Proc. Natl. Acad. Sci. USAVol. 75, No. 11, pp. 5594-5598, November 1978Genetics

Analysis of bacteriophage P1 immunity by using X-P1 recombinantsconstructed in vitro

(P1 repressor genes/PI antirepressor gene/PI dnaB analog gene/DNA cloning)

NAT STERNBERG, STUART AUSTIN, DANIEL HAMILTON, AND MICHAEL YARMOLINSKYCancer Biology Program, National Cancer Institute, Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland 21701

Communicated by Gary Felsenfeld, August 10, 1978

ABSTRACT We describe the dissection and reconstructionof a complex control circuit, the P1 immunity system, by amethod that involves inserting EcoRI-generated fragments ofP1 DNA into X vectors that can then be sequentially insertedinto a bacterial cell. Using these techniques we have isolatedX-P1 hybrid phages that express the products of P1 genes cl, c4,ant, and ban and, in appropriately constructed lysogens, con-firmed the roles played by the first three of these products inphage immunity. In addition we have'localized to particularP1 fragments the sites requisite for expression and repressionof these gene products. The analysis leads to the conclusion thatgpant acts in trans to antagonize repression mediated by gpcl,in support of' one of two proposed models for gpant action.Moreover, two features of the, immunity system are revealed:(f) a hitherto unknown component that effects gpcl repression;and (if) an unexpected ability of gpc4 to channel a superin-fecting cl'+ phage into the lysogenic state, which suggests thatgpc4 activity regulates the establishment phase of lysogeny.

Analysis of complex genetic'controls requires methods thatpermit parts of the control circuits to be isolated, characterized,and variously reassembled. We show here how the insertion ofdefined DNA segments into X vectors that can be successivelyintegrated into the chromosome of a suitable bacterium permitsthe analysis of several interacting genes by reconstruction aswell as dissection experiments. Because of the phenomenon ofincompatibility, this type of analysis would not be possible ifthe genes to be studied were located in a plasmid.The subject of our analysis is the immunity to superinfection

conferred by the plasmid prophage of coliphage Pl. This im-munity is thought to' require the presence of two repressorproteins, the products of genes ci (1) and c4 (2) (gpcl and gpc4)that are located in the separate immC and immI regions of thephage genetic map (Fig. 1). Gpcl represses the viral lyticfunctions. Gpc4 is thought to repress a second immI' gene, ant(or reb). The product of the ant gene has been postulated to acteither in trans to antagonize gpcl-mediated repression (9) orin cis to replicate DNA of the superinfecting phage even in thepresence of gpcl (i.e., it acts as a repressor bypass) (10). PhageP7, which has a ci gene functionally identical to the ci geneof P1 but differs from P1 in the specificity of its c4 repressor(9), is able to grow in a P1 lysogen because gpant of the in-fecting P7 phage is not repressed by gpc4 of the prophage.We describe here the isolation, characterization, and inter-

actions of individual X-P1 hybrids bearing EcoRI-generatedfragments of P1 (11) that express each of the known componentsof the P1 immunity system. An additional hybrid that'expressesthe P1 ban gene (12), known to be directly under cl control (13)(Fig. 1), provides'an indicator of cl repression.

MATERIALS AND METHODSBacterial and Phage Strains. The Escherichia coli strains

used were NS985 (HfrH, sup+), RW842 [HfrH XA(int-FII)gajT](14), YMC (supF) (15), K175 (supD, Xr) (10), Q1508[dnaB70(ts), thyAl (12), NS91 (C600 groPA15) (16), and N3072[HfrH, sup+, A (pro-lac) Xlii] (6). The X vector XDam15b538sr1X3c1857(ts)nin5 contains a single EcoRI site forinserting EcoRI fragments of P1 DNA (11).' Hybrid phagesobtained by inserting P1 fragments (11) are designated X-Pl:c4,X-Pl:c1, etc.; :c4 or :cl refers to pertinent genes located on theinserted P1 fragment. Other phages used were PlCm,PlCmcl.100(ts), PlCmcl.55(am), PlCmc4.32, PlCmelrs, andPlvdrsban-l. Unless otherwise indicated, all of the P1 phagescarried the Tn9 transposon (Cm). PlCmvirsant-10 andPlCmc4.32ant-17 were isolated from P1 lysogens made byinfecting strain NS985 with either PlCmvdrs or PlCmc4.32.The presence of the ant- mutation in these phages was con-firmed by their inability to make plaques on strain.K175(Plcry)(5). Mutational defects are indicated here by a minus sign su-perscript unless an allele numbei is specified.Media and Growth Conditions. Media and commonly used

microbiological procedures for P1 and N were as described (13,17). Because the attX-int region of the X vector has been re-moved' by the b538 deletion (18), homologous recombinationwith the cryptic X prophage of 'strain RW842 was used to ly-sogenize with primary X-P1 hybrids.The experiments described in this report were carried out

at an EKi-Pi containment level in accordance with the'stipu-lations specified in FCRC MUA# 1.

Construction of Xatt+imm21-P1 and Xatt+imm434-P1Hybrid Phages. The Xb538cI857(ts)nin5-P1 hybrids (X-P1hybrids) -were restructured first by recombination withXfmm21nin5 or Ximm434nin5 to replace the immX' regionwith'imm2l or imm434 and then by recombination withXb515b519Nam7cI857(ts) to replace the b538 deletion withthe two smaller deletions, b515 and b519, which leave attX andthe X int gene intact (18). The restructured hybrids (designatedXimm2l-Pl or Ximm434-Pl) can lysogenize standard strainsat 32 or 42°C.

Isolation of a X-P1:ban Hybrid. We took advantage of theability'of the P1 ban gene product to substitute for the dnaBprotein of the host (12) in order to isolate a X-Pl:ban hybrid.Strain Q1508(X) was infected with a pool of \-P1 hybrids (de-rived from 1000 plaques). The infected cells were spread onthymine-supplemented'Ll plates and incubated for 36 hr at42°C. The frequency of infected cells that survived to formcolonies (3 X 10-6) was about 30-fold greater than thednaB70(ts) reversion frequency. Phage that formed turbidplaques at 320C and clear plaques at 42°C [phage with the

Abbreviations: Cm, chloramphenicol resistance transposon Tn9; am,amber, ts, temperature sensitive; gpc4, gpcl, etc., gene product of genec4, cl, etc.

5594

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Proc. Natl. Acad. Sci. USA 75 (1978) 5595

2riE I EoRI fragments r-,n

Cmv

immlvirs

1122 7inm

immC

l /~~~~~~~/\,'flgpf2 gpc4 gpant-.- gpban gpc1

O1la ~

FIG. 1. P1 genome with the positions of the two immunity regionsand the Cm (Tn9) transposon (3,4) above the bar and pertinent genes,whose products are designated in the figure, below the bar. The extentof the deletions in the Plcry (5), Pldpro8a (6), and Pldprolla (6)prophages are indicated by the black areas. The location of EcoRI-generated P1 fragments is based in part on the map of Bachi andArber (7) and on our own marker rescue experiments using ammutations (8). The virs mutation, located in the immI region (2, 9),permits the phage to express gpant constitutively even in the presenceof gpc4. Thus, when a P1 lysogen is infected with Plvirs, gpant isexpressed, repression of lytic functions is lifted, and the infectingphage grows. For the same reason, P7, whose ant gene expression isinsensitive to repression by gpc4 of P1, will grow in a P1 lysogen.

c1857(ts) allele] were isolated from these lysogens. Infectionwith these phages (X-Pl:ban) increased by about 300-fold thesurvival of Q1508(X) at 42°C. A Xatt +imm21-Pl:ban phagewas constructed, and Q1508 lysogens containing this prophagesurvived with unit efficiency at 42°C. The X-Pl:ban hybridphage carried P1 EcoRI fragment 3(6 X 106 daltons).

Isolation of a X-Pl:cl.100(ts) Hybrid. Strain NS91(Ximm21-Pl:ban) contains a groP mutation, but Ximm434forms plaques with the same efficiency on this strain as it doeson a groP+ strain. Thus, ban gene expression (12, 16) from theXimm21-Pl:ban prophage can-suppress the inhibitory effectof the groP mutation on phage A growth. Because the P1 clgene product can repress ban gene expression (13) gpcl activitywas assayed as the restoration of the groP- phenotype to strainNS91 (Ximm21-Pl:ban). This strain was infected with a poolof X-P1 hybrid phages and the resulting NS91 (Ximm21-Pl:ban;X-P1) lysogens were tested for their ability to allow Ximm434to form a plaque. On 1 of the 30 lysogens tested, Ximm434formed plaques with '400th to 'A0th the efficiency that wasfound on the others. The X-P1 prophage present in that lysogenwas recovered and designated X-Pl:cl.100(ts).

Analysis of Phage DNA by Agarose Gel Electrophoresis.X-P1 DNA was isolated and subjected to EcoRI endonucleasedigestion and agarose gel electrophoresis as described (11),except that horizontal slab gel electrophoresis (19) was carriedout in 40 mM Tris acetate/20 mM sodium acetate/2 mMEDTA, pH 7.8 (20). EcoRI digests containing 1-2 .g of DNAin 25-50 ,ul were applied to the gel and run at 30 V for 18 hr.The gels were stained with ethidium bromide and photo-graphed under UV light.Mapping of X:X-P1 Heteroduplexes by Electron Micros-

copy. Heteroduplex formation and formamide spreading forelectron microscopic analysis were carried out as described byBroker and Chow (21); a Hitachi HU 12A electron microscopewas used at 50 kV. Heteroduplexes were analyzed by enlargingphotographic negatives X20 with a Seamco model 210 micro-fiche reader and the images were measured with a Numonicelectronic graphics calculator.

RESULTSIsolation of X-P1 Hybrid Phages that Express gpc4. Hybrid

phages were isolated by inserting EcoRI-generated fragmentsof Plcl.100(ts) DNA into a A vector (11). A lysate obtained froma pool of 500 hybrid phage plaques was usedlo lysogenize strainRW842. We expect that a X-P1 prophage that constitutively

expresses gpc4 should permit a lysogenization-deficient Plc4-phage to lysogenize. Of 50 independent RW842 (X-P1) lysogensscreened, 2 showed a 100- to 1000-fold increase in the capacityfor being lysogenized by Plc4- (PlCmc4.32). The X-P1 phages(X-Pl:c4a and X-Pl:c4b) obtained from these lysogens weremade att + int + and then integrated into another host (NS985).In NS985, as in the original host, they conferred proficiency tobe lysogenized by Plc4.32 at high efficiency. Because 90-95%of the P1 phage recovered from the NS985 (X-Pl:c4,PlCmc4.32) lysogens still carried the c4.32 allele, lysogenizationby Plc4.32 was not due to recombination between the c4+ geneof the X-P1 prophage and the c4- gene of the P1. (The re-maining 5-10% of the recovered P1 phage were Plc4+, byrescue of c4.32+ from the X-Pl:c4 prophages). Thus, theX-Pl:c4 phages express gpc4 constitutively in the prophage stateand this product can act in trans to permit Plc4- to form stablelysogens.

X-Pl:c4 Lysogens Are Immune to P1. AlthoughPlcl.55(am) formed plaques efficiently and yielded a signif-icant number of phage after infection of lysogens of eitherA-Pl:c4a or X-Pl:c4b, P1 wild type did not (Table 1, line 2).This immunity is probably a consequence of gpc4-mediatedrepression of the ant gene of the superinfecting phage, becausePlIrs and P7, whose ant gene is not sensitive to repression bythe gpc4 of P1, grew well in the X-Pl:c4 lysogens (Table 1, line2).

Expression of the ant Gene Product (gpant) by X-P1:c4Phages. Expression of the ant gene by a superinfecting P1phage induces a resident P1 prophage (2). Because ant is re-pressed by gpc4, ant expression by a superinfecting P1 phagerequires that the phage be insensitive to gpc4 (e.g., virs) or thatthe resident prophage be c4- (Table 2, Exp. A). When aPlc4-ant- lysogen was superinfected with X-Pl:c4a, the P1prophage was induced. This hybrid phage was unable to inducea c4+ prophage. We conclude that X-Pl:c4a, like P1, expressesant on infection and that this expression is subject to repressionby gpc4. The X-Pl:c4b hybrid does not express ant.

Table 1. Reconstruction of P1 immunity by using A-P1 hybrids

Genes Phage productionProphage present Pici+ Plcl- P7ci+

None None +(40) +(48) +(29)A-PI:c4 c4 -(3) +(60) +(24)X-Pi:ci cl -(3.8) +(39) -(3)A-P1:2 f2 + + +A\-Pl:c4; A-P1:2 c4; f2 -(4.3) +(52) +(33)A-Pl:cl; X-P1:2 cl;f2 +(22) +(43) +(19)Plcry cl;f2 +(50) +(60) 4-(32)X-Pl:cl; X-Pl:c4 cl; c4 -(0.4) -(0.6) -(4)Pldprolla cl; c4 -(1.7) -(1.1) -(4.5)Plcry; X-Pl:c4 cl; c4; f2 -(0.8) -(2) +(22)A-Pl:ci, A-Pl:c4A-Pl:f2 cl, c4; f2 -(2.2) -(3) +(32)Pldprolla;X-P1:2 cl;c4;f2 -(1.8) -(3) +(29)Pldpro8a cl;c4;f2 - - +P1 cl;c4;f2 -(1.2) -(1.4) +(24)P7 cl;c4;f2 + + -

The host used in this experiment was either N3072 (for Pldprolysogens) or NS985. The X-Pl:cl hybrid was Ximm434-P1:c1+, theA-Pl:c4 hybrid was Aimm21-Pl:c4b (identical results were obtainedwith Ximm2l-Pl:c4a), and the X-P1:2 hybrid was XcI857(ts)-P1:2.The Plcl- phage used was Plcl.55(am). (+), Plaque-forming effi-ciency of 0.5-1.0; -, plaque-forming efficiency of < 10-3 relative tothe plaque-forming efficiency with strain NS985. Plvirs formedplaques with near-unit efficiency on all of the lysogens. The valuesin parentheses are the phage yields per infected cell in Li broth at 120min after infection at 32°C (13).

Genetics: Sternberg et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

Table 2. Gpant production by X-Pl:c4a

Infectingphage Yield of phage/infected cell

Experiment ANS985(Plc4-ant-) NS985(Plc4+ant-)

Plvirs 24 (9ant-;15ant+) 19 (10ant-;9ant+)P1 16 (6ant-;10ant+) 0.3X-Pl:c4a 10 0.08X-Pl:c4b 0.16 0.04

Experiment BQ1508 Q1508(Plc4-ant-) Q1508(Plc4+ant-)

Plvirsban- 0.2 22 (9antC;13ant+) 16 (7ant-;9ant+)Plban- 0.4 24 (12ant-;12ant+) 0.2X-Pl:c4a 0.1 12 0.2X-Pl:c4b 0.05 0.3 0.2

Bacterial strains for phase infection were grown at 320C in Li brothwith maltose (0.2%) and, if carrying P1 prophage, chloramphenicol(25,gg/ml). P1 was assayed on strain K175(Xr) and X was assayed onstrain YMC by using TB plates (P1 will not form plaque on TBplates). Plant+ and ant- phage in the yield were distinguished onthe basis of their differential plaque-forming ability on strain K175(Plcry) (5). Experiment A: The multiplicity of phage infection was2.5 and the yield of P1 at 38°C was assayed 150 min after infection.Procedures for P1 (13) and X (17) infection have been described. Theinfecting X-P1 phage have the immunity of phage 21, and the NS985strains all carry a Ximm2l prophage. The yield of the infectingXimm2l-P1 hybrid phages under these homoimmune conditions was0.25-0.5 phage per cell. Experiment B: The multiplicity of phage in-fection was 2.5 phage per cell, and the yield of phage at 40°C was as-sayed 120 min after phage infection.

Expression of ant from X-Pl:c4a was confirmed in studieson the expression of a P1 gene (ban), which is repressed by gpclin lysogens (13). Expression of ban can be detected by the abilityof gpban to suppress host dnaB(ts) mutations and thus allowgrowth of phages such as X or Plban -, which require the dnaBproduct to replicate. In Table 2, Exp. B, we show that X-Pl:c4abut not X-Pl:c4b superinfection of a suitable P1 lysogen inducesthe prophage ban gene. Again, this ant-mediated inductionrequires that the resident prophage be c4-.P1 EcoRI Fragments Obtained from X-P1:c4a and

X-P1:c4b. DNA isolated from X-Pl:c4a and X-Pl:c4b wascleaved by restriction enzyme EcoRI and analyzed by agarosegel electrophoresis (Fig. 2). Both hybrid phages carried P1fragment 9 (2.4 X 106 daltons) which contains the wild-typeallele of P1 am3.21. In addition, X-Pl:c4a carried fragments14 (1.0 X 106 daltons) and 23 (0.18 X 106 daltons), whereasX-Pl:c4b carried fragment 22 (0.21 X 106 daltons) (data notshown for fragments 22 and 23). By EcoRI cleavage and liga-tion of X-Pl:c4b DNA, we isolated a X-P1 hybrid that containedonly P1 fragment 9. The properties of this hybrid were indis-tinguishable from those of X-Pl:c4b. Thus, fragment 9 containsthe c4 gene and a promoter sufficient for its expression. Thewild-type alleles of some ant - mutations can be rescued fromfragment 9 and other ant - mutations from fragment 14 (un-published data). Because an allele for constitutive ant synthesis(virs) is present on fragment 9 and the ant structural gene spansthe 9-14 junction, transcription of ant probably starts in frag-ment 9 and proceeds into fragment 14 (Fig. 1). If oirs definesthe site of gpc4-mediated repression of ant, this site must alsobe on fragment 9. It is unlikely that ant expression requiresfragment 23, which is present on X-Pl:c4a but is derived froma relatively distant region to the right of ant.

Heteroduplex Analysis of X-Pl:c4 Phages. Heteroduplexstructures obtained when DNA from either of the two X-Pl:c4hybrids was denatured and reannealed to DNA from the Xvector are shown in Fig. 3. An addition was found at the ex-

22,3

5.6

-

8-

944* r

10 -

11 -

12.13r-

14.4r-

15 -

16 -

19 -

FIG. 2. Electrophoresis was carried out in a 0.7% agarose slab gel.Lanes contained EcoRI-digested DNA as follows: A, from XD-srIX3vector; B, PlCmcl.100; C, X-Pl:ban; D, X-P1:2; E, X-Pl:c4a; F,X-Pl:c4b; G, X-Pl:cl.100(ts). The two common high molecular weightbands present in all of the lanes with X DNA are the two arms of theX vector (11). The minor band above these two bands corresponds tothe complex of left and right vector arms joined by the X cohesivetermini. The PlCmcl.100 DNA (lane B) that we used to clone frag-ments contains a tandem duplication of the Tn9 transposon. Thistransposon contains an EcoRI site and is located in P1 EcoRI frag-ment 4 (7). Consequently, fragment 4, which normally overlapsfragments 5 and 6, is cut by the endonuclease to give three new frag-ments, 4*, 4** (overlaps P1 fragment 9), and 4*** (overlaps P1 frag-ment 14). The P1 fragments in X-P1:c4a are derived from Plc 1.100,a phage that lacks Tn9, so the P1 fragments seen in lane E must be9 and 14 and not 4** and 4***. Although X-Pl:c4b carries a Pi frag-ment from PlCmcl.100, this fragment must be 9 and not 4** becauseit contains the c4 gene and the wild-type allele of am3.21. The sizeof the P1 fragments was determined in a separate gel with EcoRIfragments of X DNA and Hae III fragments of simian virus 40 DNAas standards (11).

pected X map coordinate (0.656) of the unique srIX3 site intowhich the P1 fragments are inserted (11). With X-Pl:c4b, theaddition formed a single-stranded loop corresponding to 2.64X 106 daltons of double-stranded DNA, as expected for frag-ments 9 plus 22 (2.6 X 106 daltons). With X-Pl:c4a, the additionassumed a stem-loop structure. The double-stranded stemcorresponded to 2.45 X 106 daltons of DNA, the approximatesize of fragment 9, and the single-stranded loop correspondedto 1.16 X 106 daltons of double-stranded DNA, the sum offragments 14 and 23 (1.2 X 106 daltons). Thus, X-Pl:c4a con-tains two copies of fragment 9 in inverted order flankingfragments 14 and 23. This arrangement of fragment 9 DNA isprobably due to the chance association of EcoRI fragmentsduring the in vitro ligation reaction because no stem-loopstructure can be generated in P1 DNA and only one copy ofEcoRI fragment 9 is seen per P1 DNA molecule by gel analy-SiS.

Characterization of a X-Pl:cl Hybrid. A X-Pl:cl.100 hybridphage was isolated, and the Xatt + imm434-Pl:cl.100(ts) hy-brid was then constructed and the Plcl+ allele was crossed intothis phage. The following properties indicate that thisXimm434-P1:c1+ hybrid phage expresses the cl gene. (i)

A B C D E F G

5596 Genetics: Sternberg et al.

Proc. Natl. Acad. Sci. USA 75 (1978) 5597

. Y" .

FIG. 3. (A) Heteroduplex between XD-srIX3 DNA and X-P1:c4bDNA. The mean (±SEM) length of the single-stranded (ss) additionloop (P1 fragments 9 and 22) corresponds to 4.07 ± 0.05 kilobases ofDNA. The short arm is 37.19 ± 0.14% of the entire X homoduplex. Thisplaces the ss loop at X map position 0.657, in good agreement with theposition of the srIX3 site (22). (B) Heteroduplex between XD-srIX3DNA and X-Pl:c4a DNA. The double-stranded (ds) stem (fragment9) is 10.47 ± 0.19% (3.8 kilobase pairs) of the length of the X homo-duplex and is located 37.2 ± 0.22% in from one end of the DNA mol-ecule (X map position 0.656). The length of the ss loop (fragments 14and 23) corresponds to 1.79 ± 0.05 kilobases of DNA. The ss standardused on this grid was OX174 DNA (5.27 kilobases). (X23,000.)

Plc4-ant- will not form a plaque on a Ximm434-Pl:c1+ ly-

sogen whereas Plc4-ant + will. This is because gpant antag-onizes gpcl-mediated repression. (ii) A dnaBts (Ximm2l-Pl:ban, Ximm434-Pl:c1+) lysogen dies at 420C and a groP-(Ximm21-Pl:ban, Ximm434-P1:cI+) lysogen exhibits a groP-phenotype [AcI857(ts) fails to plaque on this host]. These resultsindicate that ban gene expression from the Ximm2l-Pl:banhybrid is being repressed by gpcl from the Ximm434-P1:c1+phage. (iii) Plcl.55(am), although unable to lysogenize normalsup+ hosts, lysogenizes NS985sup + (Ximm434-Pl:c1+) at thesame frequency as P1 wild type (0.05-0.15). This is due to clcomplementation rather than recombination because 96% ofthe P1 phage recovered from such lysogens carried the cl.55allele. The remaining P1 phage carried the cl+ allele, showingthat this allele is present on Ximm434-Pl:cl+.The cl gene product expressed from the original

Ximm434-Pl:cl.100 isolate is temperature sensitive-i.e., P1ant- will form plaques on an NS985 (Ximm434-Pl:cl.100)lysogen at 42°C but not at 320 C. The X-P1:cl.100 hybrid andits cl+ derivative (data not shown) carried P1 fragments 7 (4X 106 daltons) and 11 (1.9 X 106 daltons) (Fig. 2).

X-Pl:cl+ Lysogens Are Immune to cl+ SuperinfectingPhage. Plcl+ and P7cl+ did not form plaques on a X-Pl:cl+lysogen although Plcl- did (Table 1, line 3). We believe thatthis effect is due to the inability of gpant produced from thesuperinfecting phage to prevent repression when gpcl is beingproduced from both incoming and resident phages.

Reconstruction of the P1 Immunity System. Table 1 (line8) shows that a lysogen containing Ximm2l-Pl:c4b andXimm434-Pl:cl+ is immune to superinfection by either aPlcl+ or a Plcl- phage. To our surprise, however, this doublelysogen was also immune to the normally heteroimmune phageP7. We call this "extended" immunity. An analogous lysogencontaining the Ximm2l-Pl:c4b prophage and the deleted P1prophage Plcry cl+ (Fig. 1) exhibited normal immunity (Table1, line 10). In contrast, another deleted prophage, P1 dprolla,which contains both the c4 region and the cl+ gene (Fig. 1),expressed the same "extended" immunity as the lysogen har-boring X-Pl:c4 and X-P1:cl+ (Table 1, line 9). These resultssuggested that an element present on Plcry and absent on

Pldprolla is required for normal P1 immunity.

We therefore lysogenized the Ximm2l-Pl:c4b,Ximm434-PI:cl+ lysogen with various X-P1 hybrid phagescontaining P1 fragments from the left 20% of the P1 map (Fig.1). A X-P1 prophage containing P1 fragment 2 (6.7 X 106 dal-tons) (Fig. 2) was found to restore normal immunity to the ly-sogen (Table 1, line 11). We suggest that this fragment containsa gene (designated f2) whose product is required for normalimmunity. The significance of this product as a component ofthe immunity system was confirmed by demonstrating that the"extended" immunity conferred by Pldprol la was abolishedby the presence of X-PL:f2. The resulting lysogen exhibited animmunity indistinguishable from that of P1 wild type (Table1, lines 12 and 14). Note that Pldpro8a, which retains fragment2, showed normal immunity (Table 1, line 13). The synthesisof gpf2 may also determine the immunity properties of lysogenscontaining the c4-deleted prophage Plcry (Fig. 1). AlthoughA-Pl:cl+ alone confers immunity to a cl+ infecting phage,Plcry cl+ does not (Table 1, lines 3 and 7). This difference canbe attributed to the synthesis of gpf2 from Plcry because aA-Pl:cl+, X-P1:2 lysogen (Table 1, line 6) resembles a Plcrylysogen in having no immunity to a cl+ infecting phage. Re-cently, two mutations, c6.101 (23) and bof (24) that affectprophage immunity have been isolated. Both are located in thefragment 2 region of the genetic map (Fig. 1). Although thesemutations remain to be fully characterized, we note that P1bof- lysogens exhibit "extended" immunity (24).

DISCUSSIONThe use of X-P1 hybrids to dissect and reconstruct the P1 im-munity system affords two major advantages over standardgenetic approaches to the study of P1 immunity: (i) the ex-pression of individual components of immunity can be studiedwithout interference from extraneous viral genes and can beshown to be independent of distant regions of phage DNA; and(ii) the interactions between individual components of im-munity located on separate DNA segments can be studiedwithout encountering the incompatibility barrier to their stablecoexistence. X vectors are particularly suited for such studies,because X hybrids bearing any of several immunities (e.g.,immX, imm434, imm2l) can be constructed and correspondingsingly or multiply immune lysogens can be readily selected.This method has allowed us not only to confirm the roles playedby elements of the P1 immunity system (c4,cl, ant) but also tolocalize sites needed for expression and control of those elementsand to reveal a new element and several new features of theimmunity system.The PIcl gene is expressed from a X-P1 hybrid prophage

containing EcoRI fragments 7 and 11. In contrast, a X-P1 hybridcontaining only fragment 7 carries at least part of the ci gene,by marker rescue tests, but does not express gpcl (data notshown). it is unlikely that EcoRI cleavage splits the cI gene intoportions contained on fragments 7 and 11 because: (i) the dis-tribution of am mutations on fragments in the immC regionplaces at least one fragment (fragment 22) between fragments7 and 11 (Figs. 1 and 2). The orientation of fragments 7 and 11relative to each other in the X-Pl:cl hybrid is inverted relativeto their orientation in P1 DNA (data not shown). Thus, frag-ment 7 contains the entire cl gene but probably lacks a site (e.g.,a transcription promoter) needed for cl expression. Fragment11 apparently can provide a substitute site for this expression.An alternative possibility, that fragment 11 contains a genewhose product is needed for cI expression, seems unlikely be-cause none of the clear mutations that affect P1 lysogeny islocated in the fragment 11 region of the P1 map (1, 23, 25).The c4 gene and a promoter sufficient for its expression are

present on EcoRI fragment 9 as is the wild-type allele of the virs

Genetics: Sternberg et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

mutation, a mutation that allows constitutive ant production.The ant gene spans the junction between fragments 9 and 14.The transcription of ant probably originates in fragment 9because the only other fragment (fragment 23) present on theX-PI:c4a phage, which expresses ant, is derived from a regiondownstream from the origin of ant transcription (Fig. 1).What is the function of gpant ? The experiments of Table 2

show that gpant can act in trans to induce a P1 prophage, asevidenced both by lytic growth of the prophage and by in-duction of a specific gene (ban) known to be subject to cI re-pression. We believe this result rules out the suggestion by Westand Scott (10) and Scott et al. (26) that the ant gene (renamedthe reb gene by these authors) makes a cis-acting protein thatallows P1 to replicate lytically in the presence of active gpcl.We favor the hypothesis that gpant antagonizes gpcl-mediatedrepression.We have shown that X-P1:c4 lysogens, although not immune

to Plcl-, are immune to P1. We believe this result reflects apreviously unsuspected role for gpant in regulating the levelof gpcl activity during the establishment of the P1 prophage.Gpc4 repression of the ant gene during the early stages of P1infection may channel the infection into a lysogenic response.Note that, if ant gene expression by the infecting phage is notrepressed by gpc4, as when the X-Pl:c4 lysogen is superinfectedby P7 or by Plvirs, then phage growth is normal (Table 1, line2). If a cell contains gpc4 and gpcl but not the product of a genecontained on P1 EcoRI fragment 2 (gene f2), it is immune tosuperinfection by Plcl+, Plcl-, and P7 ("extended" immu-nity). Because expression of the P7 ant gene is insensitive togpc4 of P1 (2, 9), the extended immunity must reflect an in-ability of gpant to overcome gpcl-mediated repression. Thepresence of the f2 gene product restores sensitivity to P7 su-perinfection. We believe that gpf2 interacts with sites thatregulate cl gene activity or with the cl gene product ratherthan with immI sites or products (Fig. 1) because we can detecta fragment 2-dependent reduction of gpcl-mediated re-pression of the PI ban gene in a cell lacking the imml region(data not shown).We thank R. Musso and R. Yuan for many enlightening discussions

during the course of this work and S. Shafer and C. Billman for diligentsecretarial assistance. This research was sponsored by the NationalCancer Institute under Contract NOI-CO-75380 with Litton Bionetics,Inc.

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5598 Genetics: Sternberg et al.