Recombination in bacteriophage T1 in the presence of host restriction.

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0022-538X/79/07-0017/08$02.00/0 Vol. 31, No.1

Recombination

in

Bacteriophage

Ti in the Presence

of Host

Restriction

JEROO S. KOTVAL,tTHOMAS V. POTTS,4 ANDJ. R. CHRISTENSEN'

Department of Microbiology, University of Rochester School of MedicineandDentistry,Rochester, New York 14642

Received for publication11December1978

Whenunmodified phage Ti infects restricting hostcellsathighmultiplicities ofinfection, there isanincrease in recombination frequency inallregions of the

Ti mapcomparedtothe level of recombination in standardcrosseswhenshort

distances are examined. The enhancement of recombination frequency is not

uniformfor all regions but isgreatestformarkersnearthecenterof themapand notsogreatfor markersnearthe ends. Crosses between markersattheextremities of the map show that there is no increase in recombination frequency under restriction conditions. An examination of phage Ti he'terozygotessuggeststhat anincrease of ends created by theprocessof P1 restriction increases recombina-tion. When Ti crosses aredone in the absence of host restriction, recombination defects inthe host havenoeffectonphage recombination andweconclude that phage Ti codes for itsownrecombinationgenes. Host recombinationfunctions

arealsodispensable for the recombination occurring during infection of restricting hostcellsby unmodified phageathigh multiplicities of infection.

BacteriophageTiissubjecttotherestriction and modification system of the prophage P1 (12).PhageTi grown on P1lysogens (T1lP) is

modified,and itplates onP1 lysogens withan efficiency of plating (EOP) of 1.0. Phage Ti grown onhostsnonlysogenic for P1(Ti .0)lacks the Pi modification andplatesonPi lysogens witha

markedly'reduced

EOP.

Ithas beenproposed(17) that the P1 restric-tionenzymebindstounmodified DNAat asite called the mediation site and cleaves at distal sitescalled cleavage sites. For each mediation

site,agivenDNAmolecule iscleavedonlyonce atany one of itscleavagesites. The restriction

enzymedoesnotappearto turn over(17). Despite the inability of Ti .0 to infect P1

lysogens successfully, under certain conditions T1.0cansuccessfully infect asubstantial frac-tion of a population of restricting cells. Thus,

under conditions ofstrongaeration and in the

presenceof nutrient medium withanincrease in

multiplicity of infection

(MOI),

there is more

than a proportional increase in the fraction of

successfully infected cells (9). If genetically

marked phage strains are used in the experi-ment, amarkedly enhancedfrequencyof recom-bination betweenmarkerscanbe observed (9).

In this paper, the successful infection of a t Present address: Department of Radiation Biology and Biophysics, University ofRochester, School of Medicine and Dentistry,Rochester, NY14642.

tPresent address: 2123Bailey Ave., Buffalo, NY 14211.

substantial fraction ofrestricting host celLs at

high MOIs of unmodified phage will be called cooperative infection, and the recombination thatis seenin theseexperiments will be called

cooperativerecombination.

TheTi geneticmapdoesnotshowauniform

recombinationfrequencyperunitlength ofDNA (15).There isahigh frequency ofrecombination perunit lengthof DNA atthe endsof themap

and alowfrequency of recombinationperunit lengthof DNA in thecenterof themap.Electron microscopic autoradiography has displayed

qualitatively similar results for Ti recombina-tion and further reveals the insertion of short pieces of labeled DNA intounlabeledchains(2).

The inferencedrawnfrom these data has been thatfree endsarerecombinogenicforTi

recom-bination (2, 15).

Phage heterozygotes have been proposed to

berelatedtointermediates inbacteriophage

re-combination (19). In three-factor crosses with

looselylinkedmarkers,ifonestudies the

segre-gation pattem of plaques

arising

from

phage

that are heterozygous for the central marker,

twokinds ofresultsarefound.Oneisthe

segre-gation oftwo genotypes which have the same

recombinantconfiguration for thetwoflanking markers. Theinferredstructurefor the

hetero-zygote producing this result can be called an

"overlap"heterozygote. Theothercommon

seg-regation resultis tofindoneparentaltypeand

a double recombinant, from which an

"insr-17

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18 KOTVAL, POTTS, AND CHRISTENSEN

tional" type ofheterozygoteisinferred. Except for very closely linked markers, heterozygosis for either of the unselected markers is

uncom-mon(19-21). A study of phageTIheterozygotes is thereforeavalidgenetictool forexploring the natureof recombinationintermediates.

In the present paperwe report studies done under conditions where unmodified Ti infects restricting hosts at high MOLs. We show that under these conditions: (i)recombinationin the

centerof the Ti map is enhanced compared to

normal Ti recombination; (ii) despite the

en-hanced recombination under these conditions, the ends of the TI molecule are notgenetically

dislinked and the enhancement of recombina-tion is due to the insertion of short,

single-stranded fragments; and (iii) these

recombina-tion events, like normal Ti recombination, do

not require any host recombination pathway requiring the recA gene product.

MATERLALS AND METHODS Phage strains. The Tlam strains and the prepa-ration ofTlam stocks have beenpreviouslydescribed (8).All of the three-factorcrossesreportedherewere betweenTi Sb HrKn+ and Ti Sb+ Hr+Kn. These markersareplaquemorphologymarkers andarewell separatedonthegenetic map ofTi soas toavoidthe problem of high negative interference (1). All eight combinations of the three markerscanberecognized ondye-containing agar plates (TDM-2) (9).

Umnodi-fiedTi stocks (Ti-0) werepreparedon Escherichia coli B andmodifiedTistocksonE. coliB(Pi) by the plate lysate method, using LB agar plates (7). All phage stocks were sedimented by centrifugation at 30,000 rpmfor90min andsuspended in nutrient broth (NB), whichhasanion concentrationfavorable forTi

attachment.

Bacterial strains. E. coli strainsB,B/i,t, B(Pi),

and B/i,t(P1) are nonpermissive for Tlam mutants andhave been described (7),ashave the amber per-missive strains KB3 and CS100 (5). KB3(P1) and

CS100(P1) were derived in this laboratory. Strain JC4583wasthewild-type strain for comparison with the various recombination-deficient strains. JC4588 (recA56), JC4584 (recB21 recC22), and SDB1006 (recA56 recB21 recC22) are isogenic with JC4583. Theyarereferred to in the text, respectively, as rec+, recA-, recB-, and recA- recB-. These were gifts of S. D.Barbour and are describedfurther byCapaldo et al. (3). These strains were madelysogenic for phage P1CM (11), which is phageP1carrying a DNA inser-tionconferringchloramphenicol resistance and was a gift fromJ. L. Rosner. TheP1CM lysogens of these strains were found togradually lose their ability to restrictTi .0during serialpropagation. Therefore, for eachstrain,asingle freshly isolated lysogenic colony wasinoculated into LB containing 12,tgof chloram-phenicol permland incubated at 37°C with aeration till aturbidculture was achieved(approximately 12 to 18 h). Those cultures which showed at least 96% of thecells capable ofrestriction were distributed in 1-mlquantities inseveral vials and stored at -70°C in 10%dimethylsulfoxide.

Media. Luria broth (LB) and NB have been de-scribed(7),ashas TDM-2(9). LB agar waspurchased from GIBCODiagnostics. NB + NaCl is NB+ 0.5% NaCl. Cyanide broth is NB + 0.5% NaCl + 10-2 M KCN.

Preparation oflog-phase cells for Ti infection. In the case ofP1CM lysogens of the isogenic set of recombination-deficient strains, one vial of the frozen stockwasthawed at 37°C, and 0.2 to 0.4 ml of the culture was diluted into 10 ml of LBcontaining 12 ,ug ofchloramphenicol per ml. This was grown at 37°C with aeration until the cells were growing exponen-tially andatiter of1x 108 to2 x108 cells per mlwas achieved. The log-phase cells were centrifuged and suspended in an equal volume ofhalf-strength NB. These cells were used for attachment of Ti for the execution ofcrosses.The preparation of log-phase B and B(P1) for Ti attachment have been previously described(16).

Procedure used for phage crosses. The tech-niques usedweresimilartothose usedby Drexler and Christensen (7). All infectionsweredone in NB, and thephagewereallowed toattach at37°C for5min. The phage-infectious cyclewas thenarrestedby the addition ofpreviously chilledcyanide broth, and the tube was centrifuged. The KCN inhibition was re-versed either by washing with NB + NaCl or by diluting out the KCN with NB + NaCl before any titrationorbyacombination of the two procedures. Thesupernatant was titrated for unattached phage by plating on KB3 in the case of Tlam crosses or on a mixture of B+B/1,t (4:1, vol/vol) in the case of three-factor crosses,usingplaque morphology markers. The pellet, which is henceforth called the infected-cell preparation, was washed by centrifugation and titrated for infectious centers on CS100(P1) in the case of cooperative crossesto measurethe modification ratio, which monitors the extent of cooperative infection. The modification ratio is the proportion of cells that release at least one modified progeny phage (9).

Above an MOI of 5 in cooperative crosses, the modification ratios are the same as yield ratios (9). We verified this for all crosses, but the data shall not be presented. For standardcrosses innonlysogenic hosts, theinfected-cell preparationwastitrated on KB3 for theTlamcrossesand on Bfor thethree-factor crosses. A portion of the infected-cell preparation was then chloroformed and titrated on appropriate hosts to monitor the number of free phage particles in the infected-cell preparation.

The washedinfected-cell preparation was then di-lutedinto atube ofNB +NaCl to giveafinal concen-tration of102 to103 yielder cells per ml.Thisgrowth tubewasincubated at 37°C for 30 min to allow phage bursttooccur. Itwas thenchilled,and1to 2drops of chloroformwereadded. Thetube was then titrated on theappropriate nonlysogenic host to measure the total progeny released upon lysis. For cooperative crosses, the growth tube was also titrated on CS100(P1) to measurethetotalyieldofmodified phage.

InTlam crosses, the extent of recombination was monitored by measuring the proportion of progeny phage thatweream'. This should measure one-half ofthe totalrecombinantfrequency, since double am recombinants are not measured. To rule out the pos-sibility that thenonpermissive character of the host J. VIROL.

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RECOMBINATION IN BACTERIOPHAGE Ti

would influence the results of these crosses, several experimentswerecarriedoutin theamber-permissive

hosts, KB3 andKB3(P1). Resultsweresimilartothose obtained in B and B(P1) (datanotshown), butmost ofourambercrossesweredone in B and B(P1).

Inthecaseofthree-factorcrossesbetween plaque morphology markers, the progeny were scored on TDM-2plates. Thebacterial cultures usedweregrown tolog phasein NB+NaCl. A mixture of4volumes of Bto1volume of B/1,twasusedtoscoretheprogeny ofstandardcrosses in nonlysogenic hosts. Four vol-umesof B(P1) + 1volume of B/1,t(Pl) wasusedto

scoretheprogenyofcooperativecrossesandstandard

crossesinP1lysogens,toavoid thescoring of unad-sorbedphageparticles. Itwasverified that theprogeny

fromcrossesin P1lysogenswereessentiallyall modi-fied, and thus would form plaques on this plating mixture.

TheTDM-2plateswerespread with 0.2 ml of the

appropriatebacterial mixture and0.1ml ofthelysate from the growth tube, diluted to give 200 to 300 plaquesperplate. The plateswereincubatedat37°C for10to12h, andtheplaqueswereidentified under adissecting microscopewith strongillumination.

Heterozygotes. To screen for heterozygotes for the Hrmarker, the growth tube from thecross was platedonamixtureof B(P1)+B(Pl)/l,t (4:1,vol/vol)

on anLBplateatadilution which wouldgive only50

to60plaquesperplate. Those plaques which showed

a sectored morphology (heterozygotes for the Hr marker) were picked into sterile NB, diluted, and spreadonTDMplates with 0.2mlofamixtureof B

+ B/1,t (4:1, vol/vol). After incubation, the plates were examined for the progeny segregated by the

heterozygousplaque.

RESULTS

Distribution of exchanges along the Ti

geneticmap.Freshmanetal.(9) observedthat forthe markers Hrand Sb,recombination

fre-quencyincontrolcrossesvaried littleasa func-tion of theMOI,whereas in therestrictinghost the recombination frequency increased

mark-edlywithMOI,andatsufficiently highMOI(10

ormore),recombinationwasabout four timesas

frequent as in control crosses. We have done

similar experiments with markers spanning var-iousregions of theTi mapand findthat there isagreatvariation fromregiontoregion. Some

typical resultsareshown in Table 1. It canbe

observed thatinthecaseofTlaml5 (gene 5) x

Tlaml3 (gene 9), which are markers in the

central region ofthe Ti map, the MOI hasno

effect onrecombination frequency in standard crosses, whereasthere isan almostfourfold in-crease in recombination frequency with MOI under cooperative conditions. On the other

hand, in thecaseofTlam4 (gene 16) xTlam30

(gene 18), whicharemarkers atone endof the

map,there isless thanadoubling of the

recom-bination frequency withincreasing MOI,under

cooperative conditions, andatmostaslight dif-ference between therecombination seen in

re-stricting andnonrestrictinghosts. Thissuggests

that theextentof the increase in recombination observedtooccurin cooperative infectionmay

berelatedtomapposition.

Tofurther investigatethedistribution of the

effect,wehavecarriedoutanextensive series of

amxamcrosses,withpairsofmarkersspanning

variousregions of themap.Somerepresentative

resultsaredisplayed in Fig.1,whereanincrease

inrecombination in cooperativecrossesis seen

as a dilation of the genetic map. It isevident

thatpairsof markersneareither endofthemap

show little or no increase in recombination,

whereaspairs of markersnearthecenterof the

mapshowamarked increase in recombination incooperativecrosses.

Crosses between amber markersnearthe

ex-tremeends of theTi mapshowthat there isno increase inrecombination frequencyin

cooper-ativecrossescomparedtostandardcrosseswhen alargeregionofthe Ti mapisexamined. Typ-icalresultsareshowninTable 2.

TABLE 1. Geneticrecombinationaccompanying cooperative infectionindifferent regions of the Tl map Controlcrossesin E.coli B Cooperative crossesinE.coliB(P1)

Phage strainscrossed am+progeny/!am progeny/

MOI total progeny MOI ratioa(x103) totalprogeny

Tlam15(gene 5) xTlam13(gene 9) 2.3 2.4 2.3 1.0 3.0

20.0 2.6 5.6 2.0 6.7

10.0 8.2 7.7

20.0 35.0 11.0

T1am4(gene 16) xT1am3O(gene 18) 1.9 3.8 2.7 0.71 3.9

3.6 4.6 5.3 1.4 6.3

14.0 5.3 10.6 6.6 5.2

13.3 10.0 6.2

aThemodification ratio is theproportionof

dually

infectedcellsthatyieldatleastoneP1-modifiedparticle. Where theMOI isinsufficienttoinsurethat all cellsareduallyinfected,the Poisson distributionwasusedto correct forcells that donotreceive bothphage parents.

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Multipleheterozygosity among progeny

of Ti crossesdoneunder conditions of

co-operative infection. An increase in

recombi-nation frequency overshort distances that

de-pendsonthemapposition,whichweobservein crossescarriedoutundercooperativeconditions,

cannotbeexplained by merelyassumingan

in-creasein the roundsofmating,since thiswould accountforonlyauniform increase inallregions

of the map. However, an increase in the fre-quency of initiation of single-strand transfers

(19) in regions of the genome in which such transfersarenornallyuncommonmight provide an explanation. Such transfers produce

struc-tures containing heteroduplex DNA, and we

soughtevidence for their existencebyexamining

the frequency and genetic structure of phage

heterozygotes.

Table3 presentsthe results ofananalysisof

heterozygotes from three-factor crosses,

be-tween well-separated markers in the center of

the map, done under standard conditions. The

frequencyofheterozygous particlesis0.1% and is comparable to the frequency reported by Trautner (20).The pattern of strandsegregation

is consistent with thehypothesisofDNAstrand

T IMAP INEco/liB

GENE NOI 2 3 5911 161I

AMBER NO 16 3 4115132910 4 30

13 .4

15 110.5 11.1 1 13.4 79 124

16 3 41 15 13 29 10 4 30

TiMAPINfco/iB(PI)

FIG. 1. Comparison ofthegeneticmapsobtained

from two-factor amx am crossesofunmodified Ti carriedoutin E.coli strains BandB(PI). Allcrosses werecarriedout athigh MOI, underconditions that promote cooperative infection. The numbers below thelineoneachmaprepresentthe proportion ofam' progeny amongthe totalprogenyproduced in each

cross.

equivalence for resolution (18), whichaccounts

forapproxinately equal numbers of overlap and insertional heterozygotes.Heterozygotes which

segregate one recombinant and one parental

strand could be the products of short single-strand insertions in the region of the Hr marker. Those heterozygotes whicharerecombinant for

the outside markers reflectheteroduplex

over-laps for the central marker (19). We noticed the inequality between the two types of possible insertional heterozygotes but have no

explana-tion for it.

The data for heterozygotes selected for the central marker from cooperative crosses are

shown in Table4.Thefrequency of heterozygous particles is increasedto0.3% ofthe total

popu-lation, consistent with an increase in

recombi-nationseeninthesecrosses.Heterozygotes

seg-regating the two patterns seen in standard

crossesaccountforonly 55% of the total

heter-ozygotes. The other 45% are heterozygous for

twoloci(41%)orallthree loci (4%) anddeserve

TABLE 2. Control andcooperative crosses between Ti markers thatarewidelyseparated onthe map

Control crosses in E. Cooperativecrosses coli B in E.coli B(PI)

Phage am+ +

strains

progeny/

crossed MOI total MOI total

progeny progeny

(%) (%)

Tlaml6 16 20 20 20

(gene 1) x Tlam30

(gene18)

Tlaml6 15 18 19 19

(gene1)x Tlam4 (gene 16)

Tlam3(gene 14 15 18 18

2)x Tlam4

(gene16)

TABLE 3. Segregatingphagetypesfound in plaques, selected asHr/Hr+heterozygotes,fromacross of modified TlSb HrKn+xmodified 5b+ Hr+ Knperformed inB(Pl)'

Segregation like that expected from overlap heterozy- Segregation likethat expectedfrom insertional

heterozy-gotes gotes

Exptno., Sb HrKn and + + +and Totalfrequency Sb Hr + and + +Kn and Totalfrequencyof

Sb+Knb + Hr + of overlap seg- Sb + + + HrKn insertional

segre-regation gation

1 3 10 1 15

0.45 0.55

2 3 4 0 8

aAtotal of 29,783 plaques from cross 1 and 15,120 from cross 2 were inspected. Frequency of Hr/Hr+ heterozygous plaques: 0.12%. Three plaques segregated the two parental genotypes and are considered to have arisenby accidental superimposition of plaques of the two parental types.

bAll

genotypesareexpressed in the order Sb Hr Kn, and the wild-type allele is designated by a "+" in the appropriate position. Thesameconvention is used in all tables thatfollow.

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TABLE 4. Segregating phage types found in plaques, selected as Hr/Hr+ heterozygotes, from a cross of unmodifiedTl Sb Hr Kn+xunmodified Sb+ Hr+ Kn performed inB(Pi)'

Segregationlikethat expected from overlap Segregation like thatexpected

heterozygotes from insertionalheterozygotes

Total

fre-Heterozygosis ~~~~~

~~~~~~~~~~~~Fre-

quencyof

feeor:gsi

Exptno. Frequency Sbr +n quency this

clas

for: Sb Hr Kn + + + and + amongsingle and SbH+ +HK

among

anion

Hr-andSb+and+Hr 9 Hr+ hetero-andSb+Kn Hr+ heterozy- + Kn single zygotes

gotes

heterozy-gotes

Selected marker 0.55

only 1 0 8 5 5

0.53 0.47

2 2 17 6 8

Segregation of one parent and one single recombinant, suggesting heterozygosis in

cis

Frequency among double heter-Sb Hr Sb + Kn + ++ + Hr + ozygotes

Kn and and Sb and Sb and + +

+ +Kn Hr + Hr + Kn

Twomarkers 0.41

1 1 1 5 3

0.47

2 1 1 2 4

Segregation of one single recombinant and onedoublerecombinant, suggesting

heter-ozygosis in trans

SbHr Sb+ Kn + + + +Hr+ Knand and+Hr and+Hr and Sb +

Sb+ + Kn Kn +

1 0 2 2 6

0.53

2 0 3 1 6

Segregationoftwosinglerecombi- Segregationoftwo Segregationoftwo

nants double recombi- parents nants

SbHrKnand+ Sb+Knand+ Sb++and+Hr SbHr+and++

++ Hr+ Kn Kn

Threemarkers 0.04

1 0 0 1 3

2 0 2 1 3

aAtotal of12,162plaquesfromcross 1and17,621plaquesfromcross2 wereinspected.FrequencyofHr/Hr+

heterozygotes: 0.31%. The sixplaques that segregated thetwoparentaltypeswereassumedtohave arisenby

the accidentalsuperimpositionoftwoplaques,andtheywere notincluded incalculatingany of theproportions

listed here.Since theparentaltypeswerethemost common twoclasses ofplaquesin the progenypopulation,

themost commonclassofsuperimposedplaqueswouldgivethissegregationresult.Therefore,few if any of the otherplaquesrecorded here could have arisen this way, except thatavery few of theplaquesthatsegregated

oneparentaltype andonesinglerecombinantcould have been of this kind.

further consideration. Of the38particles which

areheterozygousfortwoloci (double

heterozy-gotes), 18 areheterozygotesfortwolociderived

fromthesamestrand(incis).Thesecould arise

if the entire length of material encompassing

these markers were obtained from one parent

(i.e., long overlap heterozygote) orbytwoshort noncontinuous single-strand insertions at the

two regions of heterozygosity. However, 20 of

these double heterozygotes have flanking markersderived from differentparentalstrands

(in trans). These canbe bestaccountedforby assumingthattwosingle-strand insertionsgave rise tothesestructures. If the doublecis heter-ozygotesareinterpretedtoarise from

long

over-laprecombinant structures,theywould standas ananomalousclassthat isnotseeninstandard crosses, and they would not contribute to the

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disproportionateincreasesof recombination

fre-quencyincentralmappositionsthatare seenin

cooperativecrosses.Since the double cis

heter-ozygotes arealmost equal tothe double trans

heterozygotes, it islikelythatthe cis

heterozy-gotes are also produced by two independent

insertionsattheregionsofheterozygosity.

Fur-thermore, of the 93 Hr/Hr+ heterozygotes, 30 areheterozygousalso forSb,and 16are hetero-zygousforKn. If theseregionsofheterozygosity

wereindependentamongHr/Hr+heterozygotes,

about five triple heterozygotes would be

ex-pected,and in fact fourwerefound.

Effect of recombination defects of the

host on Ti crosses under normal and

co-operative conditions. Toinvestigatewhether

phage Ti codesfor itsownrecombinationgenes,

three-factorcrosseswerecarried out under

nor-mal conditionsinisogenic wild-typeand

recom-bination-deficient hosts. The results (Table 5) show thatthere isnodifference in recombination

frequency whetherthecrosses arecarriedoutin

hoststhatarerec+, recA-, recB-,orrecA- recB-.

Since recA mutations in E. coli are known to

abolishlegitimaterecombination in the host(6),

the data suggest thatphageTi codes for itsown

general recombination functions. (The possibil-ity ofan undiscovered recA-independent host

recombinationpathway allowingnormal Ti

re-combination isnotformally excluded.)

Cooperative infection differs from standard recombination not only in the conditions

re-quired for it to be observed, but also in the

resultsthat areobtained incrossesdone under

cooperative conditions. Itwastherefore possible that the host's recombination systemmight play

a role in cooperative crosses, even ifit is not

required during ordinaryTi crosses (Table 6). Inthe first sectionaretheresultsofcrossesof

T1iP in the rec+(PlCM) host, to serve as a control forthecooperative crossesinthe same

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host.The resultsareverysimilartotheresults presented in Table1forT1-0 crossed in nonly-sogenic hosts.Additionalexperiments with Ti1 P crossed in thevarious recombination-deficient

PlCM lysogens gave similar results (data not

shown).Thus,Pi lysogenyper sedoesnot influ-ence the results of the crosses, so longas the

incoming phage isnotsubjectedto restriction. Crosses of Tl*0 at high MOIs in the

rec+(PlCM) hostare presented in Table2; the

enhanced recombinationassociatedwith

coop-erative crosses (9) is clearly seen. Taking into account both the single and double

recombi-nants, the totalrecombination in the Sb toHr

region is at least three times as high in the

cooperative crosses as in the standard crosses,

and thatintheHrtoKnregionisatleasttwice

ashigh.

Recombination frequencies in cooperative

crosses in the recA-(PlCM) host are

interme-diate between those observed in standard

crosses and in cooperativecrosses done in the

rec+(PlCM)host. However,anyhypothesis that

recA-dependent host recombination pathways

are required for cooperative recombination is

refuted by the results in the

recB-(PlCM)

and recA- recB-(PlCM) hosts, which give results

similartothose obtained in cooperativecrosses

in therec+(PlCM)host. These resultsare

con-sidered furtherbelow.

DISCUSSION

Astudy of recombination in phageTi shows that thedistribution of exchange eventsin

co-operative crosses is clearly different from that seen instandardcrosses (Fig. 1). Michalke (15)

has concluded that in T1 crosses most of the recombinationoccurs nearthe ends of the mol-ecule. Fromourresults itappearsthat in

coop-erativecrosses recombinationeventsaremuch

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more uniformly distributed. In regions where

TABLE 5. Crosses of unmodified Ti Sb HrKn'xTi Sb Hr+ Kn in nonlysogenic hosts with differing

recombinationproficiencies

Recombinant progeny/totalprogenycounted Hostgenotype TotalMOI Burstsize Total phage

counted Sb+Knand Sb Hr Kn and Sb+ +and+

+Hr+ +++ HrKn

reck 18 41 2,930 0.051 0.13 0.016

15 137 2,048 0.071 0.12 0.033

recA 12 11 2,408 0.056 0.12 0.016

15 36 1,391 0.062 0.12 0.029

recB- 11 72 1,971 0.049 0.11 0.016

17 100 1,164 0.052 0.12 0.027

recA-recB 9 45 2,178 0.057 0.10 0.022

11 70 2,038 0.071 0.11 0.027

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RECOMBINATION IN BACTERIOPHAGE Ti 23 TABLE 6. Crosses of Tl Sb Hr Kn+x TlSb+Hr+ Kn inP1CMlysogenswithdiffering recombination

proficiencies

Recombinantprogeny as a proportion of to-Host genotype Total MOI Burst size Total phage

_counlted

tal progeny

Sb + Kn and Sb Hr Kn Sb + + and

+Hr+ and+++ +HrKn

Cros8se with modifiedphagea

rec+ (P1CM) 25 20.0 2,258 0.081 0.14 0.022

23 8.5 1,597 0.054 0.12 0.024

Crosses with unmodified

phageb

rec+ (P1CM) 16 3,006 0.18 0.27 0.095

23 2,704 0.19 0.24 0.12

recA-(P1CM) 19 3,872 0.077 0.17 0.051

21 1,861 0.077 0.18 0.068

recB-(P1CM) 22 2,069 0.20 0.23 0.15

24 2,903 0.21 0.24 0.13

recA- recB- (P1CM) 15 2,066 0.22 0.27 0.12

20 2,845 0.21 0.26 0.13

a

Control

for effect of

P1

prophagein theabsence of

restriction.

b

Restriction

present.

recombination is already high, cooperative crossesgivethe same results as control crosses, but inregions where standard crosses give less recombination, the recombination under coop-erative conditions is markedly enhanced. In

ad-dition,whenpairs of markers at the extremities of the Timap are crossed under standard and cooperative conditions (Table 2), there is no difference in recombination frequency and it appears thatcooperativerecombination does not

involvethereassembly of double-stranded frag-ments any more than standard recombination does. It is conceivable that the process of P1 restriction creates recombinogenic ends which enhance recombination inintemal regions of the

Timap.

Analysisofheterozygousparticles from stan-dard and cooperative crosses (Tables3 and 4) indicatesthatduring cooperativerecombination the majority of recombination intermediates

(55%) are of the typeseen instandard crosses.

However,multiple regions ofheteroduplexDNA where the flanking markersarein theparental configuration (double trans heterozygotes and

triple heterozygotes) are enhanced under re-stricting conditions

compared

torecombination instandard crosses. Such insertional

heterozy-gotesarehypothesizedtobeduetothe insertion

ofsingle-strandedDNA intotheparentalDNA

moleculetoyieldtherecombinant (19).In these

experiments, wehave selected forunreplicated

recombinant moleculesby selectingforasingle

heterozygous marker and then examined two

unselected sitesforheterozygosity.

Heterozygos-ity at thesesites wasfound frequently enough tosuggestthat, hadwebeen abletoexaminea

larger number of sites for heterozygosity, we would have foundmultiple heterozygosis in an evenlargerproportion of the plaques examined. Inaddition, we recallthat the frequency of

het-erozygosissubstantiallyunderestimates the fre-quency ofheteroduplex regions of DNA. First, one-half of the heteroduplex regions will be homozygous, because the two DNA strandswill

befromparents of the same genotype.Second, inseveralphage systems (21),there is a signifi-cant occurrenceof correction of any base pair

mismatches thatoccurwithinregions of

heter-oduplex DNA. We also note that there is a pronouncedincrease inrecombination frequen-ciesbetween markers in thecentral part of the genetic map, but that this effect isnot accom-panied by a similar increase in recombination

fiequencybetween moredistant markers. This

resultis whatwould be expectedfrom the rep-lication of the kind of recombination

intermedi-ateproposed above.

Recombination ofphage

Ti

inthe absence of host restriction appearscompletelynormal in all of therecombination-defective hosts

(Table 5).

Since recA mutations in E. coli are known to

completely abolish the recombination

profi-ciency ofthe host (6), phageTi mustcode for its ownrecombinationgenes.

TheresultspresentedinTable6demonstrate

thatcooperativerecombinationisalsounaltered

in therecA-

recBi(PlCM)

host.

Therefore,

nei-thertheRecB,C pathwaynoranyother E. coli

pathway that depends on the recA function is

required for cooperative recombination. It is known thatgenes carriedonunmodifiedTi can

express themselves under

restricting

conditions

VOL. 31,1979

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(8)

(4). Therefore, it is most likely that phage T1

recombination functionsare responsiblefor

co-operative recombination.

Inthe recA- (P1CM) host,the usual enhance-mentin recombination seen under cooperative conditions is much reduced(Table 6).Thelower level of cooperative recombination may occur

because someintermediate ofrecombination un-der these conditions issensitive tothe RecB,C nuclease, exoV, which is known to be particu-larly active in recA- strains (6). The P1 restric-tionenzyme isatype I restrictionenzyme which

acts onunmodifiedDNAbycreatinganickand thenconverting the nick intoadouble-stranded break, via two steps (13).

Cleavages

are notat

uniquesites,since there isnoevidence for unique sizeclasses(13, 17). ExoVacts onnicked double-stranded DNAor on single-stranded DNAin a

manner which creates single-stranded DNA fragments (14). Hollomanetal. (10) have dem-onstrated that invasion of a double-stranded

DNA molecule by homologous single-stranded

DNAmayinitiate theprocess ofgenetic

recom-bination. DNA subjectedtoP1restriction could conceivably provide the free single-stranded DNA which Holloman etal. show to stimulate

recombination. The free single-stranded DNA maybe astranddisplaced fromaDNAmolecule nicked by the P1 restriction enzyme or it may be a product ofpostrestriction degradation, as

for example by exoV. Since exoV degrades nicked double-stranded DNA and single-stranded DNAin astepwise manner (14),these recombinationintermediateswouldbedegraded

in a recA- (P1CM) host where the activity of

thenuclease isknown tobe increased (6) com-pared to wild type. The suggestion has been made that the complexity of type Irestriction enzymes suggests that they may have arisen through evolution from a DNA recombination

enzyme (13). Our data lend credence to this plausible role for the P1 restriction enzyme in

vivo.

ACKNOWLEDGMENTS

We thank David Figurski for helpfuldiscussions in the courseofthis workand A. J.Clark, S.Barbour, and J. Rosner

forsome of thebacterialandphagestrains used.

Supportwasprovided by Public Health Serviceresearch grantAI-02781andtraining grants DE 00003 and GM 00592.

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