Recombination of bacteriophage phi X174 by the red function of bacteriophage lambda.

0022-538X/79/02-0438/05$02.00/0

Recombination

of

Bacteriophage

pX174

by

the Red Function

of

Bacteriophage X

REIKICHIMUNEKIYO* ANDMUTSUO SEKIGUCHI

Department ofBiology, Faculty of Science, Kyushu University 33,Fukuoka812,Japan Received for publication1November 1978

Recombination ofbacteriophage 4X174 was effectively promoted when the Red function ofA wassupplied by either co-infection with A or induction ofA lysogens. Mutations in reda and redfl genesofAabolished recombinationnearly completely, whereasa mutation ingamgenereduced itonly slightly. The

Red-promoted recombination of 4X174 occurred in recA, recB, andpolAmutantsas

well as inwild-type strainsofEscherichia coli. Itwasfurtherstimulated when

OX174

mutantswereirradiated with UVlight before infection.

Bacteriophage 4X174 and its

closely

related

bacteriophage S13 are the smallest viruses to

undergo

genetic

recombination

(11, 20),

andthe

mechanism has been analyzed extensively by

both genetic and physical methods. Recently,

"figure-8" molecules

consisting

oflinked

mono-mersof two double-stranded parental genomes

wereisolatedasrecombinationintermediates (4,

23). Most4X174 recombinantsareformed

by

a

major

pathway,

which requires the host recA+

function (21, 22) but

apparently

does notneed

anyof the known4X174geneproducts (3).

In the course of studies on genetic

recombi-nation of 4X174 under various

conditions,

we

found that recombination

frequencies

of

4X174,

which are usually low compared to those of

other

organisms,

increase

significantly

when the

bacteria are co-infected with

bacteriophage

A.

Subsequent

analysis

revealed that

recombina-tion of 4X174 can be

promoted

by

the A Red

function and that the contribution of the Red

pathway

in4X174recombination isgreaterthan

that of theRecpathway.

MATERIALS AND METHODS

Bacteria. The bacterial strains used in these ex-perimentsare listed inTable1. RY14was obtained by a crossofAB2470 (5) with KD15(+X')(10).

Bacteriophage. 4X174 wild type and its amber mutants, aml8, am86, am3, and am9, were obtained from R. L.Sinsheimer, and amNl was from M. Hay-ashi. aml8 and am86 carry an amber mutation in cistron A, am3 in cistron E, am9 in cistron G, and amNl incistron H (2).

AcI857bio69 and AcI857biol6 were provided by K. Shimada. The deletion of bio69 covers both int and redgenes, andthe deletion ofbiol6 covers only int gene(9).AcI857red314 (which is reda-),AcI857redll3 (redfl-),andAcI857gam210(gam-)were gifts from Y. Sakaki (14).

Media. Thecompositions of KC broth, starvation

buffer, bottom agar, and top agarwere asdescribed by Benbow et al. (2). To facilitate adsorption ofA, KC brothwassupplementedwith 0.01 MMgSO4and 0.2% maltose andwasdesignated KCA broth.

Crosses of 4X174mutants.Bacteria were grown inKCA brothto 108 cells per mlat370C with gentle aeration. The culture was made0.003 M KCN and aerated for 10 min. Two 0.5-ml samples of2 x 108 4X174 mutantphageper mlin KCA broth containing 0.003 M KCNweremixed inamating tube in an ice bath. Anappropriate concentration of Aphage suspen-sion(usually less than 0.1ml)wasaddedtothemating tube. A 0.2-ml amount of theKCN-treated bacterial culturewasaddedtoeachmatingtube and incubated at37°Cfor15min. Then the mixturewasdiluted 100-fold intoKCA broth and incubatedat370C with vig-orousaeration. After90min ofincubation,afewdrops of chloroform wereadded to theculture, and phage

titers were determined by platingwith Escherichia coli strain HF4714(fortotal number of

OX174

phage),

strain C (for number of 4X174 wild-type recombi-nants), and strain C600 (for number of A phage). Though strains HF4714 and C were susceptible to both 4X174 andA, itwaspossibletotiter 4X174 in the presence of A since 4X174producedlargeplaqueseven afterashort timeof incubation(e.g.,3h),whereas A

didnot.InsomeexperimentsA-resistant derivatives of

strainsC and HF4714 isolated in thislaboratorywere

used,andessentially thesameresultswereobtained. Strain C600, a derivative ofK-12, was resistant to

4X174 infection and was used for titering A. Since C600 is deficientinrestrictionandmodification

func-tions,itwaspossibletotiterA grown inE.coliC. The recombination frequency between am mutants was definedasthe titer onstrainC divided by the titer on strain HF4714. Controlexperiments with one type of 4X174 mutant (selfimg) showed a frequency of less than lo-5wildtype per totalprogeny phage.

Crosses of4X174mutants in induced X lyso-gens. Thegrowth ofbacteria, treatment with KCN, and adsorption of4X174 were carried out at 330C,

whichprevented the induction of AcI857. Portions (0.1 ml) of themating mixture, without the addition of exogenousA,wereaddedto twotubes, each of which 438

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RED-PROMOTED RECOMBINATION OF OX174 439 TABLE 1. Bacterial strains

Strain Relevant characteristic Source and refer-ence C Wildtype, -X', Su- M. Hayashi HF4714 arg his leu thr pro -4X'Su' R. L. Sinsheimer

(3)

HF4712 recA13; isogenic to HF4714 R. L. Sinsheimer (3)

H560 polAI endoI thy str OX' R. L. Sinsheimer

Su- (3)

RY14 recB21 thr leu thi lac gal This laboratory aramtl pro his arg str

,XO Su+

C600 thr leu thi lac tonA oXr K.Matsubara Su+

contained 10ml of KCbroth;onetubewasincubated

at32°Candanother at40°C.Otherprocedureswere

essentiallythesame asdescribedabove.

Irradiation with UV. 4X174 phage were

sus-pended instarvation bufferat 109 phage perml and

irradiated with a 15 W Toshiba germicidal lamp to giveadose of 10J/m2 (about 10% survival).

RESULTS

Stimulation of 4X174 recombination by A co-infection. The growth of

OX174

is not

suppressedby co-infection withA.The burst size

of 4X174 in mixed infection with 4X174 and A

wasslightlyhigher than that in single infection, whereas the burst size of A was considerably reduced inthe mixedinfection. These situations allowedustoinvestigate theeffect ofAinfection

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on recombination of 4X174 in various host strains.

Table 2showsrecombination frequencies for severalambermutantscrossedpairwise inrecA+

and recA- cells withor without Ainfection. In eachcrosstherecombinationfrequencywas sig-nificantly lower in recA- cells than in recA+

cells,in agreementwithprevious results (21,22).

Infection withAcaused asignificant increase

in the recombination frequency in both recA+ and recA-cells. Almostthesamehigh levels of

recombinationwereattainedin thetwo typesof cells oninfection withA, and thusthe ratios of

recombination frequency in A-infected cells to that in noninfected cells were higher in recA-cells than inrecA+ cells.

The stimulation of

OX174

recombination by Ais furtherillustrated inFig.1.Withincreasing multiplicities of infection withA,recombination

frequencies of 4X174 in recA, recB, andpolA

mutants as well as in the wild-type strain in-creased. Itseemsthatthehost functions suchas those controlled by the recA, recB, andpolA

genes are not required for the A-promoted

re-combination of

OX174.

However, there remains apossibility that exonucleaseV (the recB and -C gene product) and polymerase I (thepolA

gene product) are concerned in the

recombina-TABLE 2. Stimulationof

OX174

recombination by A co-infectiona

Recombinationfrequency (x10-4) on:

Crossb

recA+cells recA-

cells

-A +A Ratio -A +A Ratio

am3(E)xam9(G) 4.2 57 13.6 0.56 41 73 am3(E)xamNl(H) 8.7 50 5.7 0.77 38 49 aml8(A)xam86(A) 12 -c - 0.84 59 70 am86(A)xamNI(H) 1.7 - - 0.12 10 83

am3(E)xaml8(A) 2.2 - - 0.24 17 71

'Crosses were performed as described in the text, using HF4714(recA+) and HF4712(recA-)as hosts. Forcoinfection, Aphagewasaddedtogiveamultiplicityof infection of5.

Inparentheses are the cistron for each mutant. -, Not tested.

100

50l f

0

O 5 10 15

MOI of A

FIG. 1. EffectofAinfectiononthe recombination

of fX174invarious hosts. Bacteriawereinfectedwith a mixture of4X174 mutants am3and am9 and

si-multaneously with Xc1857at various multiplicities.

Otherprocedures were asdescribed in the text. (0) HF4714(recA+);(0)HF4712(recA); (A)RY14(recB); (F) C(poLA,Su-);(U) H560(polA,Su-).

tionprocessin someindirect manner,since the

levels of recombination attained in X-infected

recB andpoLA strains are lower than those in

wild-type and recA strains.

Effects of A mutations on the 4X174

re-combination.

Phage

X

specifies

two

pathways

ofgenetic recombination: the Int

pathway

for

site-specific

recombination and the Red

path-way for

general

recombination

(6, 17).

To see

whether

one or both of these

pathways

were

involved in the

promotion

of4X174

recombina-tion, we

investigated

the effect of X mutations

onthe

4X174

recombination.

First,

weexamined the effect of mutations on

general

recombina-tion. In this

experiment

weused

XcI857,

which

produces a

temperature-sensitive

repressor;

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thus, A gene functions are induced by shifting bacteria carrying AcI857tohigh temperature (1). The recombination of 4X174 was enhanced

when HF4712 (recA) cellslysogenic for AcI857

wereincubated at40°C (Table 3). No such en-hancement occurred whennonlysogenic bacteria

weretreated inasimilarmanner.Sincethe Red

pathway is controlled by three different genes,

reda,

redf3,

and perhapsgam (18), we next

ex-amined which of the geneproductsare

respon-sible for the 4SX174 recombination. AAmutant havingamutation in theredfBgene wasunable

toenhance the 4SX174 recombination (Table 3). About 4 and 40% of the recombinationfrequency obtained by normal A was obtained with reda

andgam mutants,respectively. Second, we ex-amined the effectof absence oftheInt function. In thisexperiment, sinceaAInt-mutant cannot form a lysogen, A functions were provided by

infection. XcI857biol6, which deletesintbut car-ries redgenes, was able topromote 4X174

re-combination (Table 4), whereas AcI857bio69, which deletes both intand redgenes, wasnot.

These results suggest that recombination of

OX174

is carried out through the Red rather than theInt pathway.

Effect of UV irradiation on the 4X174

recombination.Tessman(22) showedthat the

primary recombination pathway forS13,which isdependentontherecA+ function,iseffectively promoted by UV irradiation, whereas the recA-independent secondary pathway is not. It is of interest to see whether the Red-promoted

re-combination of 4X174 is stimulated by UV

ir-radiation.

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Table 5 shows the effect ofUV irradiationon the recombination frequencies of 4SX174under

TABLE 3. Effect oftheexpression ofXgenes onthe recombinationof4X174a

Recombina-Bacterial Temp tion fre- Burs Burst size

Bacterlluencofm Brt size

strains (°C) quencyof of tX174 of A (x 10-4)

HF4712 32 0.63 71

40 0.81 52

HF4712 32 0.75 114 <0.01

(AcI857) 40 85.0 220 16.5

HF4712 32 0.77 155 <0.01

(AcI857reda) 40 3.5 177 5.5

HF4712 32 0.90 78 <0.01

(AcI857red,B) 40 0.65 209 27.0

HF4712 32 0.67 141 <0.01

(AcI857gam) 40 34.0 193 9.5

aBacteria infected with am3and amNlwereincubatedat

32or400C.

TABLE 4. Recombination of4X174 in recA cells

infectedwith various A mutants' Recombination Burst size of Aphageb frequencyof

paX174

(x10-4)

"X174

None 0.53 325

AcI857 34 330

AcI857bio69A(int red) 0.63 330

AcI857biol6A(int) 57 300

aHF4712(recA)cellswereinfectedwith amixtureof0X174

mutants am3andamNl,togetherwithvarious A strains.The multiplicityofinfection forA was 5.

bA,Deletion.

TABLE 5. Effect ofUV irradiation on the three recombination mechanisms for Xl 74a

Recombination

fre-System quency(x10-4) Ratio +UV/ -UV

Bacteria A -UV +UV

HF4714(recA') - 8.8 39 4.4 HF4712(recA) - 0.52 0.49 0.94

HF4712 (recA) + 34 74 2.2

aBacteria were infected with a mixture of am3 and amN1 which either had or had not been irradiated with UV (10 J/

m2).

threedifferent

conditions,

eachof which

permits

the

expression

ofoneof the three recombination

mechanisms.

Itwasfound that recombination of

4X174

in recA+

cells,

without A

infection,

is

stimulated

by

UV

irradiation,

whereasno

stim-ulationoccursin recA-

cells,

inagreementwith

the

result of

Tessman

(22). The

Red-promoted

recombination

of

4X174,

which could beseenin

recA-

cells

infected with

A,

was stimulated by

UV

irradiation, though

theextentof the

stimu-lationwas

relatively

low. It has been shown that

UV irradiationincreases the recombination

fre-quencyfor

phage

A (7);

thus,

the

Red-promoted

recombination of

4X174

is similar to the A

re-combination in thisrespectalso.

DISCUSSION

We haveshown that the Red function ofA can

promote the recombination of

4X174.

The

re-combinationfrequency of

4X174

increased

con-siderably

afterinfection with A orinduction of

A

lysogenes,

and this increase was associated with

expression

of theARedfunction. The

Red-promoted

recombination of

4X174

was

inde-pendent of recombinationby the hostRec

path-way, and the

efficiency

of the Red system

sur-passed

the

efficiency

of the Rec system for

4X174

recombination.

Signer

and Weil

(18)

investigated the effect of

red,int, andrecmutationsonrecombination of

Aandfound that the Redand theRecpathways

wereresponsibleforgeneral recombination ofX

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RED-PROMOTED RECOMBINATION OF

OX174

441

whereas the Int pathway functioned for

site-specific

recombination. Table 6 summarizes

their

results

concerning the roles of the Red and

the Rec pathways in the X recombination and

also the present findings obtained with

cX174.

Actions of the two systems on A and

4SX174

recombination are essentially similar. It is of

interest that the ratio of the Red-promoted

re-combination to the Rec-promoted one is higher in

4X174

than in

X.

Thisindicates that in spite

of theinherent low efficiency of the Rec system

for 4X174 recombination the Red system

pro-videshigh efficiency of recombination for both

4)X174

and

X.

The A Red system has been shown to be

controlled

by three genes, reda,

red,8,

andgam

(17). The reda gene codes for X exonuclease,

which degrades DNA progressively from the 5'

end (8, 12), and the

redf3

genecodes

for,8

protein,

which increases theaffinity ofthe exonuclease

for DNA (13). Thegam gene product has been

identified as aninhibitor of host RecBC enzyme,

whichis known to interfere with the A

recombi-nationreaction (14). The effect

of

thedeficiency

in these genes on A recombination has been

examined (16), and the result is

similar

to the

result of the present experiment with

4X174.

In

both

phages,

reda and red/3 mutations abolish

recombinationcompletely or nearly completely,

whereasagam mutation reduces itonly slightly.

Thus, in thisrespect, too, the Red system acts

on

4X174

inasimilar mannerason

A.

4oX174

has a

simple

genetic structure, and the

entire nucleotide sequence of its DNA was

re-cently

determined

(15).

This makes the virus an

ideal

organism

for

analysis

of the mechanisms of

recombination and

replication

both in vivo and

in vitro. The present

finding

may

provide

an-other

useful

means for

investigating

the

molec-ular mechanism of the Red

recombination

path-wayand its interaction with other recombination

systems.

TABLE 6. Recombinationof

OX

74and Aby the Red andRecpathways

Relative recombination fre-quency

Red Rec OX174a Ab

- - 1 <1

- + 7 36

+ - 72 83

+ + 100 100

aData taken fromTable2of thispaper:a crossof am3(E)andam9(G)mutants.

bDatataken fromSigner and Weil (18),a crossof

candRmutants.

ACKNOWLEDGMEENTIS

Wethank M. Hayashi, K.Matsubara,Y. Sakaki, K. Shi-mada,andR. L. Sinsheimerfor makingavailable to us the bacterialandphagestrainsused in this study. We also thank K.ShimadaandY. Sakaki for valuable suggestions.

LITERATURE CITED

1.Attardi, G.,S.Naono,J.Rouviere,F.Jacob,andF. Gros.1963.Productionof messenger RNAand regula-tion ofproteinsynthesis. Cold Spring Harbor Symp. Quant.Biol. 28:363-372.

2. Benbow,R.M., C. A. HutchisonIII,J. D.Fabricant, andR.L.Sinsheimer.1971.Genetic mapof bacterio-phage,X174.J.Virol.7:549-558.

3. Benbow,R.M.,A. J.Zuccarelli, G. C.Davis, andR. LSinsheimer.1974.Genetic recombinationin bacte-riophageOX174.J. Virol. 13:898-907.

4. Benbow,R.M.,A. J.Zuccarelli, and R. L. Sinshei-mer. 1975. Recombinant DNAmolecules of bacterio-phageOX174.Proc.Natl. Acad.Sci. U.S.A.72:235-239. 5. Clark,A.J., and A. D.MarguHies.1965.Isolation and characterization of recombination deficient mutants of EscherichiacoliK12. Proc.Natl. Acad. Sci. U.S.A.53: 451459.

6. Gottesman,M.E.,and R. A.Weisberg.1971.Prophage insertionandexcision, p. 113-138. In A. D.Hershey (ed.), Thebacteriophage lambda.ColdSpringHarbor Laboratory, ColdSpring Harbor,N.Y.

7. Jacob, F., andE.LWollman. 1955. Etudeg6n6tique d'unbacteriophage temper6d'Escherichia colim. Ef-fet durayonnement ultravioletsurla recombinaison g6netique.Ann.Inst.Pasteur 88:724-749.

8. Little,J. W. 1967. An exonuclease inducedby bacterio-phage A. II. Nature of the enzymicreaction. J. Biol. Chem.242:679-686.

9. Manly,K.F.,E. R.Signer,andC.M.Radding.1969. Nonessentialfunctions ofbacteriophage A. Virology37: 177-188.

10. Ono,M.,M.Kuwano,and T. Horiuchi. 1977. Genetic analysisof mutationsaffectingribonuclease fl in E8ch-erichiacoli.Mol. Gen. Genet. 153:1-4.

11.Pfeifer,D.1961.Genetic recombinationinbacteriophage *X174.Nature(London) 189:422-423.

12. Radding, C. M. 1966. Regulation of A exonuclease. I. Propertiesof Aexonucleasepurifiedfromlysogensof A

T,nandwild-type. J. Mol. Biol.18:235-250.

13. Radding,C.M., andD. M. Carter.1971. The role of exonuclease and,B proteinofphageA ingenetic recom-bination.mI.Bindingtodeoxyribonucleic acid.J.Biol. Chem. 246:2513-2518.

14. Sakaki, Y.,A. E.Karu,S.Linn,and H.Echols. 1973. Purification andpropertiesof they-proteinspecified by bacteriophageA:aninhibitorof the hostRecBC recom-bination enzyme. Proc. Natl. Acad. Sci. U.S.A. 70: 2215-2219.

15. Sanger,F.,G. M.Air,B.G.Barrell,N. LBrown,A. R.Coulson,J.C.Fiddes,C. A. HutchisonIm,P. M. Slocombe,and M.Smith.1977.Nucleotidesequence ofbacteriophage4X174DNA.Nature (London) 265: 687-695.

16.Shulman,M.J., L.M.Hallick,H.Echols,andE. R. Signer. 1970. Properties of recombination-deficient mutantsofbacteriophageA. J. Mol.Biol. 52:501-520. 17. Signer,E.1971.Generalrecombination,p.139-174.In A. D. Hershey, (ed.), The bacteriophage lambda. Cold SpringHarborLaboratory,ColdSpring Harbor,N.Y. 18. Signer,E.R.,and J. Weil. 1968.Site-specific recombi-nation inbacteriophageA. ColdSpringHarborSymp. Quant. Biol.33:715-719.

19. Sinsheimer, R.L. 1959.Purification andproperties of VOL. 29,1979

on November 10, 2019 by guest

http://jvi.asm.org/

bacteriophageOX174.J. Mol.Biol. 1:37-42.

20. Tessman, E.S., andI.Tessman. 1959. Genetic recom-bination inphage S13. Virology7:465467.

21. Tessman, I. 1966. Genetic recombination of phageS13 in

arecombination-deficientmutantofEscherichia coli K12.Biochem. Biophys. Res. Commun. 22:169-174. 22. Tessman, L. 1968. Selective stimulation ofone of the

mechanisms forgenetic recombination of bacteriophage S13. Science 161:481-482.

23. Thompson, B. J., C. Escarmis, B. Parker, W. C. Sla-ter, J. Doniger, I. Tessman, and R. C.Warner.

1975.Figure-8 configuration of dimersof S13 and 4X174

replicative form DNA. J. Mol. Biol.91:409419. 24. Zissler, J., E. Signer, and F. Schaefer.1971.Therole

ofrecombination in growth of bacteriophage lambda.I. Thegamma gene, p.455-468.InA.D. Hershey (ed.), Thebacteriophage lambda. Cold Spring Harbor

Labo-ratory,Cold Spring Harbor, N.Y.

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http://jvi.asm.org/