Restriction alleviation by bacteriophages lambda and lambda reverse.

0022-538X/81/050621-11$02.00/0 Vol.38, No. 2

Restriction Alleviation by Bacteriophages Lambda and

Lambda Reverse

PENELOPETOOTHMANt

Department of Chemistry and Institute ofMolecular Biology, University of Oregon, Eugene, Oregon 97403 Received24September1980/Accepted 29 January 1981

Deletion analysisindicated that the phage A restriction alleviation gene(s) ral resides between thecIIIand N genes. TheRal+ phenotype was expressed only whenXral+carried amodificationsuch that it wasresistant to restriction by the host specificity system. Under theseconditions, Ralfunction protected superin-fecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoPl.Ral-protectedphage DNA was notconcomitantlyK and B modified, but rather received onlythe modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoidphages tested, thehybrid phageArevhadRal activity, whereas

08Ovir

andone X-P22 hybrid did not. The restriction alleviation activity ofXrevcalled Lar, may be the same as theactivity expressed in sbcA- strains of Escherichia coli,but it wasfunctionallyseparable from exonuclease VIII activity (the product ofthe recE gene), which is also expressed in sbcA- strains.

Host modification restriction systems were firstdescribedby Arber and Dussoix (2, 11)and havebeen identified inanumber of speciesand strains of bacteria (41). Presumably, modifica-tion andrestriction have evolvedtoprotect bac-teriafrominvading foreign DNA, although ad-ditionalroles havenotbeen ruledout.The mod-ification restriction loci of Escherichia coliK-12 and E.coli B and the SB system ofSalmonella typhimuriumareallelic and include three genes

(hsdS, hsdM,

and

hsdR)

(5, 15, 24, 53). The

specificity

ofthe system is determined

by

the hsdS gene

(5).

Modification and restriction in these bacterial

species

have been

designated

typeIandarecharacterized

by

anenzyme com-plex which requires the cofactors

S-adenosyl-methionine,ATP, and

Mg2e

torestrict unmodi-fied DNA but only the

S-adenosylmethionine

cofactorto

modify

DNA

(28,

32,

37,

55).

Meth-ylation occurswithin the

recognition

sequence (45, 52), whereas

endonucleolytic cleavage

oc-cursoutside thissequence andshowsno appar-ent sequence

preference

for the actual DNA cleavage reaction (1, 23,38).

Bacteriophageshave evolved several waysto disruptthe

complex

process ofmodificationand restrictionand thus escape lethal

cleavage.

The temperatephage Mu is resistant to restriction by avariety of restriction enzymes because of

methylation

byarather

nonspecific

modification function (mom) after induction from the

pro-tPresent address:DepartmentofBotany,AJ10,University

ofWashington,Seattle,WA98195.

phage state (51). The T-even phages escape restriction becauseDNA isglucosylated (12). T3 and T7expressocrfunctions

immediately

upon infection, which preventnotonly restriction but also modification

by

EcoB orEcoK (27, 47,48). Although T3ocr-, T7ocr-, and T-even

glucosyl-ation-defective phages are sensitive to restric-tion, the rare phages that doescaperestriction aremodified. Another virulentphage,

T5,

must have some mechanism(s) to escape restriction byEcoRI, asT5 DNAhassix sites sensitiveto EcoRIinvitro (54),yetnoEcoRI restrictionor modificationoccursin vivo(8).

TheXrestrictionalleviationfunction,

Ral,

was firstcharacterized

by

Zabeau(M.

Zabeau,

Ph.D. thesis,

Rijkuniversiteit Gent, Ghent,

Belgium,

1974), whoseobservationshave been

published

recently (9, 10,58).Here Ishow that AandXrev (a recombinant between theRac

prophage

ofE. coli K-12 and X

[25])

have functions

(Ral

and Lar)whichcanpreventrestriction

by

EcoB and EcoK. In

addition,

I suggestthattheLar

activity

of

Xrev

isthesame

activity

that is

expressed

in sbcA- mutantsof E. coli

(44).

MATERIALS

AND METHODS

Strains.Phage and bacterial strainsare described in Table 1. M8521 is strain M5221 ofH. Greer (18) selected to grow on eosin methylene blue (EMB)

plates.

Media.Abroth contained1%tryptone, 0.25%NaCl,

and 0.1% yeast extract; A plate agar and top agar contained,inaddition,1.1and0.65%agar,respectively, but no yeast extract. LB plate agar contained 1% 621

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TABLE 1. Listof bacterial andphagestrains

Restriction-Strain _{tion pheno-}modifica- Othermarkers Sourceorreference type

Bacteria M72 M8002 M8521* M5222 M8521(P1) M5222(P1) Q1 Q8002 Q8003 Q8004 Q8100 Q8803 Q8209 Q8302 Q8303 Q8304 QD5003 QA8002 QA8004 QW11 M1742 M18000

MI8000AR

AB1157 JC5519 JC8679 JC8689 JC8690 JC8691 Phage

AbiolOcI857

Abiol0imm2' cl-Abio252cI857 Abio252imm2cI-Aimm43cI6T Ah bio243imm434cI6T Ah80att"°cI857

ASpi-imm22c25(OP)P22

480vir

ArevexbiolOcI857p4

Ab2intlO2red3cI857p4 Plc+CM

K K K K K,P1 K,P1 K

su-lacamtrpamstr-M72(AcIind831) M72(bio255cl857H1) M72(bio247cI857H1) B1+revertant of C600,

080R

K Ql(AcIind831)

K Qi(Aimm434T)

K Ql(Aimm21)

O C600(rm-)

O r-m-gal-met-[803]

B

rB+msB

F+[2027]

B Q8209 (Aind831)

B Q8209(Aimm43T)

B Q8209(Aimm21)

K Ymel,suIII+

K

CR63(h"°att"°cI857)AR

K CR63(h'att °imm21)AR

K recA56recB21[JC7507]

K recA- str- su-(gal-A)A

K MI742

(Ab2cI8570am5O1Sam7) K MI8000 madeAR,mal+

K AB1157suII

K recB21recC22 str31 K recB21 recC22 sbcA23 K Rec-,UV8 derivatives

K JC8679 lacking

K ExoVIII activity

E.Signer Thiswork 18 18 This work Thiswork 43

I. Herskowitz I.Herskowitz I. Herskowitz

M.Meselson via F.W.Stahl W. B.Wood viaJ.Engel W. B.Wood via J.Engel This work

This work This work 16 This work This work

J. Clark via E.Signer 21

A.Strathern

This work E.Signer J.Gillen

J.Gillen

J.Gillen J.Gillen

J.Gillen

42

W. F. Dove E.Signer E.Signer S.Hilliker E.Signer This work I. Herskowitz L. Rosner tryptone,0.5%NaCl,0.5%yeast extract,and 1.5%agar.

Low-salt tryptone top agar contained 1% tryptone, 0.6%agar,and10-3 MMgCl2. SMfor phage dilutions has been described previously by Weigle etal. (56) andwassupplemented with 0.01 MMgSO4and 0.01%

gelatin.EMBagarwas asdescribed by Campbell(7),

butwith 0.05% maltose asasugar supplement. OM

medium for scoring A deletion mutations contained

sodium citrateandwas as describedbyManlyetal. (36).

Phagestocks. Phage stocksweremade fromsingle plaquesby the confluent lysis method. The single-plaque isolations were done in the same bacterial

strains used tomakethe stocks.Modifiedphage stocks weremadeinthefollowinghosts:Q8100 forA.0,Q8803 forAh'.0,Ql forA.K, QD5003 forAh'.K, Q8209for

A.B, Q8100(P1)forA.P,andQ1(P1) forA.K,P.

Plating conditions. Plating bacteria were pre-pared daily by dilutingafreshly saturatedovernight

culture 100-fold intoAbrothcontaining 0.2% maltose andgrowingtoadensity of5x

10i

cells per ml(ifAh'

phages weretobe used,low-salt tryptone medium was used).Adilutedphage sample (0.1ml)was mixedwith 0.2 mlofplating bacteria, adsorbed at room tempera-turefor20min, mixed with2.5 ml of top agar, and poured onto 1-day-old A medium plates. A top agar

622 TOOTHMAN

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and Aplateagar were usedforA. Low-salttryptone

topagarandAplateagarwereused forh' and

080.

Plateswereincubatedat37°Cunlessotherwise

indi-cated.

Construction of lysogens.Alysogenswereformed

byspottingphageatapproximately108phageperml

onto bacteria in a A agar or LB agar plate. After

incubationat 34°C,the surviving bacteriawere

puri-fied andtested forimmunityand resistance.

To make P1 lysogens ofM5222, M8521, Ql, and

Q8100,0.2-ml portionsof platingbacteriawerepoured

onto LB agar plates containing 2.5 mM CaCl2.

Plc+CMwasspottedontothese bacteriallawns, and

single colonieswerepurified fromthe lysisareas on A

agarplates containing12.5jig of chloramphenicolper

ml.Coloniesof purified isolatesweretestedonEMB

platesfor the ability torestrictAimm4cI.Kand the

failuretorestrictAimm4cI.K,P.

Coinfections.Afreshovernightculturewasdiluted

100-fold into A broth containing 0.2% maltose and

growntoadensityof1 x108to2x 108cells permlat

37°Con areciprocatingshaker. Bacteriawere

centri-fuged and thensuspendedat108cellsperml in0.01M

MgSO4when the helperwashAand in 0.001 M MgSO4

when the helperwashe";0.2-mlportions of bacteria and 0.2-ml portions of helper phage were mixed in conical 12-mil glass centrifuge tubesat afinal

multi-plicity of infection (MOI) of5(unlessotherwise indi-cated) andadsorbedatroomtemperaturefor15min. LB broth (3 ml) was added to each tube and the infectedcells wereaeratedbyshakingat37°Cfor10

min.AsamplewasdilutedintoSM containing

chlo-roform andtitrated for unadsorbed phage. The in-fected cells were centrifuged, 0.2 ml of test phage (MOI, 0.25, unless otherwise indicated) was added,

and the pellet wasblended with a Vortex mixerto

suspendthebacteria.After furtherphage adsorption for 15 min at room temperature, the cells were (i) eitherdiluted 100-fold intoprewarmed LBbroth and

grownforasinglecycle of growthtomeasurephage yieldorplated directly withtheappropriateindicator

bacteria for infectivecenterformationorboth and (ii)

diluted into SM containingCHC13tomeasure

unad-sorbed phage. When several indicator strains were

usedtodetermine the yields of individualphagetypes,

Avirwastitratedon allindicator strainstobesurethat

the phage plating efficiencies were comparable and withintheexperimental samplingerror.

Controlinfections were done in parallel; in these

infections bothhelperandtestphagesweremodified

to determine (i) whether the Ral helper effect was

specificforstimulatingunmodified phage growthand (ii)whetherphageyields under nonrestricting condi-tionswerenormal. Eachcoinfection described below wasdone atleasttwice in thehostindicatedand, in several instances, inadditional bacterialstrains.

Singlecycleofgrowth.M8521and M5222

(lyso-genscarryingdefectiveRal+ and Ral-prophages,

re-spectively)weregrownunderbothnoninducing(33°C) andinducing(40°C)conditions and usedsubsequently atthecorresponding temperaturesto testtheir abili-tiestosupportthe growthof bothmodified and

un-modifiedsuperinfecting phages; 100-fold dilutions of saturatedculturesweregrowntoadensityof 2x 108

cellspermland thensuspendedin0.01 MMgSO4. A

0.2-mlportion of bacteria and0.2 ml ofphagewere mixedat afinalMOI of0.25andadsorbed for20min atroomtemperature.Infectedcellswerediluted 100-foldinto prewarmed LB brothatthe corresponding

temperatures and 100-fold into SMcontaining CHC13

to measure unadsorbed phage. At 10-min intervals

samples were withdrawn, treated with CHC13, and

titrated.Theendpointandphageyieldsof thesingle

cycles of growth were determined when the phage

titersreachedaplateau.

Raleffectonthemodificationof nonreplicat-ingphage. Coinfectionswere

performed

asdescribed above, except that both the helper and test phages wereaddedatMOIs of10,and afteradsorptionof the testphage A antibodywas added atafinal K of45 beforeplatingforinfectivecenters.

Isolation of Arev exbiolOcI857.

Ared-gam-phages, such as AbiolOcI857, cannot grow in

recAA-hostsandaresaidtoexhibit the Fec-phenotype (59).

To enrich for Arevin a stock ofAbiolOcI857, phage

weregrownforasingle lyticcycleinthe recA-strain MI742 at anMOI of50. Arev was isolatedata fre-quency of5 x 10-9 from this stock aslarge

plaque

formersontherecA-strainM1742.(The frequencyof isolation ofArevfromastock of

Abio252c1857

treated inparallelwas9.6x

10-'.)

Besides theFec+phenotype, ArevexbiolOcI857shared thefollowingpropertieswith

previouslyisolatedXrevphage:itgrewinP2

lysogens

(60), it grewonOMmediumplates,and it grew much morepoorlythan itsparent

(XbiolOcI857)

at

43°C.

RESULTS

Ral

function

of X prevents

restriction

of

superinfecting bacteriophage.

The data in

Table

2are

consistent

with the

hypothesis

that Xhasa

function,

Ral,

whichcan

prevent

restric-tion of

unmodified

phage

by

thehost

specificity

system.Ral

appeared

tobe

present

after infec-tions with

hybrid phages

carrying

the bio252

substitution

butnotafter infections with

hybrid

phages

carrying

biolO

(Table

2, coinfections

1

through 4). Preinfection

with

Xbio252imm2".K

and

Xbio252cI857.K

partially

blocked

restriction of

superinfecting

Ximm43.0,

increasing

the

yield

of

Ximm4'

by

factorsof143and

17,

respectively.

In other

coinfections

(Table

2,

coinfections 5

through

8),

both

Ral+

Abio252.0 and

Ral-XbiolO.0

helper

phage

yields

were

approximately

10-3, indicating

Ral+

and Ral-

phages

were

re-stricted

tothesameextent.In

addition,

the

Ral+

helper

phages

(coinfections

5 and

7)

did not

block

restriction

of

superinfecting

Ximm434.0.

Thus, restricted phages could not direct the

synthesis

of

Ral,

whichmay be thereason

why

Xral+.0

phages failtopreventtheirown restric-tion.

The

magnitude

of the Raleffect variedwith the

growth

phase,

the culture

conditions,

the

bacterial

strain,

andthe

genotype

of the infect-ing phage. Eventhe

relatively

small Ral effect shown inTable2wouldhaveasizeable selective

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TABLE 2. Expression of Ralduring coinfection Coinfection' Phage yieldsduringcoinfection

Helpereffect No. Helper phage Yield ofhelper Yield ofXimm43.K

1 Abio252imm21.K 162(88)b 4.3x lo0- (25) 143

2 XbiolOimm21.K 122 6.0x 10-3 2.0

3 Abio252imm'.K 88 5.0X10-2 17

4 XbiolOimmA.K 135 9.0X 10-3 3.0

5 Abio252imm21.0 2x 10-3(9.4X 10-4) 7.0X 10-3 (170) 2.3

6 Abioloimm21.O 4X10-3 1.0X 10-2 3.3

7 Abio252immx.0 2x10-4 5.0x10-3 1.7

8 Abiol0immA.O 1 x10-4 1.0X10-2 3.3

9 None 3.0x 10-3 (160)

aThesuperinfectingphagewas

Ximm43.0.

Coinfectionswereinbacterial strain

Ql,

asdescribed in thetext. immXhelperphages carried cI857, imm2'phageswerecI-,and thetestphagewasXimm434cI6T.Yields ofhelper phagesweredeterminedbyplatingonQ8003 (rK+ MK+,Aimm43),andyieldsoftestphagesweredeterminedby platingonQ8002(rK'mK+,A)orQ8004(rK+mK+,

Aimm2").

bYields in parentheses are from coinfections done in parallel in which the superinfecting test phage (Ximm434.K)wasmodified.

advantageafter many

generations.

Xral.0would nothavetobeabletoprotectitself inorderfor the ral gene to show aselective

advantage.

In coinfectionssuch asthosedescribed inTable2, Xral+ in thewild could preventrestriction ofa superinfectingXral+ phage carryingan inappro-priate

modification;

insuchacase,

perpetuating

yourkind is thesame as

perpetuating yourself.

Ral

activity

doesnot

require

host Recor

phage

Redrecombination functions. In the experiments shown in Table 2, Ral

activity

was assayed by the

ability

torescue

phage carrying

the immunity region of434. Since this region doesnotcarry aknown K restrictionsite, I felt thatRalmightactby

stimulating

recombination of thehelper with restrictedfragments ofDNA ratherthanby rescuing entiregenomes.Table3 shows that Ral didnot

require

host recombina-tion functionRecA or RecB. Preinfectionwith

Xral+

(in this case,

Xbio252iMm21cI-.K)

in-creased the numberofinfectivecentersformed by superinfecting AbiolOcI857.0 131-fold. Like-wise, Ral didnot actbystimulatingRedrescue ofrestricted fragments of DNA sinceboth the Ral+ helper phage

Xbio252imm2'

and the test phage

XbiolOcI857.0

wereRed-.

Ral expression in the prophage state is underimmunity control. To determine which A genes were essential for Ral function and whether Ral was expressed in the prophage state, theAcI857 lysogens carrying the

Hi

dele-tion and the bio substitutions shown in Fig. 1 were infected with

Ximm434.0

before and after thermalinduction.

Aimm434.0

was able to grow in M8521(bio255cI857H1)at 40 but not at

34°C

(Table 4), implyingthatthe Ralactivityofthe prophage wasunderimmunity control. Ral ac-tivity was not expressed in M5222

(bio247cI857)

ateither 34 or40°C. These results showed that

TABLE 3. Ral doesnotrequire host RecABor phage Red recombinationfunctionsfor activitya

No. of infective centers produced upon coinfectionofQW11 (recA- recB-) Helper phage

Helper Testphage Helper

phg etphage effect Xbio252imm2".K 0.21(0.17)b 6.8x10-4 (0.01) 131 Abiol0imm21.K 0.14 8.0x10-5 1.5

None 5.2 x 10-5 1

a Thesuperinfectingtestphage wasXbiolO.O.Coinfections were performed in strain QW11 asdescribed in the text. Helper phages carried imm"cI;thesuperinfecting phage was XbiolOcI857. Infective centerformation ofAbiolOcI857 was assayed onstrainQ8004 (rK+mK+,XimmM2),whereas helper phage infectivecenters weredeterminedonstrainQ8002 (rK' MK+,A).

'Thenumbers in parentheses arefromcoinfections done

inparallelin whichthesuperinfecting test phage (AbiolO.K)

wasmodified.

sk2 ski

int redcM ral N cI cro clIOPren QSR A

rev imm434

bro252 p4

boo255

b.olObio247 Hi bo243

ottrO .mm2l hSO

Sprl mmP22(OP)P22

rv b2

FIG. 1. Prophage map ofA indicating relevant sub-stitutionsanddeletions.The regions of DNA deleted bythe variousdeletionsandsubstitutionsare indi-catedbythe black lines. Thefigureisnot drawn to genetic orphysical scale. The bio andatt8O substi-tutionseachcarry a K restriction site, and the mor-phogenesis regionprobably carries three K sites. K restrictionrecognition sites sk2 andsklare indicated onthemap(39).

theRalfunction(s) islocatedtotherightof the bio255 substitution and is atleastpartially de-letedbythe bio247 deletion. ralmay be located to the left of the immunity 21 substitution, as

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TABLE 4. Ralexpression by a defectiveXcI857prophageafterthermal inductiona

Yields of thefollowingsuperinfectingphages be-Bacterialstrain Defective prophage Temp(00) foreandafter thermalinduction ofprophage:

AbiolOimm21.0 AbiolOimm21.K

M8521 bio255cI857H1 33 2.0 x10-4 7.5

40 0.11 (275 33(4.4)

M5222 bio247cI857H1 33 6.4 x 10-4 7.5

40 9.0x 10-4(1.4) 60(8.0)

aSingle

cycles

ofgrowth were accomplished as described in the text. The numbers in parentheses are the increases in plating efficiency at 40 comparedwith the plating efficiency at33°C.Phage yields weredetermined by plating on strain Ql(rK'mK ). The phage yield determinedon strain Ql was 0.72 that determined onstrain Q8100 (r- m-).

Abio252imm2' hybrids had Ral activity (Tables 2 and3). Altematively,the Ximm21 phagemay

haveahybrid Ral function partiallyencoded by

the immunity region of phage 21and partially encoded by the ralgeneof A.

In the experiments shown in Table 4, the

progenyphageproducedinthepresenceof Ral

hadan efficiency of plating of0.72 ± 0.06 on a

restrictinghost (Q1)comparedwiththe isogenic nonrestricting hostIQ8100),whereas the control forefficiency ofplating (Xvir.K)plated withan

efficiency of 0.90±0.04.Thisresult is consistent

with thehypothesisthatatleast 80% of the Ral-protectedprogenyarefullymodifiedorthat all

oftheRal-protectedphage lackmodificationat

onesite.

Ral alleviates restriction by EcoK and EcoB butnotby EcoPl. To determine whether Ralcould act on otherrestriction systems, the coinfections shown in Tables 5 and 6were

per-formed. Preinfection of the

rB+mB+

strainQ8209 with the Ral+ helper Xbio252cI857.B increased the yield of superinfecting unmodified Ximm43"cI.0byafactor of 46(Table 5). Clearly,

Ral could alleviate EcoB restriction as

effec-tivelyasit could alleviateEcoK restriction.

However, in theexperiments shown in Table 6, under conditions in which Ral alleviated EcoK restrictionsubstantially,nospecific effect of Ral onP1restriction wasobserved. Atahigh

tem-perature, where Ral was expressed in the

M8521(P1) lysogen, there was a 2,750-fold in-creasein theplating efficiencyof

Ximm'.P,

but

onlya7.7-foldincrease intheplating efficiency

of

Ximm32.K,

whichwasnotsignificantly

differ-ent fromthe 2.9-fold effectonthetotally

modi-fiedcontrol(Ximm43'.K,P).

Ral isnotamethylasewithbothK

spec-ificity and B specificity. Fromthe results of

the experimentsshown in Table 4,Iconcluded

thattheprogenyresultingfromaRal-protected

infectionwereK modified. Because Ralprotects DNAfromEcoB,itwasof interesttodetermine

whether Ral was a methylase which acted

in-dependentlyof the host modification and

restric-TABLE 5.

Ral

alleviates E. coli Brestrictiona

PhageyieldsofAimm43 during coinfection of anrB+mB+host Helper phage

Helper Test phage Helper Abio252cI857.B 29(11)b 0.46 (5) 46 Abiol0cI857.B 23(25) 0.004(10) 0.4 AcI857.B 43(45) 4.9 (16)

490'

ArevexbiolOcI857.B 9(45) 1.9 (9) l9od

None 0.01 (30)

a Thesuperinfecting testphage was

Ximm43.0.

Coinfec-tions wereperformedinstrain Q8209(rB+

mBs)

asdescribed inthe text.Aimm'4 carriedcI6T.Aimm4 phage yields were calculated on strain Q8302(rB' mB+, A), and helper phage yields were determined on strain Q8803(rB+mB+, AimmM4).

'Theyieldsinparenthesesareforthecorresponding coin-fections in which thesuperinfecting phage(Aimm4cI.B)was

modified.

'Thehelper effect was duetoRaland Red. dThehelper effectwasduetoLarand Der.

tion system to modify DNA at both K and B sites. Preinfection of the

rK-mK-

host

Q8100

with the Ral+ phage

Xbio252cI857.0

didnot re-sult inBand Kmodifications of the

superinfect-ing

Ximm4.0

(datanot shown). However, it is conceivable that Ral

methylated

only

the paren-talphage DNA and that this

parental

modifi-cationwas

subsequently

lost

through

rounds of

replications

under the rK

mK

conditions. To observe the effect of Ralon

parental phage,

the infections showninTable7 were

performed.

In theseexperiments,Itriedtodetermine whether

superinfecting

parental

DNA became B modi-fiedby Ral without

enlisting

the

help

of EcoB. Toscorethe modification

acquired

by

parental

phageDNA, infectionswere

performed

in

lyso-genshomoimmunetothe

superinfecting phage.

In thisway, DNA

replication

was blocked

by

therepressorofthe resident

prophage,

whereas DNAmaturation and

morphogenesis

functions were

supplied by

the

heteroimmune

helper

phage.In

conclusion,

whenthe

rK+mK+

lysogen

M8002 was

superinfected

with

Ab2intF

red-cI-p4.K

after

preinfection

with either

Ral+

orRal-

helper

phage,

theprogeny

phages

were

not B

modified,

as

they

failed to

form

plaques

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TABLE 6. Ral doesnotalleviatePl restriction Efficiencyofplatingof:f P1lysogen Temp (°C)

Aimm434.K

Aimm4.P

Aimm43.K,P

M8521(P1) 34 6.0x 10-6 4.0x10-5 0.38

40 4.6 x 10-5(7.7)b 0.11 (2,750) 1.1 (2.9)

M5222(P1) 34 3.7x 10-5 6.9x 10-5 0.65

40 7.7 x 10-5 (2.1) 4.9x10-4(7.1) 1.1(1.7)

aEfficiency ofplating

of

Ximm434

on

P1

lysogensinthe presence(40°C) andabsence

(340C)

of Ral.

Aimm434

wasAimm434cI6T. Phageweretitratedasdescribed in thetext.Indicator culturesweregrown andpetriplates were incubatedatthe temperatures indicated.Efficiencyofplatingwasmeasured withrespecttothe titeron strainQ8100 (r- m-).

bThenumbersinparenthesesaretheincreases inplatingefficiencyfor each

phage/host

combinationatthe higher temperature.

TABLE 7. Ral isnot amethylase which modifies parentalphage DNA upon injectiona Phage yields fromcoinfections of rK'mK'A lysogen Coinfec- tingphage Helper phage Yield of

superinfecting

phagein thefollowing Yield of

tion hosts: helper

r-m- rK MK'

+MB

rB + phage

1 Ab2cl-p4.K Abio252imm21.K 0.25 0.32 3.1 x10-4 13

2 Ab2cI-p4.K Abiol0imm21.K 0.32 0.38 <1x10-3 20

3 Ab2cI-p4.B Abio252imm21.K 0.09 0.09 0.02 18

4 Ab2cI-p4.B Abiol0imm21.K 9.5x 10-4 6.4x 10-4 6.4x10-4 22

a Theprocedure for the Ral rescue ofnonreplicating phage is described in the text. The MOIs of both helper phage and test phage were each 10. Helper phages carried imm21cI-; Ab2cI-p4was Ab2intlO2red3cI857p4.

Coinfections were in strainM8002,aAlysogen of bacterial strain M72. (M72 does not exhibit any MOI effect on theability to restrict unmodified phage over a MOI range of 0.25 to 15 [data not shown].)AcI-p4were scored assmallplaquesonthefollowingAimm2' lysogens: strain Q8104 (r- m-), strain Q8004 (rK+ MK+), and strain Q8304 (rB+mB+).The yields of helperphages weredetermined by plating on the A lysogen Q8002 (rK+mK').In addition,XcI-p4werescored on strain MI742, a RecA- host (data not shown). Yields wereequivalent to the yieldsfound with strainQ8004; thus,AcI-p4did not rescue the bioregion of the helper phage.

on an

rBnmB+

host. As maturation of

unrepli-cated

phage

requires

recombinationtoproduce DNAof maturable

length (46),

therewas a ques-tionastowhetherthe

putative

EcoB modifica-tion of the

parental

superinfecting

DNA could havebeen lost

by

recombinationwith the

repli-cating

helper

DNA.Thatthiswas

unlikely

was

shown by additional

experiments

(Table

7,

co-infections 3 and 4), in which the test

phage

already

carriedaBmodification. Incoinfection 3,

Ab2int-red-cI-p4.B

phage

maturedunder

re-stricting

conditions

but,

inthepresenceof

Ral,

retainedparentalmodification(oratleastagood fractiondid,astheyields calculatedon the iso-genicrB+mB+and

rK+mMK

strainswere0.02and 0.09, respectively). Since parental modification

wasretainedontheRal-protected phage

during

the necessary recombination and maturation steps, it is

likely

that any de novo Ral modifi-cation of EcoB sites on

Ab2int-redc-p4.K

(Table7,coinfection 1) would also have survived subsequent recombination and maturation ifit had actuallyoccurred. Thus,it is unlikelythat Ral is a methylase which can recognize EcoB and EcoK sites

independently

of thehost mod-ification restrictionsystem.

A-P22 hybrid ASpi-imm"2

c25(OP)'P22

and 480 do notexpress Ralfunctions. S. typhi-murium has a modification andrestriction

sys-tem (hsdSB) which is allelic to the systems of E. coli K-12 and E. coli B (53). Because the arrangementofgenesinSalmonella phage P22 is like thearrangement in A (4), it wasofinterest

todetermine whether P22 carried a Ral function in a positionhomologoustothe position of Ral in A. TheA-P22 hybrid

ASpi-immP22c25(OP)

P22 carries a substitution of P22 DNA extending from redthrough the ren genes of A (Fig. 1) (22, 50). Since this hybrid carries the P22 erf gene, it was necessary to test Ral activity on a phage whoserestrictedfragmentscould not be rescued by Erf-promotedrecombination (Table 8). The unmodified test phage chosen was

Ah'attuo.

cI857.0, which has at least two K restriction sites in DNA segments nonhomologoustotheA-P22 hybrid; one of thesesites(sk480)segregates with h80, andtheother (sk2)segregateswith immu-nity of A (39).Hence, progeny phage were scored asbeing measured by Ral if they had both the immunity of A and the host range of480. Under conditions in which preinfection by Abio. 252imm2cI-.K stimulated theformation of

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TABLE 8. ASpi-imm2(OP) 22is

phenotpically

Ral '

Infective centers made by thefollowingsuperinfecting phages during coinfections: Helper phage

Wh8att"O.O Aimm434.0.

ASpi-immF22(OP)F22.K 1.3 x 10-3 (0.21) 0.23b(0.60)

Abio252imm21.K 0.23 (0.40) NTc (NT)

AbiolOimm21.K 7.3 x 10-4 (NT) NT (NT)

None 1.1 X 10-3(0.67) 4.0x10-4 (0.90)

a Coinfections were

performed

in bacterialstrain QD5003 as described in thetext. Infective centers of helper phages were determined on strain Q8003 (data not shown, but all were approximately 1). The numbers in parentheses are the yields inparallel coinfections of the corresponding modifiedsuperinfectingphages. Infective centers ofsuperinfecting Ximm4cI6Twere assayed in strainQ8004, and infective centers of superinfecting

phageAh'att'cI857were assayed in strainQA8002. bErf-promoted rescue of

imm.

cNT, Not tested.

fectivecentersbysuperinfecting

Ah8°att*cI857.0

200-fold, ASpi-imm 2c25(OP)P hadno effect.

The rescue of

Aimm'.0

(a phage with more

extensivehomology with the A-P22 hybrid) was

probably duetoErfrescueof the immunity434

region of restricted DNA. The lack ofrestriction alleviationby the A-P22 hybrid wasnotdueto

expression of an inhibitor in the P22

immun-ity regionorto insufficientexpression ofaRal

from the P22 leftward promoter, because

Abio252immP`(OP)m,

whichcontains the P22

promoter, could supply the Ral function of X during coinfection (datanotshown).

Similarly, another lambdoid phage (480) was

tested for restriction alleviation. In the

experi-mentsshown in Table9, W80didnotexpress a

Ral function. Preinfection with 480vir did not preventrestriction of

Aimm".0

byEcoK,asthe

yield of

Aimm'

wasnotincreasedby the pres-enceof the40virhelper.

Xrev has a restriction alleviation

func-tion, Lar. Arev (isolated as a revertant of Ared gam-, which formed plaques on

RecA-hosts) carries a substitution of foreign DNA

between b2 and cIII which codes for a new

attachment site, as well as an integrase and

recombination function (Fig. 1)(17, 59).Arevhas rescued therecEgenefrom E. coli andexpresses

its product, exonuclease VIII (ExoVIII), upon

infection (13), which gives risetothe

Der'

phe-notype (Redin reverse) and theability of this

"Red"gam-phagetoplateunder RecA-

con-ditions. The recE gene is expressed in sbcA mutants of E. coli (49). sbcA- mutants were

selectedasmitomycin C-resistant, Rec' revert-ants of recB recC- strains (3) and have the

additional phenotype that they exhibit a

sub-stantiallyincreasedplating efficiencyofA.0(44). ThesedatasuggestedthatArevwasprobably

created by arecombination event with E. coli

DNA which picked up the recE gene and a

nearby"int"gene,indicatingthatthe sbcA locus

may be part of a cryptic prophage (17). The

TABLE 9. 480isphenotypically Rala Phage yields on coinfection Helper phage

Yield of helper Yield ofXimm434

4080

105(135) 0.02(12)

None 0.04(55)

aThesuperinfectingphagewas

Ximm4.0.

The

coin-fectionswereperformedinbacterial strainQD5003as described in the text. Yields in parentheses are for

parallel coinfections where thetestphagewas modi-fied. Ximm43 wasXimmmcI6T, and

080

was p80vir. Theyields ofAimm43 were determinedonthe O'R host strain Ql, and

080vir

were assayed on strain QD5003.

increased plating efficiency of A.0 on sbcA-strains indicates that this cryptic prophage might also express a Larfunctionanalogousto the Ral function of A. To test this hypothesis, plaque-forming phagewereselectedon arecA

-host fromastock of the Ral- phageAbiolOcI857 (see above). This Xrev exbiol0 had the same propertiesaspreviously isolated Arev phages(17, 59): (i) it grew in recA- andpoLA- bacterial strainsand P2lysogens (60), (ii) it

plated

onOM mediumplates containing citrate, indicating that it hadanetdeletion ofphage DNA. (iii) andit grewpoorlyat ahightemperature

(430C).

Xrev exbiolOcI857 appeared to have a Lar function which

prevented

restriction ofthe su-perinfecting Ah

'bio243imm434.0

phage (Table 10). The numberof

rK+mK'

cells infected with

Xh80bio243imm434.0

whichformedinfective

cen-tersincreased50-foldinthepresenceofthe Arev helperphage, when Xh

imm434

infectivecenters

wereselected for(Table 10).Thiswas

probably

aminimum Lar effect since coinfection with the Arev

helper

phage

reduced the

probability

of infective center formation

approximately

five-fold when the

superinfecting phage

wasmodified (Table 10). These

Ah8'imm'

phages

probably

retained the bio substitution, as no

Ah'-revimm

43

recombinantswerescoredasinfective

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TABLE 10. Arevisphenotypically Ral+a

Infective centers and yields producedbythefollowing phagesduringcoinfection

Helperphage Xh`bio243iM4"b

ArevimM41

Infective Yield Infective centers Yield

centers

XrevexbiolOcI857.K 0.05(0.08) 35(27) 0.02(0.02) 0.7(0.7) None 0.001(0.38) 208(116) <5.3x 10-5(<3.1x 10-4)

aThesuperinfectingphagewas

AhJ°bio243imm4

'.0.

Coinfectionswere

performed

asdescribed in thetext. Ah5'bio243imm4' carriedthecI6Tmutation.Thenumbersinparenthesesarefromparallelcoinfectionswhere thesuperinfecting phagewasmodified. Infective centersweredeterminedon

rKW

mK'

hosts.

bInfective centers were determined on strain QA8002 (AR,

Ah"°cI857).

Plaqueswerevery tinydue to the heteroimmune prophage exclusion exhibited by certain Xh52 prophages against certain

Ah'

superinfecting phages (P. Toothmanand D.Forbes,unpublisheddata).Noinfective centers weredetectedonstrainMI8000

XR (recA ,Alysogen),indicatingthat theAh imm4&4infectivecenters onstrainQA8002carriedbio243 andnot rev.Thehelpereffect was due toLar.

'Infectivecentersandyieldsof revimm43 recombinantswereassayedonstrainMI8000(Rec-, A).Thehelper

effectwasdue toDer.

centers onthe appropriate selection host (data

not shown). It is unlikely that the

Ah8sbio243imm434

progenywererescued by the Der recombination function, unless for some

otherreasonEcoKfailed to restrict both thesite

inthe bio substitutionand the site(sk2)between bio243 and imm434. However, if such were the case, Xhsebio243imm43 should have behavedas

ifit had only one restriction site (skl), and it wouldnothave beenrestrictedsoefficiently (i.e.,

only 50-fold insteadof1,000-fold) (39).

Lar and Der haveverydifferenteffectsonthe average burst size of the phages which they

rescue, where averageburst is defined asyield per infective center. The average burst of

Xh8simm434,

Lar-protected phage was large

(equivalenttothe burstofthemodifiedcontrols) (Table10). Theaverageburst of theXrevimm43 recombinantsrescuedby Derwassmall, approx-imately 1(Table 10). This differencemayreflect

the times that Lar and Deractduringinfection (early and late, respectively). However, from these data it isimpossible totell whether Der acted before or after Lar or evenwhether Der

actedin thesamecellsasLar.

Table 11 presents further evidence that Lar

andDerareseparatefunctions. Gillenetal. (14)

isolated Rec-UV-sensitive revertants of

recB-recC- sbcA mutants in an attempt toisolate mutations in the recE structural gene. Three

such revertantswhich containedno detectable

ExoVIIIprotein are listed inTable 12. Two of

these (JC8689 and JC8690) simultaneously

re-verted to the stringent restriction phenotype. Thesemutantsmaybe sbcA+revertantsor

dele-tions of recEandlar.However,JC8691 retained itsrestrictionalleviationactivity despite its loss of ExoVIII. Thus, Lar activity expressed in

sbcA-strains isseparablefrom,oratleast does

notrequire,ExoVIII activity.

TABLE 11. Restriction alleviationactivity ofsbcA-strainsofE. coli isnotduetoExoVIIIactivitya

Bacterial strain EfficiencY

of

plat- tion

alle-viation

JC5519(recB-recC-) 2.0x10-5(0.50) 1 JC8679(recB-recC-sbcA-) 1.3x10'(0.56) 6.5x103 JC8689(Rec- revertant) 6.0x10-5(0.34) 3 JC8690(Rec-revertant) 6.0x10-5 (0.38) 3 JC8691(Rec-revertant) 1.3x10-2(0.38) 6.5x102

a Phage were titratedasdescribed in the text.Aimm43 was Aimm4cI6T. The numbers in parentheses are theplating

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efficienciesofAimm44.K.Restriction alleviation was measured withrespecttotheplating efficiencyonstrainJC5519. Bac-terial strainsJC8689,JC8690,andJC8691 were Rec- revert-antsofstrainJC8679 and containednoenzymaticallyactive ExoVIIIprotein(JaneGillen,personalcommunication).

TABLE 12. Ral isexpressed inAB1U57a

Phageyields during

coinfections Helper

Helper phage Hle

Yield of Yield of effect helper Ximm434

Abio252cI857.K 17(16) 0.02 (9) 20 AbiolOcI857.K 15(20) 0.001(6) 1

ArevexbiolOcI857.Kb 32(32) 1.1(7) 1,100

None 0.001(55)

aThe superinfecting phage wasAimm434.0. Coinfections

wereperformed in strain AB1157 as described in the text.

Yields ofhelper phagesweredeterminedon strain Q8003, and

yieldsofAimm43 weredeterminedon strain Q8002.Aimm3 carriedcI6T.Yields in parenthesisarefrom parallel coinfec-tions wherethesuperinfectingphage wasmodified.

'The coinfectingphagesupplied Derfunction as wellas

Lar.

TheArevisolated by Zissler et al. (59) and the Arevisolated byGottesmanet al. (17) also

sup-plied

Lar during coinfections and alleviated EcoB restriction.

Ral

activity

does not require transacti-vation ofhost Lar activity. To test whether

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GENETICS

Ral

activity requiredhost Larfunction,the coin-fection experiments describedinTable12were done. The coinfection host was AB1157. The observationsthat sbcA - mutantscannotbe iso-lated inAB1157(49), thatXrev cannot lysogenize AB1157efficiently(17), and thatAB1157is Rac-(35) all suggestedthat AB1157was deletedfor the cryptic prophage containing recE and lar. Recently, Kaiser andMurray (25) have shown that Xrevis formedbyrecombination with the Rac prophage, and these workers have con-firmed that the Rac prophage is deleted in AB1157. AsTable 12shows, bothAbio252cI857 and Xrev exbiolOcI857 express restriction alle-viation activityincoinfectionexperimentsin an AB1157genetic background. Therefore, the re-striction alleviation activity expressedinE. coli K-12 strains after infection byAbio252cI857or

Xrev

exbiolOcI857 does not result from the de-repression of the hostLarfunctionunder sbcA control.

DISCUSSION

Ralexpression is under controlof thecIgene inA and isexpressed early during infection by Xral+-modified phage. Unmodified phagewhich superinfect a Xral+-infected cell aremuch less likelytoberestricted,and theprogenyofthose phage which do escape restriction arefully or almostfully modified.

TheRalfunction ofA canpreventrestriction of unmodified phage DNA by the EcoK and EcoBenzymes.Ral doesnot seem tobe a meth-ylase which can recognize and methylate the

appropriate

bases within the

A(N)8TGC

recog-nitionsequence foundinbothEcoK and EcoB sites(26,31,40). If thiswereso,the nonreplicat-ing phage whichmaturein the presence ofRal

should

haveboth EcoK and EcoB modifications

(Table

7). Thus, the

specificity

ofmodification whichoccursinthe presenceofRalisthe spec-ificity defined

by

thehostmodification and re-striction system.

TheobservationthatRalcansavephagefrom restrictioninthe absenceofrecombination func-tions is consistent with the observation of Za-beau et al. (58) that Ral actsto rescue entire genomes, since it protectsnonhomologousphage P2 fromrestrictionatlowMOIs.

Under Ral- conditions, theRec and Red re-combination systemsseemtodiffer in theability torecombinehelper phagewithrestricted frag-ments. Thesimplest interpretationof the data inTable8is that theX-P22

hybrid

doesnothave aRal functionanalogoustothe Ral function of

A,

whichcanprevent restrictionof nonhomolo-gous DNA, at least under the conditions in which the XRal function is active. The Red function of this

hybrid (Erf

of P22) can

effi-ciently rescue the immunity region of phage

Aimm434,

indicatingthat Red canrecombine re-stricted fragments andhelper phages. There is no evidence that Rec can perform similarly, sinceinfective centers (which shouldbethe most sensitive indicator of such a phenomenon, as opposed to phage yield) are not increased sig-nificantlyby a

Xred-ral-

helper,comparedwith nohelperatall. This may be due toexpression of inhibitorsofrecBC nuclease,such asgam,by theunmodifiedtest phage used in these experi-ments,or, morelikely,it is anintrinsic property of the Recrecombinationsystem(perhaps,

sim-ply, restricted fragments are better substrates for nuclease digestion than for recombination).

In vitrostudiesonthe kinetics ofmodification andrestriction of unmodifiedDNA byEcoBand EcoKhaveshownthat it ismodified slowly and restrictedrapidly (55). SeveralwaysRal might stimulate modificationattheexpenseof restric-tion are as

follows.

Four stages have been rec-ognizedinthe restriction process. First,

S-aden-osylmethionine

is bound to the enzyme which undergoes activation; this form of the enzyme binds

nonspecifically

to DNA. Next, ATP is bound, and theenzyme undergoesa

conforma-tionchange, binds

specifically

andtightlytothe unmodified DNA, and, in the continued pres-ence of ATP, restricts the DNA (20, 57). Ral might interact

directly

with the restriction

en-zyme

complex

to

produce

anr-m+

phenocopy,

resembling the hsdR mutant EcoK-18, which forms short-lived

recognition

complexes

that cannotbestabolized by ATP andcannotrestrict

unmodified

DNA

(DNA.O),

butstill can meth-ylateit (6, 20). Debrouwere etal.

(9)

have sug-gested that the

Ral

effect maybe mediated

by

adirectinteractionwith rho. Evidence for such an

interaction

is all

indirect,

but it is an inter-esting model.

Invitro,EcoK and EcoB restrict unmodified DNAin the presence of

S-adenosylmethionine,

ATP, and

Mg2+,

butEcoK and EcoBcan

modify

DNA.0

if ATP and Mg2+ are left out of the reactionmixture(19, 37).Thus,

potentially,

Ral mightact to

stimulate

modification

by

lowering

ATP

concentrations

orMg2+ concentrationsor

bothin the

cells.

Another

possibility

isthatRal may be a

DNA-binding

protein

that interacts with DNA at the EcoB and EcoK

recognition

sitesto change thelocal structure of the DNA and make the DNAa morefavorablesubstrate formodificationoralessfavorablesubstrate for restriction.

The Lar

function

expressed by

Arev and sbcA -strains maypreventrestrictioninafashion anal-ogoustothe Ral function of A. The observation that A can rescue the reverse functions

(an

in-tegrase, a

general

recombination function

VOL. 38,1981

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[Der], and a restriction alleviation function

[Lar])

from many strains of E. coli suggested

that revispartof a lambdoidprophage.

Subse-quently, KaiserandMurray (25) demonstrated

thatrevishomologousto the Racprophage(33,

35), which isvery closeto the sbcA locus (34). Thus, the recE geneis involved in(i)

recombi-nation during transfer ofrac+ by Hfr mating with rac-recB- orrac- recC-recipients (35),

(ii) recombination proficiency in recB-

sbcA-and recC sbcA strains(30, 49),and(iii)

recom-binationproficiency byArev(13).Itis notknown

whether theLar expressedin sbcA- strainsand

byXrevis codedforbyagenedistinctfromrecE.

However,itis clear that

ExoVIII,

whichiscoded

forbythe recEgene (29),is notrequiredforLar

activity.The recEgeneproductwitha

molecu-lar weight of 1.4 x

105

may contain separate

domains for theexoVIIIandLaractivities (13).

It remains to be determinedwhether recEand

larareseparategenes.

ACKNOWLEDGMENTS

I thank Ira Herskowitz for histhoughtful tutelage and

providinganatmospherewhereideasandgraduatestudents flourished. I thank Jane Gillen for bacterial strains, Jane

Gillen,Marc vanMontagu,MarcZabeau,andJeffSchellfor

discussion andcommunication oftheirunpublished data,and KimPersons andPaulaBortle forpreparationof the

manu-script.

Thiswork wassupportedbyaPublicHealthService re-searchgrantAI11344to I.HerskowitzandbyaPublicHealth

ServiceBiochemistry TrainingGrant to theDepartmentof

Chemistry,UniversityofOregon.

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