compared for the super-coiled DNA isolated from C-la(pP4 virl)-l and for DNA ex-

tracted from purified P4 virl phage virions. Three differentrestriction enzymes were used. The identical restriction pat-terns of the two species of DNA (Fig. 3) establishes the exis-tence of P4 virl as a plasmid.pP4 yirl Is Also Integrated. The fact that phage P4 virl can

be maintained as a plasmid does not rule out the possibility thatcopies might also be integrated within the host chromosome,especially because the int (integration) function of P4 virl is

12 3 4 5 6 7

kb11.311.0 -.7.06.3 -a5.7-4.4 -

ChromosomalDNA

| Ad Relaxed P4 DNA|--I Linear P4 DNA

SupercoiledP4 DNA

L P4 yirl phage DNA pP4 yirl plasmid DNA

FIG. 3. Restriction enzyme digest patterns of DNA from CsCl gra-dient-purified P4 virl phage and from ethidium bromide/CsCl gra-dient-purified plasmid DNA resolved by agarose gel electrophoresis(see Fig. 2). The Sal I digest of P4 virl phage DNA (slot 3) shows twoadditional minor bands that are not evident in the Sal I digest of P4virl plasmid DNA (slot 7). These extra bands result from separationof the cohesive ends of some of the phage DNA molecules. This doesnot occur in the case of the purified, supercoiled plasmid P4 yirl DNAbecause its cohesive ends are covalently closed. Slots: 1, HindI; 2,EcoRI; 3, Sal I; 4, uncut; 5, Hindu; 6, EcoRI; 7, Sal I.

probably not repressed (13). To examine this possibility, DNAfrom lysates was electrophoresed and analyzed by blot hybrid-ization with radioactive phage P4 DNA. As expected, strain C-la(pP4 virl)-l yielded bands of both supercoiled and relaxedP4 DNA (Fig. 4 Right, lane 3). Of particulatinterest here is thefinding that this strain also showed, like the wt P4 lysogen con-trol (lane 2), a positive blot for P4 DNA in the location of hostchromosomal DNA. Thus, strain C-la(pP4 virl)-l appears tomaintain P4 virl DNA in both episomal and integrated states(though not necessarily in the same cell).To demonstrate that pP4 virl DNA is not merely trapped in

the chromosomal DNA, the total extracted DNA was treatedwith restriction enzyme. If pP4 virl is not merely trapped inthe chromosomal DNA, but is instead integrated, we wouldexpect formation of the same host-pP4 junction fragments as

from lysates of strain C-la(P4 wt).Accordingly, the total DNA extracted from these strains was

treated with restriction enzyme EcoRI. This enzyme cuts ma-

ture linear phage P4 DNA (from virions) at three sites, givingrise to four fragments in the map (Fig. 5) order A (68.6%), B(25.2%), C (4.0%), and D (2.2%) (31, 32). In the circular formof these molecules (spontaneously formed in vitro) the A and

1 2 3 1 2 3

FIG. 2. Agarose gel analysis of cleared lysates. Plasmid DNA wasisolated as described (29) and scaled up by a factor of 10. Electropho-resis of DNA was done in horizontal 0.7% agarose slabs. Species ofplasmid in each track: 1, pBR322 (4.4 kb) (30); 2, pJS4 (5.7 kb) (the cossite containing EcoRI-HindIII fragment of P4 was cloned intopBR322); 3 and 4, pJS9 (6.3 kb) and pJS11 (7.0 kb), respectively (pJS4plasmids with different portions of tn5); 5, pJS6 (11.3 kb) (pJS4 plas-mid with an intact Tn5 insertion); 6, pP4 virl (11.0 kb); and 7, pP4 virl(11.0 kb) purified from ethidium bromide/CsCl rather than from acleared lysate.

ChromosomalDNA

.....Relaxed pP4vin1 circles

-- Integrated P4

pP4u rl supercoils

FIG. 4. Hybridization of 32P-labeled phage P4 DNA with undi-gested DNA from cells lysogenic for P4 wt and cells carrying pP4 virl.(Left) Ethidium bromide-stained gel. (Right) Blot hybridization auto-radiograph. Slots: 1, C-la nonlysogen; 2, C-la(P4 wt); 3, C-la(pP4 virl)-1.

C-la

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a

Cos

RI iRI0 (2.2%)

A(6846%) B(252% S

RI RI RI RI RI RI RIto A+D---t r 2.

Cos

FIG. 5. EcoRI cleavage maps of P4 DNA. (a) iUnear phage P4DNA. (b) Circular phage P4 DNA. (c) Prophage P4 DNA.

D fragments will be fused through their cohesive ends (unlesspreviously heated). The expected replicative form for pP4 yirlDNA from cells would be covalently closed and, hence, shouldalso show fragments A + D, B, and C. In addition, if pP4 virlis also integrated at the P4 att (attachment) site, located in theregion 66.0-68.6% from the left end of the P4 genome (13),fragmentA + D will be split andjoined to the host chromosome(see Fig. 5). The fragment A + D would then be replaced bytwo new fragments, X1 and X2 (the prophage-hostjunction frag-ments), whereas fragments B and C should still be found.

Fig. 6 Right shows Southern blots ofEcoRI-digested lysates.Strain C-la(pP4 virl)"1 retained the fragment A + D expectedfor a circular plasmid molecule, and it also yielded characteristicprophage-host junction fragments X1 and X2 (lane 3). This resultshows that at least some of the pP4 yirl DNA molecules areintegrated at the normal host location for prophage P4 wt. Wehave not yet determined what fraction of cells in a C-la(pP4

1 2 3 4 1 2 3 4

Chromosomal r XDINA

_~~~~~~X2

-c

FIG. 6. Hybridization of 32P-labeled phage P4 DNA with EcoRIdigests of circular P4 DNA and DNA extracted from cells lysogenic forwtP4 andcells carryingpP4 yirl. (Left) Ethidium bromide-stained gel.(Right) Blot hybridization autoradiograph. Slots: 1, C-la nonlysogen;2,.C-la(P4 wt); 3, C-la(pP4'vilr)-1; 4, circular P4 phage DNA. Longerexposure times of the autoradiograph more clearly show the presenceof band X2 in slot 2 and band C in slot 3.

virl)-l culture maintain the integrated form of P4 virl, or theplasmid copy number.¶

DISCUSSION

We have shown that a single spontaneous mutational event iscapable of allowing satellite bacteriophage P4 to undergo a sta-ble new mode of propagation-plasmid maintenance. Our re-sults also demonstrate that this plasmid, pP4 yirl, under ap-propriate conditions, will display other-developmental optionsseen for different types ofreplicons: (i) integration into the hostchromosome (e.g., temperate bacteriophages A and P2, inser-tion elements such as ISJ, IS2, IS3- and IS4, and oncogenicmammalian viruses); and (ii) lytic replication dependent on geneproducts supplied by a heterologous helper genome (e.g., ad-eno-associated satellite virus and the murine leukemia system).The mutant phage P4 yirl was isolated as a spontaneous im-

munity-insensitive variant of P4 wt (1). We postulated that inthe presence of ongoing unrepressed P4-specific DNA repli-cation, without helper phage to make lysis possible, there wouldbe a shift in P4 and host developmental pattern's favoring theestablishment of plasmid maintenance. Accordingly, we haveisolated a strain ofE. coli in which P4 virl is stably maintainedas a plasmid, and we have shown that extrachromosomal su-percoiled P4 DNA is readily recovered from such cells. Ouranalyses indicate that pP4 yirl DNA (plasmid P4 yirl DNA),aside from being a closed supercoil, is identical to linear DNAisolated from the mature P4 yirl phage virion.

The conditions that establish stable pP4 virl-containing cellsare different from those permitting other phages such as A andP1 to exist as a plasmid. The unique nature of pP4 yirl stemsfrom the helper dependency of its wt parent; i.e., satellite bac-teriophage P4 can complete its lytic cycle only in the presenceof a- helper virus such as P2 (1). Thus, a mutation to virulencein phages A or P1 produces a phage that always kills the hostrather than one that exists as a plasmid (34, 35). In contrast, wehave shown that spontaneous mutation to virulence allows formaintenance of P4 as a plasmid and for survival ofthe host. Theplasmid-forming derivatives of phage A, called A dv (defectivevirulent), also differ from pP4 virl because they arose sponta-neously by illegitimate recombination causing deletion of mostof the A genome (36). An understanding of these differences isof practical importance because of the recent use of satellite P4as a cloning vector in E. coli, Serratia, Rhizobium, and Kleb-siella. Introduction of the sid (capsid size determination) mu-tation results in a P4 irl sidl "phasmid" vector (10) with prop-erties both useful and different from other vectors: the capacityto carry --27,000 base pairs of foreign DNA, high efficiency ofinfection as a phage vector and subsequent stable maintenanceas a plasmid vector, easy isolation of encapsidated phasmidDNA after superinfection of clones with helper phage P2, andlimited inadvertent spread as a phage due to inherent depend-ency on a helper for lytic development.

It appears that all known lytic regulatory functions of phageP4, which act in trans on a heterologous helper, are also ex-pressed by pP4 yirl. Hence,. the plasmid will respond imme-diately to superinfection by an appropriate helper because it ispoised on the brink of lytic development. Therefore, superin-fection with helper bacteriophage P2 invariably results in a largeand rapid P4 burst (25-30 min versus 50-60 min for P4 infectionof a P2 lysogen). Exploitation of this fact allowed for develop-

I Since submission of this paper, we have determined by the meth-odology ofTimmis et al (33) that pP4 virl is maintained as a high copynumber plasmid with an average copy number of 70 per cell 3hr afterinitial infection of strain C-1055 (unpublished results).

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ment of a sensitive plate assay (the patch test) for cells carryingpP4.

pP4 yirl would not be expected to repress its int functionbecause of the presence of the virl allele. For this reason wetested whether phage P4 yirl was not only carried as a plasmidbut also integrated into the host chromosome. Southern blotsof restriction digests of total DNA show both forms to be pres-ent. In contrast, cultures lysogenic for P4 wt contain only theintegrated form: We do not yet know the copy number pP4virl,1 whether it continually hops between extrachromosomaland chromosomal locations, and whether both forms are alwayspresent in the same cell.The close evolutionary relationship between defective vi-

ruses and plasmids is supported by our observation that a singlespontaneous mutation in satellite phage P4 endows it with theability to propagate as a plasmid. On the other hand, the elab-orate mechanism by which P4 modifies and utilizes the productsof a heterologous helper might suggest that this mode of de-velopment is the primary course in nature (4, 7, 9-12). Ac-cordingly, just as a lytic phage might be derived from temperatephage by loss of capacity to integrate and to repress, P4 mighthave been derived from a temperate ancestor by loss of abilityto produce a capsid and lyse the cell. In fact, such a viable hybridphage has been recently constructed by joining the late (mor-phogenic and lysis) functions of helper phage P2 to the "essen-tial" region (replication and regulation) of P4 (37). However,although this hybrid contains all known P4 regulatory functions[E (derepression of helper P2), 8 (activation of helper lategenes), psu (polarity suppression) and sid (capsid size deter-mination)], it cannot "direct" in cis (as it does normally in trans)the assembly of small P4-size capsids derived from helper geneproducts.

Outside of the laboratory environment, circumstances lim-iting the availability of helper virus might provide a positiveselection for the plasmid mode of development. Indeed, hadthe original selection been for a plasmid rather than for a phage,phage P4 might have been discovered as a plasmid. Moreover,pP4 yirl can be mobilized for transfer by the F episome, whichfurther reflects its plasmid-like attributes (unpublished results).The fact that all known phage P4 gene products are expressedin a coordinated fashion during maintenance as a plasmid sug-gests that they may play an important role in this mode of de-velopment. In particular, we have found that the phage P4 psugene, considered to be "nonessential" for lytic phage growth(38), appears to be required for stable maintenance of P4 virlas a high copy number plasmid: its product, acting in cis as atranscriptional antitermination factor, allows high-level reacti-vation of gene a of P4, whose product is required for DNA rep-lication (unpublished results).

Phage P4 was isolated from a colicinogenic strain -of E. coli(1). Such an origin is suggestive as to why P4 ts/3 mutants, con-taining lesions in a region of the genome considered "unessen-tial" for lytic phage growth, act to allow expression of a proteincausing aberrant cell division and subsequent shutdown ofmac-romolecular synthesis (ref. 15; unpublished&results). Charac-terization of the P gene product (and related functions from thenonessential region of defective phage P4) should provide adefinitive answer as to its colicinogenic origin.We are grateful to Bernard Davis for editorial suggestions. We also

thank Richard Novick, Fevzi Daldal, Dan Fraenkel, Janet Geisselsoder,Susan Ely, Jane Weeks, and Vince Cryns for criticism and encourage-ment of this work. David Shore and Se-Jin Lee are also acknowledgedfor preliminary characterizations ofpP4, Vince Cryns for having greatlyimproved the efficiency of the patch test, Rosalba Lagos for the pP4

copy number, Se-Jin Lee for determining the effect of the psu producton gene a, and Vernard Coulter for technical assistance. This researchwas supported in part by research Grants GB41769, PCM-04017, andPCM-06276 from the National Science Foundation, by Biomedical Re-search Support Program Grant S07-RR-05381-19 and Grant GM-27143from the National Institutes of Health, and by Grant 72 from the Swed-ish Medical Research Council. One of the authors (R.G.) was also sup-ported in part by Career Development Award AI-00084 from the Na-tional Institute of Allergy and Infectious Diseases.

1. Six, E. & Klug, C. A. C. (1973) Virology 51, 327-344.2. Lindqvist, B. (1974) Proc. Natl Acad. Sci. USA 71, 2752-2755.3. Six, E. & Lindqvist, B. (1971) Virology 43, 8-15.4. Goldstein, R., Lengyel, J., Pruss, G., Barrett, K., Calendar, R.

& Six, E. (1974) Curr. Top. Microbiol Immunol 68, 59-75.5. Six, E. (1975) Virology 67, 249-263.6. Six, E. & Lindqvist, B. (1978) Virology 87, 217-230.7. Geisselsoder, J., Youdarian, P., Deho, G., Chidambaram, M.,

Goldstein, R. & Ljungquist, E. (1981)J. Mol Biol. 148, 1-19.8. Lindahl, G. (1974) Mol Gen. Genet. 128, 249-260.9. Souza, L., Calendar, R., Six, E. & Lindqvist, B. (1977) Virology

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