Novel bacteriophage lambda mutation affecting lambda head assembly.

JOURNAL OFVIROLOGY, Feb. 1979,p.782-788 Vol. 29,No. 2 0022-538X/79/02/0782/07$02.00/0

Novel Bacteriophage Lambda Mutation

Affecting

Lambda

Head

Assembly

C. P. GEORGOPOULOS,I* R. BISIG,2 M. MAGAZIN,2 H. EISEN,2 AND D. COURT3 Departmentof Microbiology, UniversityofUtah, Salt LakeCity, Utah84132';DepartmentofMolecular

Biology, UniversityofGeneva, Geneva,Switzerland2;andDepartmentofMolecularBiology,National CancerInstitute,Bethesda,Maryland200143

Received for publication21June 1978

Anovel phage X

mutation,

called dc10, which interferes with properX head assembly hasbeen isolatedandcharacterized. PhageX

carrying

this mutation is (i) unabletoform plaquesat 30 or37°C but doesso at42°C and (ii) unable to form plaquesat42°ConpN-constitutive hosts. Both

properties

aredue todclO since all phage revertants for one phenotype simultaneously lose the other phenotype and vice versa. The dclO mutationhas been mapped in the Bgene and has been shown to be dominant over the corresponding wild-type product. At 30°C the dclO mutation results in the formation of abnormal petit X heads made upof pE, pB,pC, andpNu3. UnderpN-constitutive conditions, the dclO mutationresultsin theformation of abnormalpetitXheadsmade up ofpE, Xl, andX2only.A modelto explainthe dataispresented.

The bacteriophage A headis an icosahedron of about 60 nm in diameter. On the basis of genetic and biochemical analysis it has been shownthattheprotein products ofatleast nine phage genes (pA, pW, pB, pC, pNu3, pD, pE, pFI and pFII), possiblytwo moredefined by the defective mutations Nul andNu2, and the prod-uct of at least one bacterial gene (groE) are

required for proper head assembly (1). Ofthe headgeneproducts, onlythose of genesB, D, E, and FIIarefound unmodifiedin

purified

infec-tiousvirions. Inaddition, theproducts ofgenes B,C, and E,arefoundinmodified forms. One of these (B*) results from cleavage of intact pB duringassembly. Similarly,theproductof gene Eisfoundasseveralspecies,onepE thoughtto be theunmodifiedproduct,and two others (Xl andX2)whichresult fromcleavageof aprotein formed eitherbyfusionofthe Eand Cproducts or by fusion of cleaved E and Cproducts (6). Thegene Cproduct is foundonlyinthe form of Xl and X2. Theprotein products of the remain-ing head genes are not found in the finished virion. During A infection, a small head struc-ture, called petit

A,

about 2/3 the diameter of the mature phage head, can be seen in the electron microscope. Recently, it has been shown that thepetitA can encapsidate A DNA in vitroandsubsequently be joined to a phage tail to give rise to an infectious virion (9, 12), suggestingthat petitA is aprecursor to forma-tion of the phage head. The various forms of petit A resulting from infection with various phage mutants in the head assembly pathway

haverecently beendescribed, andaschemefor the formation of mature petit X has been pro-posed (7, 10, 12, 14).Briefly,itappears that the

Nu3gene

product

plays

therole ofa

scaffolding

protein, inasmuchasit isrequired forthe assem-bly of the B and C proteins into the petit A structureand issubsequently removedupon for-mationofXI and X2. Theprocessing of Cand E to form Xl and X2 does not require the presenceof theBprotein, whereas, the process-ing of pBtopB* dependsonthepresence ofan active C gene product. The hostgroE gene is essentialforall oftheseproteinprocesses totake place. Once petit X heads containing pE, pB, pB*, and Xl and X2 have been formed, DNA encapsidation can proceed with the final addi-tions of the D and FIIproteinstoformafinished head structure. The addition of afinished tailto this structure produces an infectious phage A unit.

In this paper we report on the isolation and characterization of a novel phage mutation, calleddc10, which interferes with proper A head morphogenesis. We show that dclO is a cold-sensitive mutation in the B structural gene, that itmakesphage growth sensitive to high levels of Nprotein, and that it is trans dominant over the wild-type B gene product.

MATERIALS AND METHODS

Bacterial strains. The Escherichia coli strains M72 (sup"), TC600 (sup+2) and Ymel (sup+3) were usedas indicator bacteria. A series of W3101 (sup') bacteriacarrying various i434cI+dgalprophages were 782

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LAMBDA HEAD MORPHOGENESIS 783

akind gift of A.Campbell. StrainDC510 isaK-12 his-strr(chiDpgl)Asupoderivativecarrying more than one prophage of the type

iecI857Nsus7sus53nin5

(sus [suppressorsensitive] is identical toam[amber]) and wasusedinisolating strain DC331 which carries the dclO mutation.

Phage strains. The various lambdoid phage strains used in this work were from our collection exceptphagecarrying the susa8 mutation in the Nu3 gene, which was a gift of Helios Murialdo. T4 wild type andT4rII169 were agift of Richard Epstein.

Media. T-broth(TB) is 1%tryptone-0.5%NaCl; T-agar is TB plus 1% agar. L-broth (LB) is 1% tryptone-0.5% yeast extract-0.5% NaCl; L-agar is LB plus 1.5% agar. Low-sulfur M9, for labeling with 35SO42 (NewEnglandNuclear),contains per liter:7g ofNa2HPO4, 3 g ofKH2PO4, 0.5 g ofNaCl, 1 g of NH4Cl,11mgofCaCl2,7mg ofMgSO4.7H20,1mg of

thiamine-HCl, 4 g ofmaltose, and all amino acids except methionine and cysteine as described before (4).Adil is10-2MTris(pH 7.5) and10-2MMgCl2. H-buffer is10-2 M Tris(pH 7.7),10-3 MMgSO4,and10-4 Mdithiothreitol.

Bacterial andphageplatings. Platingswere as

previously described (4).

Curing of DC331of residentprophages.Phage i2lcI+b2 at 107/ml was spotted on top ofa lawn of DC331 bacteria and incubatedovernightat30°C. Bac-teria whichsurvived the infectionatthecenterof the spot were grownand tested for ability topropagate phage iAcIat30°C.About 10% of thesebacteriawere

abletoplate phage iAcIat300Candwerejudgedtobe cured of their residentprophagessincetheywerealso unable toplatephagesieOsus29andi21Esus4.

Electron microscopy of lysates. TC600 bac-teriagrowing in TBsupplementedwith 0.2%maltose

at 4 x 108 cells/ml were infected with phage

ixcI60Nsus7sus53nin5Ssus7dc10

atamultiplicity of5

perbacterium and grownateither30 or420Cfor2h. The culturewasconcentrated20-fold, lysed with chlo-roform, andcentrifugedat10,000xgfor5min, anda

portion of the supernatantwaslayered on a carbon-coated grid and negatively stained with 1% uranyl

acetatesolution. Sampleswereobserved inaPhilips

300electronmicroscope at anaccelerating voltageof

60kV.

In vitro complementation. Cultures of

M72-(iWcI857Esus4)

andM72(iAcI857Jsus27)weregrown in TB at 300C to about 3 x 108 cells/ml, induced by heatingto420C for20 min, andsubsequentlygrown

at370Cuntillysed. Portions of eachlysateweremixed with eachother and withalysate of TC600 bacteria with phage iXcI857Nsus7sus53nin5dcl0 described above for electronmicroscopyandincubatedat370C for 2 h. Production ofviable phage was scored on TC600 bacteria at30°C.

Phagecrosses.Forphagecrosses, bacteriaTC600

were grown in TB-mal to 2 x 108 cells/ml, concen-trated20-fold inAdilandinfectedatamultiplicityof 5phagesof eachtype perbacterium. After20min at roomtemperature forabsorption,the infected cultures

were diluted 10,000-foldinTB andgrownat300Cfor 90min,atwhich timechloroformwasaddedto assure

lysis. Forcomplementationorrecombinationstudies

inwhich oneparentresidesasaprophagein theform

M72(iMc1857),bacteria were pregrown at 30°C in TB-mal,infected at amultiplicity of 5 with phage of the other type, shifted to 420C for 8 min to induce the resident prophage, diluted 10,000-fold into TB and grownat370C for 90 min. For measuring complemen-tation, the progeny phage was assayed on TC600

(W'cI+)

at 420C to detect the iAdclO parent and on TC600 (iAcI+) at 300C to detect the i2l parent. For measuring recombination, the i21sus' recombinants wereassayed on M72(iAbiol0cI857AH1) at300C.The iAdc+ recombinants from thecrosseswith thei434cI+dg prophageswere assayed onTC600 (i43cI+) at 30°C. Preparation ofpetit X heads. TC600 bacteria were grownin M9-lowsulfur at300C to about 2x108 cells/ml, concentrated 20-fold in 10-2 MgCl2 and in-fected withphageat amultiplicity of5per bacterium. After20minat roomtemperatureforadsorption, the infected bacteriawere diluted 20-fold into fresh M9-low sulfursupplemented with 0.2%glucose, 0.5 mCi of carrier-free35SQ42 wasadded, and the cultures were incubated at the desired temperature for 2 h. Cells wereconcentratedbycentrifugationat10,000xg for

5min,resuspended in0.25mlof H-buffer, lysed with chloroform, treated with 5 ,ug of DNase I for 5 min at 370C, and centrifuged at 10,000 x g for 10 min to remove cellular debris. Samples of the supernatants were layered on top of a 10-ml 10 to 30% glycerol gradient in H-buffer underlaid with 0.5 ml of CsCl solution ofdensity 1.62in10-2 M Tris (pH 7.5). Gra-dientswerecentrifugedat36,000 rpm for 75min in a BeckmanSW40 rotor, and 20 fractions were collected from the bottom and counted for radioactive material. Under these conditions the petit X head preparations

areusually found in fractions 7 to 12.

SDS-polyacrylamide gel electrophoresis. The procedure for preparing the samples and for

discontin-uous sodium dodecyl sulfate (SDS)-polyacrylamide slabgel electrophoresis were previously described (15).

RESULTS

Isolationof theDC10morphogenetic mu-tation.

(i)

Selection of bacterial strain DC331. Bacterial strain

DC510

(iANsus7-sus53cI857nin5)n

>Iwasgrown at

320C

in TBto

about5 x 108cells/ml. The culture was induced for 15 min at

430C

and plated at

400C.

Most cells die at this temperature

(40°C)

due to de-repression of the prophagelyticgenes.However, temperature-resistant survivorswerefoundat a frequency of 3 x 108. This selection was origi-nallyused in thehope ofidentifyingamongthe temperature-resistant survivors bacterial mu-tantswhichinterfered with theexpression ofthe Q geneproduct of phageX.Suchmutantswould not be able to express phage late functions in the absence ofN andQfunctional geneproducts. Strains carrying such

phages

are thought to survive at a high temperature with the phage existingas aplasmid (3).Atotal of50survivors from 10 independent cultures were tested for abilitytoplate phage

i434cI.

Only onesurvivor, calledDC331, provedunabletopropagate

i434cI

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784 GEORGOPOULOS ET AL.

phageat any temperature.

Since phage i43cIis derived fromphageAwith the exception of the immunity region (11), the block exerted by DC331 bacteria on phage growthis eitherat the level ofphageadsorption or at asubsequent intracellularstep.

(ii) Plating

properties

of DC331. Table 1

shows the

plating

properties

of various

phages

onDC331bacteria andaDC331 derivative cured of the resident

prophages.

The fact that both

i21b2

and

ijhAcI

phage

plate

well on DC331

bacteria suggests that the block exerted

by

DC331 is not atthephage adsorption level but at a subsequent intracellular event. The fact that phage T4rII169 does not propagate on

DC331 bacteria suggests that the lambda rex geneproduct isexpressed constitutivelyat370C. Furthermore, the

inability

of some lambdoid phagederivatesto propagate onthishost isdue

TABLE 1. Plating propertiesofvariousphageson strainDC331a

Efficiency ofplating"at38°Con: Bacteriophage

DC331 DC331 cured

iAcI60 <10-6 1.0

i'vir <10-6 1.0

i4.UCI <10-6 1.0

i21cI

0.8 1.0

i21cIPsus3 1.2 x10-4 <1o-6 i2`cIOsus29 0.12 <10-6

i21Ssus7 0.5 <10-6

q80cI

0.9 1.0

hAi80cI

0.65 1.0

T4 1.0 1.0

T4rII169 <10-6 1.0

aStrains DC331 and DC331 "cured" (cured from

prophageA)werepregrownin TB-malat42°Cbefore platingat37°C.

bEfficiency of plating denotes the number of plaques formedonDC331 relativetoDC331 cured. In the case ofphagei'cIOsus29 andi21cIPsus3 the effi-ciency of plating is relative to that on strain TC600(sup'2).

to a prophage-specific mutation since DC331

bacteria cured of their A prophages support

phageXgrowthnormally (Table 1).

(iii) Rescuing of the dclO mutation from DC331 bacteria. To study in detail the

pro-phage mutation(s) which interferes with A

growth, we tried to rescue a phage derivative

carrying this mutation from DC331 bacteria. DC331bacteriawereinfected with phage i43cII

at420Cand thesmallnumber ofphageprogeny

wereplatedonTC600(i43cI+) bacteria at420C. Twotypesofplaquewerefound from thiscross,

small and large. Upon further testing it was

found that the large plaqueswere

indistinguish-able from the iAcI857Nsus7sus53nin5 parent strain. Tenout oftensmall plaques examined, surprisingly, proved unable to propagate on

TC600, Ymel, or M72 bacteria at 30 or 370C.

One of those plaques, called iAcI857Nsus7-sus53nin5dclO,waschosen for furtherstudy.

Characterization of the dclO mutation.(i) Platingpropertiesof the dclO mutation. To facilitate phage growth and studies, we

at-tempted to cross the dclO mutation into a

N+iYcI60

background. In doing the cross

iXcI60N+ x iXcI857Nsus53nin5dcl0 we found

out that it was impossible to isolate the iAcI60N+dclO recombinant at anytemperature. It appears, then, that the combination of the

dclO mutationwithawild-type Ngeneis lethal.

Table 2 demonstrates this fact. Phage iAcI60Nsus7sus53nin5dclO, from the above

cross, is unable to propagate on strain M72

(iAbiolOcI857AH1) at 42°C, at which tempera-ture the N gene product is produced

constitu-tively. It appears that both the cold-sensitive phenotype and the N-sensitive phenotype are

due to the same mutation, inasmuch as all

(50/50) "revertants" on TC600 at 300C simul-taneously become abletogrowat420Con

M72-(iWbiolOcI857AH1).

The plating properties of

one such revertant, called Rl, are also shown

in Table 2.Furthermore,all (50/50) plaque

for-TABLE 2. Growthpropertiesofphage carryingthedclOmutation

Efficiency of plating'on:

M72 Phage

yield'

on

Phage M72at: TC600at: (iAbiolOcI857AHl) TC600at:

at:

300C 370C 420C 300C 370C 420C 420C 300C 370C 420C

iAcI60Nsus7sus53nin5dclO

<10-7 3x10-6 0.65 5x10-8 2x1O-7 1.0 6x10-8 0.06 0.75 6.2

iAcI60Nsus7sus53nin5dc+R1c 0.55 0.90 0.80 0.9 1.2 1.0 0.85 12.2 26.8 18.5 aEfficiency of plating denotes the number of plaqueson agivenhost andtemperature

relative

tothe numberofplaques formedonTC600at420C.

bPhage yield denotes the number of viable phageprogeny perinfected bacterium.Themultiplicityof infection was 0.1 phage

per bacterium.

'Isolation asplaqueformerofiAcI60Nsus7sus53nin5dclOonTC600at300C.

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mersat42°ConM72(iAbiolOcI857AHl)bacteria

simultaneously become able to propagate at 30°Constrain TC600. A reconstruction experi-ment, lysogenizing M72

(supo)

bacteria with phage iAcI857Nsus7sus53nin5dcl0, produced a

lysogenic strain whichwasunabletoform colo-niesat42°C.Inthisrespectitdiffers from strain DC331andweareforcedtoconclude that in the originalDC331strainatleastanother, and

pos-siblymore,prophage mutation exists in addition

todclO, whichallowsthe bacteriatosurviveat

42°C.Apparently, this additional mutation(s) is not carried by phage iXcI857Nsus7sus53-nin5dclO.

(ii)delOphenotype is dominantoverwild

type. Theresultspresented inTable 1suggest thatatleastone prophage mutation present in

strain DC331 caninhibit the growth of phageX

in trans. Tofindoutif thisproperty is also due tothepresenceofthe dclOmutation,wetested

the ability of dclO mutation carrying phageto block lambdoid growth intrans. Thiswasdone

by mixedly infecting bacteria with phage iAcI60Nsus7sus53nin5dclOandavarietyofother

lambdoid phages. Table 3 shows that the effect of the dclO mutation is more pronounced in

mixed infection experiments with

i'

and i434 phages, whose Ngeneproductsare

interchange-able, and less in mixed infection experiments withi2' phage, whose N function hasadifferent

specificitythan that ofiAN function (2). (iii) In dclO infection tail activity butno

head donoractivity is found. Since bacteria infected with

iecI60Nsus7sus53nin5dclO

phage lyseatall temperatures, itappeared likely that thedclOmutationspecificallyaffectsalatestep

during the phage cycle, atthelevel of heador

tail morphogenesis orboth. This was testedin vitro by mixing lysates of dclO-infected cells

with lysates of iAcI857Esus4 (tail donors) and iAcI857Jsus27 (head donors). Complementation occurred only with the iAcI857Jsus27 lysate (data notshown), suggesting that the block

ex-ertedby the dclOmutationon phage growthis

atsomestepduring head assembly, either at the

level of petitXformationor atthe DNA pack-aging step.

(iv)Electron microscopy of dclO lysates. Electron microscopic examination of lysates fromdclO-infectedcellsconfirmed andextended

the above conclusion. Itwasfoundthatat300C the infected cells contained a normal

comple-ment of tails and that petit A heads were the

only class ofAheadsseen (datanotshown).At

420C, however, in addition to tails and a few

normal heads, abnormal head structures and polyheadswerevisible (datanotshown). Inthis respect the delO lysate resembles infection of

gro+

supo

bacteria by Bsus (13) phage or

infec-tion ofgroE bacteria byAwild-type phage (4).

From these observations it appears likely that

the step during head assembly affected by the dclOis at the level ofproperpetit A assembly

andnot atthesubsequent DNA packagingsteps. (v) Recombination studies with the delO mutation. The above-mentioned conclusion

wasstrengthened by the recombination studies

shown in Table 4.The recombinationfrequency between the dclO mutation and BsuslO was

lower than the frequencies obtained in crosses

with ambermutantsinthe othergenesrequired

for headmorphogenesis.Furthermore, the wild-type allele of the dclO mutationwas rescuable

from an i43'dg deletion which ends between

genesBand C(dg514) butnotfroma deletion

which endssomewhere ingeneB(dg635).

(vi) Complementation studies with the delO mutation. The above conclusionwas

ver-ified inaseries ofcomplementation experiments

between iAcI6ONsus53nin5dclO and various i2l

derivativephage carrying mutations in the head assembly pathway. Itwasfound (Table 5) that

dclOphage complemented allknownhead mu-tants except mutantsin geneB. The lowlevels

ofphage produced in the successful complemen-tationtestsareduetothe partialdominance of

the dclO mutationunder theexperimental

[image:4.505.64.460.548.662.2]

con-ditions.Thus,itappearsthatthe dclOmutation TABLE 3. Mixedinfections

Totalphage yield TC600cells infected with:

300C 370C 420C

Parent A

iAcI60Nsus7sus53nin5dclO 0.02 0.42 5.3

iAcI60 13.5 88.4 39.1

i434cI 8.6 47.5 20.6

i21cI 19.2 63.4 32.7

Parent A+parentB

iAcI60Nsus7sus53nin5dc1O+iAcI60 0.04 0.36 4.1

iXcI60Nsus7sus53nin5dclO+i44cI 0.05 0.73 6.7

idcI60Nsus7sus53nin5dclO+

i21cI

0.38 2.9 24.6

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TABLE 4. Recombination studieswith the delO mutation

I Parent A Parent B i"sus+recombinants(%)

iXcl60Nsus7sus53nin5dcl0

i2"Asus32

2.3;2.4

iecI60Nsus7sus53nin5dc10

i2"BsuslO 0.08;0.19

iAcI60Nsus7sus53nin5dclO i21Csus42 0.18;0.25

iAcI60Nsus7sus53nin5dclO i21Nu3susa8 1.7;1.1

iecI60Nsus7sus53nin5dc10

i2'Dsusl5 2.8;1.7

Parent B

II ParentA iAdc+progeny

dg deletion endpointin gene

iAcI60Nsus7sus53nin5dclO i4dg651 A 2.1x 102

iAcI60Nsus7sus53nin5dcl0 i4dg615 A-B 1.5x 102

iAcI60Nsus7sus53nin5dclO i4dg635 B 6.7x 10'

iAcI60Nsus7sus53nin5dclO i4dg514 B-C 3.4X 105

iAcI60Nsus7sus53nin5dc10 i4dg614 C 6.4X 105

[image:5.505.58.452.90.245.2]

iAcI60Nsus7sus53nin5dclO i4dg652 D 3.9x 105

TABLE 5. Complementation studies with thedelO mutation

ParentA Parent B Yield of parent B

i2'Asus32 7.4x 106

i2'BsuslO 6.2x 106

i2lCsus42 5.8x 10"

i2'Nu3susa8 4.3X 106

i21Dsusl5 6.7 x 105

M72(iAcI857Nsus7sus53nin5) i2'Asus32 9.8x 108

M72(iAcl857Nsus7sus53nin5) i21BsuslO 7.5x 10"

M72(iAcI857Nsus7sus53nin5) i2lCsus42 7.0x 108

M72(iAcI857Nsus7sus53nin5) i21Nu3susa8 6.2x 108

M72(iWcI857Nsus7sus53nin5)

i21Dsusl5 6.5x 108

M72(iWcI857Nsus7sus53nin5dclO)

i21Asus32 1.3x 108

M72(iAcI857Nsus7sus53nin5dclO) i21BsuslO 8.1 x 10'

M72(iAcI857Nsus7sus53nin5dclO) i2lCsus42 4.4x 10"

M72(iAcI857Nsus7sus53nin5dclO) i21Nu3susa8 5.7 x 10"

M72(iWcl857Nsus7sus53nin5dclO)

i21Dsusl5 6.3x 108

is in the Bstructuralgene.

(vii) Abnormal

petit

X made

during

infec-tion

by

dclO

phage.

PetitX

fractions,

labeled

with 5SO4-2, were prepared from bacteria in-fected with phage carryingthe dclO mutations under various experimental conditions. The re-sults are shown in Fig. 1 and 2 and can be summarizedasfollows. Underconditions where the amount ofN gene product isreduced, e.g., infecting TC600 (sup+2) bacteria with iAcI60-Nsus7sus53nin5dclO, the majority of the petit A heads contain both B and C product in the expected amounts. Some of the C product is processed toXl and X2, and someof the B to B* cleavage takes place at both 37 and 42°C. Surprisingly, under N-constitutive conditions, e.g.,infectinganM72(iAbio10cI857AH1) lysogen pregrownat42°Cfor 4h, very littleBprotein is found associated with the petit N. However, C protein notonly wasfound associated with the petit X structures but wasprocessed to Xl and X2in asubstantialportion of them. This

obser-vationdefinitely accounts for the inability of the phagecarryingthedclO mutation to grow on N-constitutive hosts, although the mechanism of action by which normal amounts of N gene product result in exclusion of the B protein from petitAstructureremains unknown.

The composition of petit X heads made by phage iAc160Nsus7sus53nin5 after infection of TC600 or M72(ibiolOcI857AHl) at 30, 37, or 42°C was essentially that of wild-type petit A heads, i.e., normal amounts of E, B, B*, Xl, and X2proteinswerefound.

DISCUSSION

A novel morphogenetic mutant, called dc10, located in gene Bofphage X has been isolated andpartiallycharacterized. The presence of the dclO mutationinterferes with properphage head morphogenesis both at low temperature and in the presence of constitutive levels ofN protein. Both properties are the result of the dclO mu-tation since reversion to the wild-type

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LAMBDA HEAD MORPHOGENESIS 787

typefor either sensitivityresults insimultaneous loss of the other character. The step in head morphogenesis where theB gene product acts is

T 6_b A E

to

I3-0

E eL 0

2-U) on

5 9 13 5 9 13 5 9 13 FRACTION NUMBER

FIG. 1. Glycerol gradient profilesofpetit A

prepa-rations labeled with 35SO4-2 prepared as

previ-ously described (4). TC600 bacteria infected with iAcI60Nsus7sus53nin5Ssus7dcl0phage at: (A) 30°C;

(B)37°C; and (C) 42°C. M72 (iAbiolOcI857AHl),

pre-grown at 42°C for 10 generations, infected with

iAcI60Nsus7sus53nin5Ssus7dcl0 phage at: (D)300C; (E)37°C; and (F) 420C.

A B C

anearly one,involvingtheaddition ofthe B and Nu3 products to the petit X head (14). It is knownthattheC gene product can be processed inpetitX heads to Xl and X2 in theabsenceof functionalBproduct. Onthe other hand, theB gene product can be transported to the petitX headstructureintheabsence of C, but no pBto pB* processingor pNu3 exit fromthe petit Acan take place (7, 10, 14). In this respect, the com-positionof thepetitA indclOinfectionsis inter-esting. Under pN-limiting conditions, e.g., growth ofiAcI857Nsus7sus53nin5dcl0phageon

a sup+2host (TC600), themajority of thepetit AmanufacturedaremadeupofpE, pB,pC, and pNu3. Some processingof pB to pB* and pC to Xl and X2takes place, especially at 42°C, but the processing is never complete. Thus, it ap-pearsthatalthough the dclOmutationisinthe Bgene, it somehow interferes also withcomplete processing of theC protein to Xland X2, espe-ciallyat30°C. UnderN-constitutive conditions, however, e.g., growth ofiAcI857Nsus53nin5dclO phage on M72 (iAbiolOcI857AHl) bacteria, the majority ofthepetitA heads manufactured are composed ofpE and Xl and X2. Surprisingly, no B protein is found associated with petit A

D

,& A ,& .

B-_

_--,

_*B

-fift -Xi

-X2

E F

jAA AA

-C

- ~- E

-Xl

- ~~~~~X2

Nu3-

-Om -1 -Nu3

-_a

IPw

11I 1 1 1 1 1 II 1

5 7 9 4 6 8 67 8

I I I I I11

[image:6.505.53.247.124.269.2]

5 7 5 7 7 9

FIG. 2. SDS-polyacrylamide gel electrophoresis ofthe""S-labeledpetitApreparationsshown in Fig. 1. The percent acrylamidewas10. Wild-typeAphage preparationswereusedtomonitor thepositions oftheB,B*, E,Xland X2proteins, andpetitA heads derivedfrom groEA44

(iMcI857Ssus7)

lysogens fortheC andNu3 proteins.

29,1979

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[image:6.505.55.450.358.622.2]

788 GEORGOPOULOS ET AL.

structures atanytemperature. Under these

con-ditions, quantitative processing of pCtoXland X2 takesplace. Thus itappearsthat the BdclO

protein, when not present in the petit A struc-ture, doesnotinterfere with thepCprocessing. In thisrespect, the dclO infection is analogous toBsus infection ofa

gro'sup°

host. Toexplain

the fact that underpN-limiting conditions the BdclO product is transported into the petit A

structurebut under N-constitutive conditions is not, the following model is proposed. The B product,tobe properly assembled into the phage prohead, hastobe acteduponbyahostfactor.

The nature of this action and the subsequent modification of the B protein, if any, is not

known. The host factor could be thegroEgene

product, which has recently been identified on

SDS-gelsas apolypeptide chain of65,000 (5, 8).

Thepresence of the dclO mutation in thegene

product somehow interferes with the proper

modification of the B protein by the host

com-ponent, but does not completely eliminate it. Thus, under N-limiting conditions the levels of Bprotein within thecellarerelativelylowand

the slowed modificationprocesscaneventually

handle the B "processing" so that enough B

geneproduct is assembled into the petitA.Under

N-constitutive conditions, however, higher BdclO levelsaremanufactured,notall ofwhich

canbeprocessed within the time of infectionso

that the BdclOproductcannotbe assembledto the petit A structure atall. This, in turn, may

imply that the B product is transported into petitXincomplexes which contain multiple pB subunits and that the unprocessed BdclO

pro-tein interferes with the functionorassembly of

theprocessed pBorBdclOprotein. This scheme

explains whynoBdclOprotein is found in petit

A under pN-constitutive conditions. The

pres-ence of the dclO mutation may have an

addi-tional effect in slowing down the processing of both the B and Cproteins aswitnessedby the

factthat underlimiting conditionsnotallthepB

to pB* processing and the pC to Xl and X2 processing takes place evenat 420C. The cold sensitivity associated with the dclO mutation could be duetosuchaneffectonthepB and pC

processing.

There are three lines of evidence that give

partial support to the abovescheme, proposed toexplain the effect of the dclO mutation. First,

wehaveshown previously (4) thatsome of the

A mutants, called E, which can overcome the

groE block, mapin thephage Bgene,implying an interaction between this gene product and

the host, groE gene. Second, some evidence

exists that thegroE bacterial product and the B

gene product interact as witnessedby the fact

that they cosediment ina 25Scomplex (Helios

Murialdo, personal communication). Third,

some of thegroEbacterial mutants inour col-lection can be shown, under certain temperature conditions, tomimic the effect of the dclO mu-tation. That is,when infected withwild-typeX phage, the majority of the petitX which accu-mulatecontain normal amounts of Cproteinbut very little or no B protein. Furthermore, some groE bacterialmutants canbe found which

ac-cumulatepetitAwhere thepBtopB* and pCto

XlandX2

processings

are

incomplete.

Thismay

imply that the groEgeneproduct

plays

adirect rolein thecleavageprocesses.

ACKNOWLEDGMENTS

Financial supportwasprovided by grant3-519-75from the Fonds National Suisse de la RechercheScientifique and by Public Health Service grant GM23917 from the National Institute ofGeneral Medical Sciences.

Wethank Sherwood Casjens for reading the manuscript and EthelEldredge for typing it.

LITERATURE CITED

1. Casjens, S.,and J.King.1975.Virusassembly.Annu. Rev. Biochem. 44:555-611.

2. Friedman,D.I.,G. S.Wilgus,and R. J. Mural.1973. GeneNregulator function of phageAimm2l:evidence thatasiteof N action differs fromasiteof N recogni-tion. J.Mol. Biol. 81:505-516.

3. Friedman,D.I.,and M. B.Yarmolinsky.1972. Preven-tion of thelethality of induced A prophage byanisogenic Aplasmid.Virology50:472-481.

4. Georgopoulos, C. P., R. W. Hendrix, S. R.Casjens,

and A. D.Kaiser. 1973. Hostparticipationin bacterio-phage lambda headassembly. J.Mol. Biol. 76:45-60. 5.Georgopoulos,C.P.,and B. Hohn. 1978. Identification ofahostprotein necessary for bacteriophage morpho-genesis (the groE gene product). Proc. Natl. Acad. Sci. U.S.A.75:131-135.

6.Hendrix,R.W.,and S. R.Casjens.1974.Protein fusion: a novel reaction in bacteriophage A head assembly. Proc.Natl.Acad.Sci. U.S.A. 71:1451-1455.

7.Hendrix,R.W.,and S. R.Casjens.1975.Assembly of bacteriophage lambda heads: protein processing and its genetic control in petit A assembly. J. Mol. Biol. 91: 187-199.

8. Hendrix,R.W.,and L.Tsui.1978.Role of the hostin virus assembly: cloning of the Escherichia coli groE gene and identification ofits protein product. Proc. Natl. Acad.Sci. U.S.A.75:136-139.

9. Hohn, B.,and T. Hohn.1974.Activityof empty head-likeparticlesforpackagingof DNA ofbacteriophageA in vitro. Proc.N0tl.Acad.Sci. U.S.A. 71:2372-2376. 10. Hohn,T., H. Flick,and B.Hohn. 1975.Petit A, a family

ofparticles from coliphage lambda infected cells. J. Mol. Biol. 98:107-120.

11.Kaiser,A.D., and F. Jacob.1957.Recombination be-tweenrelatedtemperatebacteriophages and the genetic controlofimmunity and prophagelocalization.Virology 4:509-520.

12.Kaiser, D.,M.Syvanen, andT.Masuda. 1975.DNA packaging stepsinbacteriophage A headassembly. J. Mol. Biol.91:175-186.

13.Kemp, C. L., A. F.Howatson, and L. Siminovitch. 1968.Electronmicroscope studies of mutantsof lambda bacteriophageI.Generaldescriptionandquantitation of viralproducts. Virology36:490-502.

14. Ray, P., and H.Murialdo.1975.Therole ofgeneNu3in bacteriophage lambda head morphogenesis. Virology 64:247-263.

15. Russel,M. 1973.ControlofbacteriophageT4 DNA po-lymerase synthesis.J. Mol.Biol.79:83-94.

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