JOURNAL OF VIROLOGY, Aug. 1979, p. 288-298 Vol. 31, No. 20022-538X/79/08&0288/11$02.00/0

Functional Relationship Between Bacteriophages G4 and4X174

W. E. BORRIAS,* M. HAGENAAR, R. VAN DEN BREKEL, C. KUHLEMEIJER, AND P. J.WEISBEEK

Department ofMolecular Cell Biology and Institute ofMolecular Biology, State University of Utrecht,Padualaan 8, Utrecht, The Netherlands

Received for publication 29 January 1979

Mutants of bacteriophage G4 were isolated and characterized, and their mu-tations were mapped. They constitute six different genes, namely, A, B, E, F, G,and H. The functional relationship with bacteriophage OX174 was determined bycomplementation experiments using amber mutants ofOX and amber mutants ofG4. Bacteriophage 4X was able to use the products of G4 genes E, F, G, and H.In bacteriophage G4, however, only the OX gene H product was functional.

Bacteriophage G4 is a small isometric phageisolated in 1974 and is considered to be relatedto bacteriophage 4X174 (15). The two phagesare similar in size and shape and contain single-stranded DNA genomes of approximately thesame size, the G4 genome being 191 nucleotideslonger than the OX genome (17). They code fora similar series of about 10 proteins, as shownby their protein patterns after gel electrophore-sis; the electrophoretic mobilities of most of theproteins differ only slightly (15, 31). The moststriking difference between G4 and 4X174 is inthe first step of the process of DNA replication,namely, the initiation and the synthesis of thecomplementary strand. The complementarystrand synthesis begins in G4 at a single uniquesite both in vitro (45) and in vivo (22). In qX174the minus strand synthesis starts at multiplesites in vitro (27). In vivo, a preference may existfor one of the initiation sites (22, 46). Also, morehost proteins are needed for the synthesis ofcomplementary strand DNA of 4X174 than forG4, both in vitro (30, 43) and in vivo (12, 26, 33,34).G4 was isolated as a 4X-like phage to elucidate

the evolutionary relationship between the re-lated bacteriophages 4X174 and S13 (15). Im-portant parameters in this respect are theamount of serological cross-reaction, genetic re-combination, and complementation, all three pa-rameters being consequences of the base se-quence of the DNAs. In terms of evolution, it isimportant to know how much a protein canchange and still perform the same function. Al-though 4X174 and S13 show a rather high de-gree of base sequence divergency (11, 14), 4X174particles are inactivated by S13 antiserum (44),and in vivo both phages do recombine (38) and

complement each other in all but one of theirgenes (25).Comparison of the nucleotide sequences of G4

and 4X174 (17) showed an average of 33% nu-cleotide changes between the two phages. Re-combinant DNA molecules have been found(39). However, marker rescue between 4X174single-stranded DNA and G4 DNA fragmentswas found at only a very low level, and only inthe overlapping D-E region (25a). No markerrescue was found between 4X174 replicative-form (RF) I DNA and G4 single-stranded DNAfragments (21). This does not mean that recom-binant DNA molecules are very rarely forned,but that something else can go wrong on thelong way from the formation of a recombinantDNA molecule to the appearance of a recombi-nant viable phage.

In this work the relationship between 4X174and G4 in vivo was analyzed. We have recentlyobserved an in vitro relationship between OXand G4 in that the 4X174 gene A protein nicksnot only 4X DNA but also G4 RF I DNA at aspecific place in the viral strand at what provedto be the origin of replication. Also, the G4 Aprotein nicks both G4 and 4X174 RF I DNA (S.Langeveld, personal communication). Here wedescribe the induction, isolation, and character-ization of conditional lethal mutants of G4. Agenetic map was constructed, and functionalrelationships between the G4 and 4X174 geneswere analyzed by complementation experimentsbetween conditional lethal mutants of bothphage types.

MATERIALS AND METHODSMedia. The media used for the growth of phage

and bacteria have been described previously (6, 7).288

RELATIONSHIP BETWEEN G4 AND OX174 289

The same media were used for bacteriophages G4 and4X174. It should be mentioned here that G4 phagedoes not grow in Escherichia coli grown in 3XDmedium.

Bacterial strains. E. coli C (BTCC 122) was thestandard suppressorless (sup') host for titration andgrowth of wild-type (WT) and temperature-sensitive(ts) phage and for the complementation and rescueexperiments.

E. coli HF4712 (SUPUAG) (4) was used to grow andassay the amber and tourmaline (to) nonsense mu-tants.

E. coli HF4704 (sup' thyf uvrA) (4) was used forthe preparation of isotopically labeled phage-specificproteins and for the measurement of phage DNAsynthesis.

Shigella paradysenteriae Y6R (SUPUAG) was usedto make spheroplasts (6).

Bacteriophage. OX174 WT and mutants am86,aml8, am42, am3, and am9 were gifts of R. L. Sin-sheimer (2). 4X174 mutants amH57 and amNl weregifts of M. Hayashi (20); to8 is an amber mutantisolated in this laboratory (7). G4 WT was obtainedfrom G. N. Godson.

Induction and isolation of G4 mutants. Themethoxyamine mutagenization procedure has been de-scribed previously (9). To 1 volume of WT phage(concentration, 1010 particles per ml) 4 volumes of a1.25 M methoxyamine mixture was added. The reac-tion was halted by a 10OX dilution in 0.05 M Tris (pH8.0), with acetone added at a final concentration of 2%.The mutagenized WT phage was then screened forinduced mutants. Amber mutants were picked up withthe double-layer technique as turbid plaques at 300Cafter the mutagenized phage were plated together withE. coli HF4712 (SUPUAG) on plates that had beenoverlaid previously with E. coli C (sup'). The mutantsamA4, amB3, amB10, amG17, and amHll were iso-lated after a 10-4 inactivation at 370C, and the mutantsamA2, amF20, amG6, amG10, amG13, and amH17were isolated after a 10-2 inactivation at 250C. ThetsFl mutant was picked up after the 10-4 inactivationat 370C as a small plaque on E. coli C after 5 h ofincubation at 300C. The nomenclature of the mutantsis based upon the gene assignment (see below).The HNO2 mutagenization procedure (C. A. Hutch-

inson III, Ph.D. thesis, California Institute of Tech-nology, Pasadena, 1969) was used for the isolation ofan amber mutant in the lysis gene. To 1 volume ofWTphage (concentration, 1012 particles per ml) 9 volumesof 1 M mutagenization medium was added. The reac-tion was halted by dilution in 1.0 M Tris buffer, pH8.0, at 00C. Next, an enrichment procedure was appliedto the sample which had been inactivated to 5 x 10-3survival; this consisted of one growth cycle in E. coliHF4712 (SUPUAG) at 370C, followed by seven succes-sive growth cycles in E. coli C (sup') according to thefollowing procedure. The multiplicity of infection was0.1, and at 10 min after infection, G4 antiserum wasadded. At 60 min after infection, the infected nonlysedcells were centrifuged down, washed with TKB me-dium, and lysed with Iysozyme (final concentration,100 ,ug/ml) at 370C. Finally, one more growth cyclewas given in E. coli HF4712 (SUPUAG), and the super-natant was screened for amber mutants with the dou-

ble-layer technique at 370C. In this way amE2 wasisolated.Antiserum inactivation. To 9 volumes of phage

(concentration, 108 particles per ml) 1 volume of G4and -X antiserum was added, and the inactivationafter 5 and 10 min at 300C was determined. The Kvalues were about 2 for the OX antiserum and about0.4 for the G4 antiserum.Complementation. The method has been de-

scribed previously (8). The rescue experiments inwhich the G4 and OX WT phage were tested to deter-mine whether they could provide OX and G4 functions,respectively, were performed in the same way as thecomplementation experiments.

Protein synthesis. Preparation of labeled extractsof E. coli HF4704 (sup') after infection with G4 ambermutants, followed by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, was performed as de-scribed for OX174 (41), with the exception that 50 ,uCioflabeled compound was always used. Tritium-labeledproteins were made visible by fluorography (5), and35S-labeled proteins were made visible by direct auto-radiography of dried gels.DNA synthesis. The measurement of [3H]thymni-

dine incorporation into trichloroacetic acid-precipita-ble material has been described previously (8).Preparation of DNA. RF DNA was prepared by

the method of Jansz et al. (24), except that chloram-phenicol was added 4 min after infection. The circularclosed form (RF I) was isolated by a CsCl gradientcontaining ethidium bromide (28).

Single-stranded DNA was prepared directly fromthe lysate. To 1 ml of phage lysate (titer, at least 10"particles per ml in a low-salt solution), 0.25 ml ofRNase (1 mg/ml; preincubated for 12 min at 8000)was added. After 1 h of incubation at 370C, 0.05 ml ofproteinase K (10 mg/ml; preincubated for 30 min at370C) was added for another 30 min at 370C. Thismixture was extracted three times with distilledphenol previously saturated with 0.01 M Tris-0.001 MEDTA, pH 7.0. The water layer was immediately freedfrom phenol by extraction with chloroform-isoamylalcohol (24:1, vol/vol), followed by filtration over aSephadex G100 column equilibrated with 0.05 M Tris-0.005 M EDTA, pH 8.0. The DNA fractions were useddirectly in the fragment assay.Preparation of DNA fragments. WT double-

stranded G4 RF I DNA molecules were completelydigested with HindII restriction enzyme of Haemo-philus influenzae Rd, and subsequently the fragmentswere fractionated by gel electrophoresis (40), givingfive bands, A through E (16), of which band D consistsof two different fragments of the same size. The frag-ment bands were excised from the gel with a scalpeland pressed through a syringe. The fragments wereeluted with 0.05 M Tris-0.005 M EDTA, pH 8.0, andprecipitated by adding 2 volumes of 100% ethanol and0.1 volume of 3 M sodium acetate (pH 5.5) and coolingovernight at -20°C. After centrifugation for 30 min at23,000 rpm and -5°C, the fragments were taken up insmall volumes of 0.05 M Tris-0.005 M EDTA, pH 8.0.

Spheroplast assay. Spheroplasts of S. paradys-enteriae Y6R were prepared by the method of Guthrieand Sinsheimer (18), except for the use of PAM me-dium instead of nutrient broth (19). For the assay, 0.1

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290 BORRIAS ET AL.

ml of DNA was mixed with 0.1 ml of spheroplasts.After a 15-min adsorption period at 25°C, 0.8 ml ofPAM medium was added, and the suspension wasgrown for 2 h at 25°C. Then 0.1 ml of CHC13 wasadded, and the suspension was analyzed for progenyphage.Mutant mapping. The G4 amber mutants were

physically mapped with the bioassay for DNA frag-ments (42). Fragments of G4 WT RF DNA made bycleavage with the HindII restriction enzyme were an-nealed to mutant single-stranded DNA. These partialhybrid molecules were used to infect spheroplasts ofS. paradysenteriae Y6R. After a 2-h growth of theinfected spheroplasts at 25°C, the phage yield wasdetermined on E. coli HF4712 (total yield) and E. coliC (WT). The rationale behind the experiment wasthat if the mutation being studied was covered by afragment carrying the WT allele, the fraction of WTparticles in the yield after spheroplast infection (thatis, the ratio ofWT to total) would be increased mark-edly.

RESULTSIsolation of mutants. In general, a temper-

ature of 30°C was chosen for the isolation ofamber mutants because of our experience with4X174, in which several amber mutants are alsots, apparently because the suppression mecha-nism in the Sup host inserts a non-WT aminoacid, giving rise to a missense protein. At firstwe tried to isolate mutants by the method ofdirect mutagenization of specific DNA frag-ments (9). To our surprise, this method did notwork with G4. Later the reason for this becameclear, namely, that the mutagenized DNA hadbeen passed through spheroplasts of E. coli K58at 300C, as was the normal procedure for OX174.However, under these conditions, G4 mutantDNA, and to a lesser extent also G4 WT DNA,shows a very poor activity. The infectivity of theDNA can be improved drastically by usingspheroplasts of S. paradysenteriae Y6R at 25°C.Both host and temperature contribute to theimprovement (see below). Meanwhile, we iso-lated mutants by random mutagenization ofWTphage particles with methoxyamine or HNO2.All mutants formed plaques at 20°C, one of thecharacteristics of G4 (15).Serology. Figure 1A shows the inactivation

of G4 and 4X174 WT with antiserum againsteither 4X or G4 phage. Clearly, G4 phage wasnot inactivated by OX antiserum, in agreementwith earlier experiments (15). 4X phage, how-ever, was inactivated by G4 antiserum. The G4antiserum therefore cannot be used to discrimi-nate between 4X and G4 phage. Figure 1B showsfor several G4 mutants that the mutants reactedin the same way with the G4 and OX antisera asthe G4 WT did; i.e., there was good inactivationwith G4 antiserum and no or little inactivationwith 4X antiserum. Only amB3 and amE2 wereslightly more sensitive to OX174 antiserum than

Time (min)

FIG. 1. Antiserum inactivation of (A) 4X and G4WT phage and (B) G4 mutants. To 9 volumes ofphage (108 particles per ml) I volume of antiserumwas added, and the inactivation at 30°C was deter-mined. Solid lines, 4OX174 antiserum; dashed lines,G4 antiserum. (A) Symbols: 0, dIX174 WT; E, G4 WT.(B) Symbols: , amA4; 0, amB3; A, amE2; V, amF20;A, amGl7; *, amHl7.

the other G4 mutants were, but their inactiva-tion pattems still resembled those of G4.Complementation. Complementation ex-

periments revealed that our collection of G4mutants comprised five complementationgroups. However, on the basis of our protein gels(see below), it was apparent that group V (Table1) contained mutants belonging to two differentgenes. That they nevertheless constitute onecomplementation group can be explained as aconsequence of polarity. Representatives of allsix genes are included in the experiments de-scribed below.Our criteria for intergenic complementation

are a burst size greater than one and at least 10times greater than the self burst size. Table 1shows the result of mutual complementation ofrepresentatives of each of the six genes. In antic-ipation of their gene assignments, throughoutthis paper the mutants are named by the genein which they are located. The letter stands forthe corresponding gene in 4X174 (2), and thenumeral stands for the isolation number. Themutants complement each other, except amG17and amH17. We ascribe this to a polarity effectof gene G on gene H, as described for the relatedphages S13 (37) and 4X174 (3).Complementation of the other G4 mutants

with these six representatives gives the distri-bution shown in Table 2.Gene products. To determine in which pro-

J. VIROL.

RELATIONSHIP BETWEEN G4 AND OX174 291

TABLE 1. Complementation between representatives of six genes of bacteriophage G4a

Comple- Burst size after complementation with:mentation Mutant

group amA4 amB3 amE2 amF20 amG17 amH17

I amA4 0.20 26 77 28 2.5 95II amB3 0.04 106 23 21 163

III amE2 1.2 37 152 78IV amF20 0.40 25 14V amG17 0.03 0.04

amH17 0.01a E. coli C (sup') cells were grown with aeration to log phase, centrifuged, and resuspended in fresh medium

to a concentration of 10' to 5 x 10' cells per ml. At 10 min after the addition of KCN (final concentration, 0.003M) the phage mutants were added at a multiplicity of 3 each in the mixed infection and of 6 in the self bursts.After a 10-min adsorption period the unadsorbed phage were inactivated by incubation with G4 antiserum for10 min. The adsorption mixture was diluted 104 times in prewarmed medium, and after a 1-h growth periodCHC13 was added (except in the case of amE2). The yield of mutant particles was determined by plating on E.coli HF4712 (SUPUAG). The experiment was done at 30°C.

TABLE 2. Distribution of the G4 mutants over thegenes

Comple-

tion Gene Mutant(s)group

I A amA2, amA4II B amB3, amBlO

III E amE2IV F amF20, tsFlV G amG6, amGlO, amG13, amG17

H amHll, amHl7

tein the mutants of each gene are defective, weanalyzed their protein patterns under restrictiveconditions. E. coli HF4704 (sup' uvrA) cellswere treated with mitomycin C (to prevent lysisand to reduce host protein synthesis) and in-fected with mutant phage. [3S]methionine or

[3H]leucine was added 25 or 40 min after infec-tion. After the infected cells were washed andlysed, the labeled extracts were electrophoresedon 15% acrylamide gels. For representatives ofeach complementation group the results afterautoradiography ([35S]methionine) and afterfluorography ([ H]leucine) are shown in Fig. 2and 3, respectively. Two different radioactiveamino acids were used to diminish the chance ofmissing a protein. The G4 viral proteins are

given a gene assignment that is based only uponcomparison with the known molecular weightsof the 4X174 proteins. Our interpretation ofthese protein patterns is as follows. (i) In amA4no distinct protein band is missing. (ii) In amB3a distinct band between proteins G and D ismissing. This is probably the B protein (31).Also one of the low-molecular-weight proteins(LMW-4; Fig. 2A) is missing. (iii) In amE2 no

distinct protein band is missing. (iv) In amF20the F protein band is missing. There is a newband (molecular weight, approximately 38,000)

between proteins F and H. (v) In amG17 the Gand H protein bands are missing. Also missingis a distinct band, X, just above the G proteinband. (vi) In amHll the H and X protein bandsare missing. There is a new band (molecularweight, approximately 15,000) between proteinsB and D (Fig. 2).The mutants amA4 and amE2 with protein

patterns indistinguishable from those of the WThad to be characterized further.Characterization of amA4. On most of our

gels no protein comparable to the OX gene Aprotein was consistently detectable, not even inthe G4 WT-infected cells. On the basis of a fewprotein gels, it seemed that amA4 might bemissing the A protein. Therefore, we had to useother methods to prove this point. In 4X174,gene A mutants show two properties which areeasy to test, namely, a complete asymmetry incomplementation and a complete lack of repli-cation under restrictive conditions (35, 36). Weinvestigated both aspects for mutant amA4. Totest the complementation behavior, we isolateda ts mutant in gene F (tsFl) and determined inmixed infections of this tsFl mutant and amA4at restrictive conditions the yield of each mutantin the progeny phage burst. As a control, amB3was also tested in this way. The results areshown in Table 3. It is clear that the burst fromthe mixed infection with amA4 and tsFl con-sisted completely of the ts mutant, whereas inthe control both amB3 and tsFl were propa-gated. amA4 therefore complements asymmet-rically.The DNA synthesis at restrictive conditions

was measured by the incorporation of [3H]thy-midine into trichloroacetic acid-precipitable ma-terial in cells pretreated with mitomycin C tosuppress host DNA replication. As a controlamB3 and G4 WT were included in the experi-ment. The experiment was done in the presence

VOL. 31, 1979

292 BORRIAS ET AL.

x- _t 4.S ,;,;1 G- _

B - a*LW-

L.MW-2LMW3 :. J

LMW-4 -

FIG. 2. Autoradiograph ofelectrophoresis on a 15% acrylamidegel of35S-labeled extracts ofphage-infectedcells. Log-phase E. coli HF4704 (sup' uvrA) were pretreated with mitomycin C at 300 pg/ml, washed,resuspended in fresh medium (final concentration, 4 x 108 cells per ml), and aerated for 5 min at 37°C. Thesecells (0.5 ml) were infected with phage (multiplicity of infection, 10 to 50). At 25 min after infection, 50 ltCi of[35SJmethionine was added, and 95 min later the infection was stopped. The cells were centrifuged, washed,and resuspended in 30 ILI of lysis buffer. These suspensions were kept for 4 min at 1000C and then used for gelelectrophoresis. The electrophoresis was at 200 V. Migration was downwards. Superscript a, Molecularweights (M. W.) according to Godson (14); superscript b, molecular weights according to Burgess and Denhardt(10); superscript c, molecular weights according to Siden and Hayashi (32); superscript d, molecular weightsaccording to Gelfand and Hayashi (13). LMW-I through -4 are low-molecular-weight proteins.

and absence of chloramphenicol. In the presenceof chloramphenicol the synthesis of single-stranded DNA is inhibited, but the synthesis ofprogeny RFDNA is not affected (23, 36). Figures4A and B show that amA4 did not synthesizeDNA whether chloramphenicol was present or

not. The WT and amB3 under both conditionsshowed considerable amounts ofDNA synthesis.Therefore, both asymmetric complementation

and the lack of DNA synthesis under restrictiveconditions demonstrate that amA4 is a gene Amutant. This also supports the assumed func-tional similarity between the G4 and 4X174 Agenes.Mutant amB3 showed the replication pattern

that is expected of a mutant blocked in thesynthesis of single-stranded DNA. Again, this isin agreement with the expected function of gene

1, .. ... i.,

.:.., %.'-. , ;%:i"t ;k..

4, :.

A PI

J. VIROL.

RELATIONSHIP BETWEEN G4 AND 4X174 293

0 r- -st In V-

g < m E 0It E E E E E

:' 4.4f ,:": .. ...o *

G4

F-

H-

X-

G-B-

D-LMW-1 -

LMW-2-LmW-3

LMW-4

FIG. 3. Fluorograph of electrophoresisacrylamide gel of 3H-labeled extracts offected cells. The method described in theFig. 2 was used, except that [3HJleucine u

40 min after infection at 30°C. The elect)was at 300 V.

B when compared with 4X174.Characterization of amE2. The p

which led to the isolation of amE2 (seaimed at the isolation of a mutant ingene. One would expect that such awould not lyse its host under restrictiitions and that the number of intracelluparticles would increase with time to a ]

is the same or even higher than that ofWe measured this under conditions oflow multiplicity of infection. At high muwe monitored the infection with a niand a spectrophotometer. We found t120 min after infection at 370C the celllyse and that the optical density at 70(still increasing, probably because thecells increased in size. The cells infecte(WT showed lysis and a simultaneous

in optical density at 700 nm by 12 min afterCo infection.

At low multiplicity we determined the burstsize under restrictive conditions (in E. coli C at30°C). At 60 min after infection, the burst size,measured after CHC13 treatment of the infectedcells, was about 50 for the mutant and 500 for

oX the WT. This is contrary to our expectation thatthe burst size of the mutant should be at least of

_ ; the same order of magnitude as that of the WT.The reason for this could be that, analogous tothe D-E overlap in 4X174 (1), the nonsense

H codon in the E frame causes a missense codon in

the D frame, the latter not leading to conditionallethality but to a lowered capacity to reproduce.In view of the facts that (i) amE2-infected cellsdo not lyse at the normal time, (ii) a burst sizeof 50 under restrictive conditions is very high foran amber mutant, and (iii) amE2 is very easy to

-G grow to a high-titer lysate, we conclude thatB amE2 contains a nonsense mutation in the lysis

gene.

- D Localization. Representatives of each of thesix genes were mapped with the bioassay for

DNA fragments (42) (see above). We tested onlythe HindII restriction enzyme fragments (Table4). Mutants amA4 and amB3 are covered byfragment C, amE2 is covered by fragment E,amF20 is covered by fragment B, and amG17and amH17 are covered by fragment A. Anothergene A mutant, amA2, maps on fragment D2.The order of the G4 HindII fragments is C, E,D1, B, A, D2 (16). The mapping results, togetherwith the known order of these restriction frag-

on a 15% ments, reveal the order of the genes. Taking intophage-in- account that gene G exerts polarity on gene H,legend to the gene order is A, B, E, F, G, H, A. Thus, thevas added gene order in G4 is similar to that in 4X174.rophoresis This is in agreement with the order of the genes

established by comparison of the nucleotide se-

quence of the G4 DNA with the N-terminalrocedure amino acid sequences of the G4 WT proteins'e above) (17). Figure 5 shows the location of the muta-the lysis tions on the G4 gene map.

i mutant Functional relationship between G4 andve condi- 4X174. Is there any functional relationship be-lar phagelevelthat TABLE 3. Complementation of tsFlaF the WT.high andltiplicityicroscopehat untilIs did notnm was

,infectedd with G4decrease

Burst size Burst sizeAmber mu- (mixed infection) (unmixed infections)

tantts Amber ts Amber

amA4 23 <0.1 1.3 0.11amB3 16 18 1.3 0.01

aThe experiment was performed as described inTable 1, footnote a, except that the temperature was37°C. Analysis of the burst size was on E. coli HF4712(amber and ts) and on E. coli C (ts).

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294 BORRIAS ET AL.

15 30 45Time (min)

60 80 5 1*5 30 45Time (min)

FIG. 4. Measurement of DNA synthesis. E. coli HF4704 (thyf uvrA sup+) was grown in TPG mediumcontaining 200 ug of Casamino Acids per ml and 2,ug of thymine per ml to a concentration of2 x 10' cells perml and treated with mitomycin C (50 pg/ml) for 25 min at room temperature in the dark to block host DNAsynthesis. The cells were centrifuged and resuspended in fresh TPG medium containing 200 jig of CasaminoAcids per ml and 0.5 jig of thymine per ml, with 35 ytg of chloramphenicol (CAM) per ml (B) or without CAM(A). After a 10-min incubation at 30°C, [3H]thymidine (41 Ci/mmol) was added to a final concentration of 5,uCi/ml simultaneously with the phage (multiplicity of infection, 5). DNA synthesis was measured by theincorporation of[PHithymidine into trichloroacetic acid-precipitable material. The precipitation was accom-plished by adding 100 ug of bovine serum albumin as a carrier to a 0.5-ml sample. After 20 min in the cold, theprecipitate was spun down and washed twice with cold trichloroacetic acid, solubilized in 1 ml of 0.55 NKOH, and counted for radioactivity in a liquid scintillation counter. A correction was made for noninfectedcells. Symbols: 0, WT; O, amB3; A, amA4.

TABLE 4. Mapping of G4 amber mutants with restriction enzyme fragmentsaHindII restriction fragment

MutantA B C DD E Noneb

amA2 8.3 x 10-5 1.2 x 1O-5 5.9 x 10- 1.3 x 10-2 7.8 x 10-5 6.6 x 10-5amA4 2.9 X 10-5 8.7 x 10-4 4.6 x 10-2 2.3 x 10-4 2.5 x 10-5 2.3 x 10-5amB3 5.5 X l0-5 2.9 x 10-3 1.9 X 10-2 2.8 x 10-4 5.5 x 10-5 3.2 x 10-5amE2 2.3 x 10-5 1.7 x 1O-4 2.3 x lo-5 2.9 x lo-5 2.9 x 10-2 6.2 x 10-5amF20 1.2 x 10-4 2.2 x 10-2 5.1 X 10-4 7.4 x 10-4 1.2 x 10-3 1.3 x 10-4amG17 1.9 X 10-2 4.8 x 10-4 1.1 X 10-3 1.4 x 10-4 1.1 X 10-4 5.9 x 10-5amH17 1.2 x 10-2 1.2 x 10-4 1.6 x 1O-5 1.5 x 10-5 6.2 x 10-5 1.2 x 10-5a WT HindII restriction fragments were annealed to single-stranded DNA of amber mutants representing

each of the six G4 genes. The partial hybrid molecules were added to spheroplasts of S. paradysenteriae Y6R,and after 2 h of growth at 25°C the yield was analyzed. The ratio ofWT to total phage was determined for eachmutant-HinduI restriction fragment combination.

'Values in this column represent the reversion frequency of the DNA preparation.

tween G4 and OX; i.e., can G4 complement adefective OX gene and vice versa? We first askedwhether OX WT could provide any functions toG4. Strains with mutations in individual G4genes were tested together with OXWT in mixedinfections of E. coli C at 300C. The yields were

analyzed at 20 and 300C by using the double-layer technique to discriminate among G4 ambermutants (turbid plaques at 200C), G4 WT (clearplaques at 20°C), and OX WT (clear plaques at300C). In all cases tested OX WT failed to give

any rescue of the G4 mutant. Next, the recipro-cal test was done by using the corresponding OXmutants and G4 WT. Although analysis of theyield was not as clear-cut as in the first case

(because the yield consisted mainly of G4 WT,which grows at all conditions where the 4Xmutants grow), it was evident that G4 WT res-

cues 4Xam3 (gene E) and 4XamNl (gene H).Both of these OX mutants comprised at least20% of the progeny, as measured by the double-layer technique at 300C.

A-CAM

El

B+CAM

10-

9

8

.7

6

>s.

.D 4

I1') 3.

2.

0.

60

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RELATIONSHIP BETWEEN G4 AND OX174 295

FIG. 5. Localization of the G4 mutations. The G4mutations were mapped with the bioassay for DNAfragments by using HindII restriction fragments. Theinner circle represents the G4 gene map as deter-mined by Godson et al. (I 7). The outer circk showsthe HindII restriction enzyme map (16, 17). ForamF2O and amHll the sites of mutations within thefragments concerned were determined more accu-

rately from the sizes oftheir amberprotein fragments.

We conclude that G4 WT is able to rescue atleast two OX mutants, whereas OX WT fails torescue the G4 mutants. Of the OX mutants thatwere rescued the best by G4 WT, fXamNl (geneH) was tested for complementation with G4amber mutants (Table 5). In those cases whereno complementation could be expected, 4Xam3(gene E) was used instead of OXamN1 (gene H).It should be noted here that complementationbetween a mutant in the lysis gene E and amutant in any other gene constitutes true com-

plementation only when the non-E mutant isrescued, because the lysis function is not neededfor the synthesis of viable phage. If SX and G4proteins are interchangeable, then one wouldexpect that complementation would occur be-tween the OX mutant in gene H and the G4mutants in genes A, B, E, and F, but would notoccur with G4 amber mutants in gene H norwith the gene G mutant, which is polar on geneH. Mutants of G4 genes G and H were tested incombination with OXam3 (gene E). Table 5shows that 4XamN1 (gene H) complementedremarkably well with G4amA4, resulting in asignificant increase of 4XamNl, whereas com-plementation between 4XamNl and G4 mutantsin genes B and F was very poor. Complementa-tion with the G4 mutant of gene E cannot bemeasured because of the very leaky character ofthis lysis mutant. As expected, mutants in G4genes G and H did not complement with4OXamN1 (gene H), but G4amH17 was rescuedby rXam3 (gene E). When each burst of themixed infections (Table 5) was analyzed for the

yield of each mutant in it, it was found that theincrease in the yield was due to an increase inthe OX mutant and not to an increase in the G4mutant, except in the case 4Xam3 (gene E) xG4amH17. In this case the G4 H mutant showedan increase compared with unmixed infection.Yet 80% of the yield consisted of the 4X mutant.These complementation experiments againshow that G4 is able to provide certain functionsto 4X. We conclude that (i) the defective OXgene H is complemented best by a G4 ambermutant in gene A and (ii) the only G4 functionwhich OX can provide is the gene H function.This was tested further by complementing theG4 amber A mutant with OX amber mutants inseven different genes and by complementing a4X amber A mutant with representatives of thesix G4 genes (Tables 6 and 7). Both G4amA4and 4Xam86 did not appear in the progeny sincegene A mutants were not rescued. Therefore,where complementation was found, the higherburst size from the mixed infection (comparedwith the yield from unmixed infections) wascompletely due to an increase in the yield of thenon-A mutant. In this complementation assay itwas possible to test accurately which 4sX proteinwas functional in G4 infection and vice versa,except in the case of A proteins. It is clear fromTable 6 that G4 proteins E, F, G, and H canfunction in the OX infective cycle, but that theG4 proteins B and D cannot. Table 7 shows alsothat 4X proteins B, F, and G cannot function inthe G4 infective cycle, whereas OX protein Hcan do so. Therefore the only protein which isdemonstrably cross-functional in vivo between4X and G4 is the gene H product.

TABLE 5. Complementation betweenOXI 74 and 04eBurst size Burst size

Mutants tested (mixed infec- (unmixed in-tion) fections)

4,X174b G4 OX174 G4 OX174 G4

amNl (H) amA4 17 0.96 0.48 1.4amNl (H) amB3 1.3 0.49 0.48 0.49amN1 (H) amE2 <0.05 5.5 0.34 4.9amNl (H) amF20 1.2 0.37 0.41 2.3amNl (H) amG17 0.49 0.01 0.48 0.04amNl (H) amH17 0.45 0.05 0.48 0.05am3 (E) amG17 2.2 <0.02 0.16 0.43am3 (E) amH17 5.4 1.1 0.16 0.32a The complementation was performed as described

in Table 1, footnote a. Analysis of the burst aftermixed infection was done by plating the progeny onE. coli HF4712 at 300C (G4 and OX mutant and WT)and at 200C (G4 mutant and WT) and on E. coli C at300C (G4 WT and oX WT) and at 200C (G4 WT).Corrections for efficiency of plating were made.

b The letters within parentheses represent the4X174 gene (see reference 2).

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296 BORRIAS ET AL.

TABLE 6. Asymmetric complementation between4X174 and G4a

Burst sizeMutants tested Burst size (unmixed infec-

(mixed tions)infection)

*X174 G4 *X174 G4

am86 (A) amA4 0.97 1.4 2.4to8 (B) amA4 0.90 0.55 1.6am42 (D) amA4 1.6 1.2 1.6am3 (E) amA4 5.8 0.12 1.4amH57 (F) amA4 10 0.85 1.5am9 (G) amA4 10 0.20 1.6amNl (H) amA4 18 0.40 1.4

a See Table 5, footnotes a and b.

TABLE 7. Asymmetric complementation betweenOXI 74 and G4a

Brt Burst sizeMutants tested Buzet (unmixed infec-

(mixed tions)

*X174 G4 infection) OX174 G4

am86 (A) amA4 0.97 1.4 2.4am86 (A) amB3 0.10 0.07 0.03am86 (A) amE2 4.4 0.12 4.8am86 (A) amF20 0.02 0.07 0.01am86 (A) amG17 0.04 0.07 0.01am86 (A) amH17 5.3 0.10 0.03a See Table 5, footnotes a and b.

DISCUSSIONMutants in six different G4 genes were isolated

by direct mutagenization of G4 WT phage par-ticles. The mutants have been verified as G4mutants in three ways: (i) by their ability togrow at 200C, a property that no 4X mutant (orWT) possesses, (ii) by their inactivation patternwith G4 and 4X antiserum, and (iii) by analysisof the viral proteins.The antiserum inactivation patterns show

that the best discrimination between 4X and G4mutants is the reaction with the OX antiserum;4X antiserum does not inactivate G4 WT andmutants or does so only slightly, whereas itinactivates OX WT and mutants at a muchhigher rate. G4 antiserum inactivates OX parti-cles at a rate which is comparable to that of theG4 antiserum reaction with the different G4mutants (Fig. 1).The results of the complementation experi-

ments and the protein gel patterns show thatour collection of G4 mutants represents sixgenes. The assignment of mutants to genes B, F,G, and H was done on the basis of missingprotein bands on the gels, by analogy with theOX protein pattern. Assignment to gene A wasdone on the basis of a completely asymmetriccomplementation behavior and lack of DNA

replication under restrictive conditions, and as-signment to gene E was done on the basis of ahigh burst size under restrictive conditions andthe absence of lysis of the infected cells.Our G4 protein pattern on a 15% acrylamide

gel is slightly different from the one shown byShaw et al. (31), in that the G4 H protein runsslightly faster than the 4X H protein at lowvoltage (200 V) and the G4 G protein runsslightly slower (Fig. 2), which is in agreementwith the molecular weights stated by Godson(15). There are at least four proteins which runfaster than the D protein, which we called thelow-molecular-weight proteins (LMW) 1through 4. In our gels amB3 lacks, besides the Bprotein, the LMW-4 protein. The nucleotide se-quence of G4 does not show an extra proteininitiation site (17) in gene B, so amB3 may be adouble amber mutant with the second ambermutation in another gene, or gene B may bepolar on another gene. amHll lacks, besides theH protein, the very distinct protein band X, andin some gels it also lacks three proteins whichrun between H and X proteins. These extramissing proteins have molecular weights of atleast 20,000. The length of the possible extraproteins, as concluded from the nucleotide se-quence (17), is at most 101 amino acids. Thiscannot make up a protein of 20,000 molecularweight, so these missing proteins may be phage-induced host-coded proteins.Mutations of each gene were mapped by the

bioassay for DNA fragments (42). Their positionon the G4 gene map (17) is given in Fig. 5. Theexact position of each mutation within its restric-tion fragment was not determined. The positionof the mutations amF20 and amHll within theF and H genes, respectively, can be deducedfrom the size of the amber protein fragments.

In the spheroplast assay the G4 DNA ap-peared to be much more active in S. paradys-enteriae Y6R than in E. coli K58 (about 100times). It is very likely that E. coli K58 has arestriction enzyme that is active on G4 DNA. Itis surprising that the yield after spheroplastinfection was so much higher at 250C than atelevated temperatures (our standard tempera-tures for 4X single-stranded DNA are 30 and370C), especially for the mutant DNAs, whereasthis does not hold true for the infection of viableparticles in normal cells. The yield of WT phagefrom infected spheroplasts at 25°C is about 100times greater than that at 370C. For mutantsthe difference can be 103 to 104. Our only expla-nation for this is that the G4 DNA may beintracellularly much more temperature labilethan the OX DNA. The time of exposure toinactivation may be longer for amber mutants

J. VIROL.

RELATIONSHIP BETWEEN G4 AND 4X174 297

under sup+ conditions than for the WT.Comparison of the nucleotide sequence ofOX

(29) with that of G4 (17) makes it clear that theoverall organization of the G4 genome is thesame as that of 4X174. One important questionis whether the proteins in OX and G4 that aresupposed to have the same functions (based oncomparable schemes for overall replication andmaturation) are able to complement each other.Complementation experiments between a G4amber mutant in gene A and 4X amber mutantsin seven different genes showed that the OXamber mutants in genes E, F, G, and H werecomplemented by the G4amA mutant. So thereis a functional relationship for the genes E, F, G,and H. Complementation experiments betweena 4)XamA mutant and G4 amber mutants ineach of six genes showed that only the G4 ambermutant in gene H was complemented by OX. Sothe only protein which is cross-functional in vivois the gene H protein.The results of the G4-4X rescue and comple-

mentation experiments leave many problems tobe solved and are in some aspects contradictory.Some of these problems are listed below. (i)G4amH17 is complemented by 4Xam86 (A) and4Xam3 (E), but not rescued by fX WT. (ii) Astriking aspect of the rescue experiments is thatthe yield of G4 WT particles was not affectedvery much by coinfection with a <0X amber mu-tant, whereas the yield of OX WT was lowereddrastically (by a factor of 10) by coinfection witha G4 amber mutant. (iii) The OX mutants ingenes F and G are complemented by G4amA4,but G4 mutants in genes F and G are not com-plemented by 4Xam86 (A). Several phenomenamay contribute to these problems: (i) phenotypicmixing-during the G4-4X coinfection phageparticles may be formed with a mixed G4-4Xcoat; perhaps the extent of mixing of the G4 and4X coat proteins influences the capacity of theseparticles to adsorb to the host cell; (ii) it isknown that the infective cycle in G4 is shorterthan that in 4X. Maybe the replication cycles ofG4 and 4X are so much asynchronized that atthe moment that, e.g., G4 needs a OX function,the OX protein is not in a high enough concen-tration at the right place within the criticalperiod.We found that several OX functions could be

provided by G4, whereas only one of them (geneH function) was cross-functional in vivo. Al-though the two phages are clearly related, theirproteins differ considerably.

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