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Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging.


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Vol. 41, No. 2 JOURNALOFVIROLOGY, Feb. 1982,p.657-673



of the Bacterial

groM Gene Product in





Arrest at the Level





DepartmentofGenetics, Instituteof BiologyIII, Universityof FreiburgimBreisgau, D-78Freiburgim Breisgau, WestGermany,1 and Departmentof Microbiology, Biozentrum, Universityof Basel, CH4056


Received 14 June1981/Accepted18September1981

The multiplicationofbacteriophage T7 is blocked in Escherichia coli M. The

genetic determinant of thisability(groM) to inhibit T7growthwas transferred to an E. coli K-12 recipient by means ofconjugation. We determined at which

precisestep T7maturation is blocked.Phage-directed protein andDNAsynthesis

as well as degradation of host DNA were not qualitatively affected. Instead of

infective phages, only preheadswereproduced. These, however,werematurable

invitro. The newlysynthesized phageDNAaccumulated inaconcatemericform

and maturedfrom its tetramericor


forms (very fast


DNA) only into its dimeric form(fast-sedimenting DNA)orlonger forms.Thefollowingstep,

i.e., the maturation of the dimerictounit-length DNA, wasnotobserved. Since

the concatemeric form of T7 DNA accumulated in spite of the presence of

maturable preheads, it is likely that the maturation process was blocked atthe

level of DNA packaging. As intermediates in the packaging process, we found someprehead-DNAcomplexes.Weinterpretedtheseas trueassembly intermedi-ates (or breakdown products thereof), since the attached DNA was still in its

concatemeric form. This shows that the very first DNA packaging step, the

binding of theprogenyDNAtothepreheads, wasobviouslynotblocked. Rather,

alaterstep, suchasthefilling of the preheads withT7 DNA orthestabilizationof

completely packaged particles


the finalcuttingof theconcatemers into

unit-size length),was inhibited.

Theinfection of certain bacterial strains with bacteriophagesmayleadto an"abortive" phage reproduction. The mechanisms of this inhibition of viraldevelopmentare numerous.(i) Bacterial restriction enzymes may fragment the parental phageDNA. Inthiscase,thevegetativecycleof the virus isinterruptedatitsverybeginning (2). (ii)For a variety ofreasons, an incompatibility

may exist between the host bacterium and the phage(19). For example, abacterial gene prod-uct necessary for phage maturation may be

altered so that the host phage interaction is disturbed. Growth oftheuninfected bacteriumis not remarkably affected by this change. Addi-tional dispensable genetic elements in the host,

such as Ffactors, may also cause inhibition of phagemultiplication,asinthe case of

T7-infect-ed male strains ofEscherichia coli (6). In this

case,the early phage ofthe vegetativecycle of T7 proceeds normally but is followed, 10 min

tPresent address: Department of Microbiology, Biozen-trum,University of Basel, CH-4053 Basel, Switzerland.


(p.i.), by a rapid decrease of all metabolicprocesses.

Anumber of female strains ofE. coli


disturbed development of phage T7 have

beenisolated and described byHausmann(11). One of these (E. coli M) did not show any

restriction of the parental phage DNA, but a

reduced burstof 0.5 phage per cell. Thequestion arises of whether the abortive phage reproduc-tion is due to a host factor coded by the host

chromosome or by a plasmid. That the defect

wasdueto ahost cell determinantwas previous-lyshown by the fact that the defect (groM) could

be transferred to E. coli K-12 by conjugation withan F' E. coli M derivative (M. Reck, Ph.D.

thesis, University of Freiburg im Breisgau, Frei-burg, WestGermany, 1976).Theprecisenature

ofgroMisyet to be determined. Inthis paper, weinvestigatedthe step at which the

morphoge-netic pathway isstopped inthe K-12 groM

(K-12-M) strainafterT7infection. Particular




pathway leads to the accumulation of abortive 657

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phage particles. In a second paper (A. H. U.

Kuhn, H. Tutte, and E. Kellenberger,

manu-script inpreparation), we will show that the level of intracellular K and Mg is lower in infected K-12-M than in K-12 and that the morphogenetic block isovercomebyraising the outside

concen-tration of Mg.


Bacterial and phage strains. E.coliBE(sup'),K-12 (W3110), and B40-1(supF) were used as normal hosts. E.coli Mwas described by Hausmann (11). E.coli K-12-M, obtained by M. Reck, was cured of F' by acridine orange and was insensitive to the male-specif-ic phages M12 andQpbutretained theGroM- pheno-type. Phage T7-Ha has recently been described and compared with other T7 strains (36). The T7 mutant in gene 5, amH30, was described by Hausmann and Gomez (13), and that in gene 10,aml3, was a gift of F. W. Studier. M9 minimal medium (1) supplemented with amino acids (1% [M9A] or 0.1% [M9a]) was used.

SDS-polyacrylamide gel electrophoresis. The

experi-mental equipment for polyacrylamide gel electropho-resis in the presence of sodium dodecyl sulfate (SDS) was similar to that described by Studier (34). Infection and the treatment of samples were essentially as described by Korsten et al. (18). Host bacteria (3 x 108 cells per ml) were UV irradiated to inhibit host protein synthesis, infected with a phage multiplicity of infec-tion (MOI) of 5, and incubated at 37°C. At various times, 50 ,uCi of[35S]methionine (Amersham Corp.) permlwas added. Two minutes later the cells were heated to100°C in SDS, and the proteins were electro-phoretically separated on a polyacrylamide gradient (10to18%) slab gel. Autoradiographs of the dried gels were densitometrically analyzed with a Joyce Loebl Chromoscan.

Degradation of prelabeled host DNA. Five

microli-tersof a freshovernightculture of E.coli 12-M, K-12, andBE grown in M9a was diluted into 5 ml of fresh

M9 medium. One microcurie of [3H]thymidine (19 mCi/ml; Amersham) was added, and the culture was shakenat37°C until the cellconcentration reached 2x

108 cells/ml. The cultures were centrifuged (Sorvall typeSS-34 rotor) at 2,000 x g for 5 min, and the cell pellets were suspended in 5 ml offresh, prewarmed

M9amedium. Phage was addedat an MOI of 10. At

different times after infection, portions of 0.5 mlwere

taken, treated with egg white lysozyme for 20 minat

4°C (200 ,ug/ml), and precipitated overnight in 10% coldtrichloroacetic acid (TCA)containing 7% NaCI.

Eachsamplewasfiltered through aGF/C filter (What-man), and the filters were washed and dried. The filters were placed into Insta-Gel (Packard Instrument Co., Inc.) scintillation liquid, and the TCA-precipitat-edradioactivity retained by the filterwas determined

inaPackard-2425 scintillation counter.

PhageDNA[3H]thymidinelabeling. Freshovernight cultures ofE.coliK-12-MandE.coliBEwerediluted 1/50 into M9 medium. The cultures were grownto a

concentration of2 x 108cells/mlat 37°C. Toinhibit

host DNA replication, the cells were irradiatedwith UV light for 30 s, at a distance of 50 cm, with a

Westinghouse Sterilamp 182-20(main emission band

at 254nm).

After 2 min of aeration at 37°C, the culture was

infected with T7phage at anMOI of 5. Six minutes afterinfection, 0.1 p.Ci of [3H]thymidine per mlwas

added (Amersham; 50 Ci/mmol), and 5-ml portions

were withdrawnatthetimes indicated. The isolation procedurewasessentiallyasdescribedby Center(3).

Theportionsweredilutedinto 5 ml ofanice-cold Tris buffer(100 mM, pH 8) containing 15 mM EDTA and 150 mM NaCl andcentrifugedat12,000x gfor 5min

at4°C. Each pelletwassuspendedin 0.5 ml of 50 mM Tris-hydrochloride (pH 8) buffer with 5 mM EDTA and 100,ug of egg white lysozyme per ml. After 20 min in ice, 0.1% N-lauroylsarcosine (Sarkosyl; Sigma

Chemical Co.) was added, and the temperature was

raisedto30°C. After 5 min of this treatment, 50 ig of pronase per ml(Calbiochem)wasadded. Afteratleast 50min,200,ul of each samplewaslayeredontop ofa

13-ml linear 5 to 20% sucrose gradient and sedimented inanSB-283 rotor of an IECB-60centrifugeat140,000

x g for 2 h at 4°C. After centrifugation, 0.5-ml fractions were collected from the bottom of thetube, and 0.1 ml of each fraction was tested for TCA-precipitable radioactivity, asdescribed above.

Isolationofpreheads. Fortheisolation ofpreheads,

wefollowed essentially the method described by Mi-yazakietal. (26). Five-liter cultures of E. coli BE and ofK-12-Mweregrownto adensity of 5 x 108 cells/mlat

37°C in M9A medium. The culture was infected with T7phage at an MOI of 5. Cells were harvested at13 and 15 minp.i.,respectively. The infected cellswere

immediately spun downat8,000 x g inanIEC-DPR 6000 centrifuge for 20 min at 4°C. The pellets were

suspended in 20 mMTris-hydrochloride buffer (pH8) containing 0.1 M NaCl and 2.5% glycerol. The culture waslysed byadding 500 ,ug of egg whitelysozyme per ml andafew drops of chloroform. After incubationat

37°C for 15min,50 p.g of DNase per ml(Calbiochem)

wasadded, and the lysate wascentrifuged 12,000x g for20 minat4°C. The supernatant fractionswereput intopolyallomer tubes and centrifuged at 4°C inan A-237rotorofanIEC-B60ultracentrifugeat10,000x g for2 h. Eachpelletwassuspended in 1 ml of 20 mM Tris-hydrochloride, pH 7.5, containing 0.5 M NaCl and1 mMMgCl2 and storedovernight, and 200 pl of this extract was layered ontop ofa12-mi 5 to20%

sucrose gradient. Gradients were spun in an SB-283

rotor in an IEC-B60 ultracentrifuge for 150 min at

140,000 xg,and 0.5-ml fractionswerecollected and analyzed by electron microscopy.

Isolation ofpossible packagingintermediates. Fresh overnight cultures of E. coli K-12-M and BE were diluted 1/50 intoM9amedium. Then 100-mlportionsof these cultureswere grownto aconcentration of3 x

108cells/mlat37°C. T7phagewasaddedat anMOI of 5;4minlater,0.1 ,uCi of"4C-labeled amino acids per ml (Amersham; 58 mCi/matom) and 0.2 ,Ci of [3H]thymidine per ml(Amersham; 45 Ci/mmol) were added. At the times indicated, the cultures were

harvestedbycentrifugationat6,000x g for 20minat

4°C,and thepelletswerewashed andsuspendedinto 5

mlof 20mMTris-hydrochloride buffer, pH 7.9, con-taining 200mMNaCl and1mMEDTA. The bacterial debriswasremovedbycentrifugationat12,000xg for

20minat4°C,and 3 mlof thesupernatantswaslayered

on top ofadiscontinuous sucrose-D20 gradient

ac-cording to Moncany et al. (27). The gradient was

prepared byplacing successive layersontop ofone J.VIROL.

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another,asfollows: 4 ml ofa sucrosecushionwitha

density of 1.32,4ml withadensity of 1.28,5mlwitha

density of 1.24, 5 ml withadensity of1.20, 6mlwitha

density of1.16,and 8 mlwithadensity of 1.12. Each

of thesucrosesolutionswasin 20 mM

Tris-hydrochlo-ride, pH 7.8, 1 mM EDTA, 200 mM NaCl, 1 mM

spermidine, and 1 mMdithiothreitol andwasprepared withheavywater(Merck & Co.). Thegradients were

centrifugedat4°C for 7 h inanSB-110rotorofan

IEC-B60ultracentrifugeat13,500 x gat4°C. Fractions of

0.6 ml were collected, assayed for acid-precipitable radioactivity, and analyzed by electron microscopy.

Electron microscopy. (i) Embedding and thin section-ing. Ten-milliliter cultures ofE. coli BE or K-12-M were allowed to grow in M9amedium at 37°C to a

density of3x 10'cells/ml.T7phagewasthenaddedat anMOI of 5. Atvarious timeintervals,2-mlsamplesof

theculturewereprefixedby the method of Van Drielet

al.(37)with 5%acrolein (Fluka) and 4% glutaraldehyde

(Polysciences) at 0°C for 10 min. The prefixed cells

were sedimented inacentrifuge, and the pelletswere

washed, agar embedded, osmified, postfixed,

dehy-drated by ethanol, and embedded in Epon 812 as

described by Wunderlietal.(39). Afterpolymerization

at50°C for24 h and70°Cfor 1 week, the embedded materialwasthin sectionedon anLKB-8800A

Ultra-tome III. The sections were collected on

carbon-coated collodion films on 200-mesh copper grids (Balzers).Thesectionswere stained with uranyl ace-tate(Fluka; 4%)for 1minandlead citrate(Merck)for 1 min, accordingtoReynolds (28). Micrographswere taken inaPhillipsEM 300electronmicroscope atan

accelerating voltage of80kV,usingKodak-LR film.

(ii) Negative staining. One drop of fractions from

sucrosegradientswasdialyzed againstextraction

buff-erand then applied to collodion-carbon-coated grids

for 1min. After washingon adrop ofdistilledwaterfor 10s,the gridwasputon adropof 1% uranylacetate,

pH 4.6,for 1 minfornegative stain. After theexcess

stainwasremoved, the gridsweredried andexamined

withaPhillipsEM 300electronmicroscope.

(iii)Observation ofDNA-protein complexes.The


on pentylamine glow discharged carbon films (5).

Then 15,ul of the fractionswasspreadonthegrids and

stained with a2% uranyl acetate solution and shad-owedby platinum. The grids were examined witha

PhillipsEM 300electron microscopeatanaccelerating

voltage of80kV.


Effects ofgroMonthe physiology ofT7

multi-plication. Asafirststepindetermining thecause

of the blockofphage development in T7-infect-ed E. coli K-12-M cells, we followed bacterial

growth and lysis by the turbidity of the culture andby PFU. Turbidity stopped increasing after about 5min p.i. and stayedconstantforatleast 2 h. Lysis did not occur, and only 0.5 phage/cell was detected after the cells were lysed by egg

whitelysozyme and chloroform at20min p.i. Thefateofhost DNAwasfollowed by

moni-toring the degradation of [3H]thymidine-prela-beled DNAinT7-infected E. coli K-12-M cells andcompared with that in T7-infected K-12 and

BE cells (Fig. la). To prevent any interference by newly synthesized phage DNA, we chose a

T7mutant phage defective in DNAsynthesis (T7 amber mutation in gene 5). The degradation of the host DNA began at about 5 min after the onset of infection, continued for 10 min, and reached a plateau at 60% of the initial 3H

label-ing. There was no significant difference in this respect between the wild type and the E. coli K-12-Mstrain. In another experiment, the synthe-sis of phage DNA was measured by incorpo-ration of [3H]thymidine, which was added simultaneously with the phage (Fig.lb). In K-12 and BE, the synthesis started about 5 min after infection and leveled off at 15min p.i., the time atwhichlysis occurred.Wealso observed some

incorporationof[3H]thymidinein the T7-infect-ed K-12-M cells, but at a greatly rT7-infect-educT7-infect-ed rate.

The total amount of synthesized DNA reached only about 20% of that found in infectedE. coli BE and E. coli K-12 cells.

Phage-directed protein synthesis in

T7-infect-edE. coliK-12-M and BE cells was also studied.

Figure 2a shows the autoradiographs of

[35S]methionine-labeled phage proteins

separat-ed by SDS-polyacrylamide gel electrophoresis.

At the indicated times, the label was added to T7-infected cultures during 2-min pulses. We

observed thatphage protein synthesis in

K-12-M-infected cells started normally about 2 min

p.i. with the earlyproteins, but after 8minp.i. the rate of synthesis decreased slightly. We

compared the phage-directed ,protein synthesis of K-12-M cellswith thatofB cellsby

electro-phoresis and obtained similar patterns (Fig. 2b).

Noprotein bandwas missing in thecase of

T7-infected K-12-M cells, but the rate of protein synthesis was slightly reduced compared with that in BEcells. Studiesontheincorporationof


amino acids into acid-precipitable material indicated that the phage proteins

syn-thesized inK-12-Mcells reached only about 30% of the amounts in K-12 or BE cells (data not

shown). A transcriptional block in the K-12-M

cells isthus not verylikely, sincephage-directed

protein synthesis

wasonly slightly reduced.

Weconcludethat, after infectionof E.coli K-12-Mby T7, host DNA isdegraded,



is produced, andphage-coded proteinsare syn-thesized. We felt that the observed reduction of

synthesisrates was notsufficient toexplainthe

maturation block, and another cause must be

responsible for the abortive outcome of the

phage infection: only a much more specific action could explain the nearlycomplete block


Analysis ofviralDNAaccumulatedininfected K-12-M cells.TheT7progeny DNAwaslabeled



as described in Materials

andMethods.Attheindicatedtimes, samplesof

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5 100 15 220

Time after infection

K-12 -M




0 5 10 12 15


Time after infection

FIG. 1. T7-induceddegradationof host DNA andphage-directedDNAsynthesis. (a)Host DNA(E. coliBE,

K-12,orK-12-M) wasprelabeled over10generations (37°C)asdescribed in MaterialsandMethods.Infection

(MOI=5)wascarriedoutwithanambermutantdefective forT7DNApolymerase(gene 5product).The

TCA-precipitableradioactivitywasassayedattheindicated times. (b) Exponentially growing cultures ofE.coli BE, K-12, and K-12-Mataconcentration of 2x 108 cells/mlwereUVirradiatedtoinhibit synthesis of bacterialDNA.

After2min ofaerationat37°C, wild-typeT7phage (MOI=5) and[3H]thymidine(1 ,uCi/ml)wereadded. After

various times, samplesweretakenand assayedfor acid-precipitableradioactivity. Symbols: (0) K-12-M, (A)

BE,and(0)K-12cells afterT7am5infection; (*) uninfectedK-12-Mcells.

the infected cultures were washed, lysed, and

layered on top of5 to 20% sucrose gradients.

The sedimentation profiles of the phage DNA harvested 10 min afterinfection of BE cells by

T7 (Fig. 3a) showed that onlypartof thephage

DNA cosedimented with the '4C-labeled T7 DNAused as a marker. Mostof the DNA was

observed as faster-sedimenting material (FS

fractions), which we interpreted as a

concate-meric formofphage DNA (16). Theamount of theseconcatemers decreasedstronglyat14min p.i. (Fig. 3a). At this time the situation was

similartowhatweobserved in T7-infected K-12

cells(Fig. 3c).

In T7-infected K-12-M cells, the DNA sedi-mented in a broad peak ranging from unit-size

T7 DNAto concatemeric forms when observed

at 10 min p.i. (Fig. 3b). Ten minutes later, the


E >

100--C _0c



10 a 50 I



K-12 -M






-16 -E



12-0 o w



I.- 4-2



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1215 5

19 8 17 4

.. a -


1.3 10


(N R cO co 0 (N R co

?- Ir T-

r-I I I



a N




15 8


BE K12-M



FIG. 2. SDS-polyacrylamide gelelectrophoresis.(a) Timecourseofsynthesisofphage-codedproteinsafter infection ofK-12-M. The numbers of the abscissa indicate the duration of the [35S]methionine pulse, and the numbers of the ordinate refer tocorresponding 17 genes(fordetails, seereference12).(b)Bandingpatternof phage-encodedproteins synthesized between 8 and10minp.i.in K-12-M cells(lowerline)and in BE cells(upper


sedimentation profile indicated no increase of

theunit-sizedDNA(Fig. 3b). Weconclude that the faster DNA (i.e., the concatemeric forms)

was converted into unit-sizedDNA in infected

BE and K-12 cells, but not in E. coli K-12-M cells. Here,theconcatemericformswere

appar-ently accumulated without further processing. TheFSfractionsappeared to be heterogeneous,


the observation that concatemeric

forms ofverydifferent




This finding was further


with the

pulse-chase experimentsdescribedbelow. From our data we cannot decide if the observed small

amount of unit-sized DNA was derived from

concatemers or whether it had never entered

such aform. We also observed onlyDNA con-catemers in E. coliBE cellsinfectedbyamutant

ingene 10(defectiveforthe maincapsid protein,

661 VOL.41,1982


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075 o s





1 5 10 15 20 25 1 5 10 15 20 25





10 15 20 23


10 15 20 25



FIG. 3. Analysis of phage-directedDNAonneutralsucrosegradients.3H-labeled T7 DNAsynthesizedin E.

coliBE, K-12, and K-12-Mcellswasanalyzedon5to20% neutralsucrosegradients.US(unitsize)indicatesthe fractions with[14C]thymidine-labeledT7 DNA usedas amarker. Thepeak fraction is indicatedbyan arrow(for

actualdata,seepanelscandd).The DNA that sedimented faster thanunit-sized DNA isindicatedbyFS.(a)T7

wild-type DNA synthesized in BE cells and analyzed 10 (0) and 14 (0) min p.i. (b) T7 wild-type DNA

synthesizedinK-12-Mcellsandanalyzed10(0)and 20(0)min p.i. (c)T7wild-typeDNAsynthesizedin K-12cells

andanalyzed14min p.i. (0 *). [14C]thymidine-labeledDNA extracted frommatureT7phagewasusedas a

marker(0). (d)T7 amlO DNAsynthesized in BE cells andanalyzed14minafter infection(0). [14C]thymidine-labeled DNA extractedfromT7+ phagewasusedas amarker(0).

gplO[35]) (Fig. 3d). We conclude that the pres-ence of preheads may be correlated with DNA


Tocorrelate thestepsof the DNA maturation pathway with thedifferent forms of DNA,

vege-tative T7 DNA was studied by pulse-chase

ex-periments as described in Materials and

Meth-ods. [3H]thymidine (0.2 ,uCi/ml)was addedtoa

T7-infectedE. coli BEor K-12-Mculture 6 min

p.i. at 37°C. Two minutes later the label was

chasedby adding 2mgofcold thymidineperml.

Atdifferenttimes after the chase, the viral DNA

wasextracted and analyzedonneutral 5to20%

sucrosegradients (Fig. 4). One minute afterthe

chase(Fig.4a), the phageDNAin E.coliBEwas

recovered mainly in the concatemeric form

(fraction I); asmallerpartcomigratedwith 14C-labeled T7 marker DNA(fraction II). Four

min-utes after thechase (Fig. 4a), both forms were

present, but the sedimentation profile clearly showed that most of the T7 DNA was now in

unit-length size. In the normalhost,the


intounit-sized DNA within 3 min.The

concate-meric fraction(I)mayhave beenheterogeneous,

withtwopeaks(fractionsIaand Tb)indicatedby



15 H


E 0. u




0. 1



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l__l__ l_______l the gplO- mutant of T7 which is unable to

a b assemble


preheads (Fig. 3d). The

a BE concatemeric fractions of T7 DNA synthesized


Qin the K-12-M cells appeared to be

heteroge-neous, as found with the normal host BE (Fig.




indicate ashift from the65Sto


,#1\ \ the 45S fraction after a 4-min chase



l \ l9 These observations



that the

newly synthesized phageDNA can mature only


tetrameric intodimericconcatemers.To


whether DNA maturation is blocked at astepof nicking the concatemers at unit-size

,, , intervals, we analyzed extracted DNA on

alka-5 10 15 20 23 line 5 to 20% sucrose gradients. Figure 5a shows

ll theresults with T7-infected BE cells harvested 1


a bI




5 10 15 20 25


FIG. 4. Maturation ofphageDNA studiedby pulse

chase. [3H]thymidine incorporation intophage DNA

wasperformedasfollows. Six minutes after infection,

0.2 puCi of [3H]thymidine per ml was added to the culture and 2minlater chased with 2mgof

nonradio-activethymidine perml. Then 500-,l1 samples were

taken9minand 12min p.i. and submittedtosucrose

gradient centrifugation as in Fig. 3. A T7-infected culture of BE(a)andaninfected culture of K-12-M(b)

wereanalyzed after1(0) and4(0) min of chase.The

[14C]thymidine-labeled phageDNA extracted from T4

(open arrow) and T7 (closed arrow) was used as a marker.

the cosedimentation of 14C-labeled T7 and T4 DNA, whichweusedasmarkers(Fig. 4b). The

sedimentation constant of T7 unit-sized DNA has been determinedtobe 31S, and that of T4 DNA has been determined tobe 61S (4). The sedimentationconstants of the materialpresent

inthepeaks offractions Taand lbof Fig. 4bwere

therefore65S and45S, assuminganideal

linear-ity of the gradient between fractions 6 and 20. This corresponds approximately to tetrameric anddimeric forms.

In T7-infected K-12-M cells, phage DNA

ac-cumulated inconcatemeric forms(Fig. 4b).

Al-mostno unit-sized DNAwasrecovered, even4

min after the chase. We conclude that phage

DNA in T7-infected K-12-M cells remained in concatemeric forms,very similar tothecase of




E 1


._ E



3 I _

a b l

b a b oK-12-M

2 \



71 *>d9 <

I_ I

1 s 10 15



20 25

FIG. 5. DNAsedimentationpatternin alkaline

su-crose gradients. The pronase-treated samples (see

Materials andMethods)wereincubated together with

[04C]thymidine-labeled T7 DNA,usedas amarker, in a0.3 MNaOH and0.7 MNaCl buffer for2 h. Then 200


sucrosegradient. Centrifugationandtreatment of the

sampleswere asdescribedin Materials and Methods.

T7 DNAwasanalyzed after1minof chase in BE cells

(a) andafter 4min of chase in K-12-M cells (b) (0).

The open symbols refer to corresponding profiles

obtained under neutralconditions; thearrowsindicate

fractionscontaining [04C]thymidine-labeledT7 DNA.







25 _


VOL.41, 1982



K- 12 -M

I .1

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min after the chase. The phage-directed DNA was found in concatemeric forms (fractionI)and

ina major peakof unit-length DNA(fractionII).

Fraction I was composed mainly of

fast-sedi-menting molecules (fraction Ib), butsome very

fast sedimenting molecules were also present

(fraction Ia). By comparing the two profiles in

Fig. 5a, wenoted that, after alkalinetreatment, partof the radioactivity of fractionI wasshifted tofractionII;this demonstrates thatsomeof the concatemeric DNA was present in a nicked

state. However, the majority of concatemers

had no nicks. The phage DNA fromT7-infected

K-12-M cells was also analyzed after alkaline

treatment (Fig. 5b). One part of the DNA

sedi-mented as concatemers; another part sediment-ed as unit-sized DNA. Alkaline treatment in-ducedashift from theconcatemers tounit-sized phageDNA. This demonstrates that some of the

accumulatedconcatemers hadnicksatdistances of aboutone T7 genomelength.

Intracellular morphology of T7-infected E. coli K-12-M cells. Thin sections ofE. coli


cells infectedby T7wild type at 10 minp.i.are shown

in Fig. 6a. The first newly assembled preheads seemed to be preferentially located at the

pe-riphery ofanucleoid-like DNA pool. They be-came filled with DNA ina very compact form, whichthen appeared asdarkly stainingparticles ("black particles"). Freshly assembled

pre-heads continued to appear and to mature into black particles (Fig. 6b).

Adifferentsituation wasshownby T7-infect-edK-12-Mcells (Fig.7a). Asintheabovecase,

the intracellular preheads appeared at the

pe-riphery of theareaof DNA(nucleoid and

phage-DNA pool). No black particles appeared after continued incubation. Apparently nopackaging occurred, despite thepresenceofnewly synthe-sized T7 DNA. We will call the accumulated head-related structures M-preheads (Fig. 7b). These M-preheads contained a small electron-scatteringcoreandwere similartothepreheads during earlystagesofT7infection(Fig. 6).Ithas to be emphasized that electron microscopy

re-vealsmorphological similarities but is obviously notabletodistinguishtruepreheads-whichare

maturable-from prehead-like, but abortive,

structures. We canalso notexclude that these

particles originally contained packaged DNA

which was releasedartificially during the

proc-essing forembedding procedures. To test if the

M-preheadswere trueprecursors, we


aninvitropackaging test, described below.

Isolation and in vitro packaging of preheads accumulated in T7-infected K-12-M cells. The

arrest of virus maturationinT7-infectedK-12-M cellscould be theconsequencesofthefollowing

causes: (i) the accumulated preheads are abor-tive structures, i.e., areunable tobecome filled

with DNA; (ii) the vegetative phage DNA is incompetent for this process; or (iii) the

packag-ing process itself is affected. To test the first

hypothesis, we submittedthe M-preheadstoin

vitro packaging according to Miyazaki et al. (26).

To exclude particles other than preheads which occur in minor amounts that are not

detectable by electron microscopy, we isolated the M-preheadson a sucrosegradient. The

cor-responding peak contained preheads of various

aspects (Fig.7c). Asmall amount (about1%) of the particles had a roundish outline and thick

shells and contained excentrically placed cores

(Fig. 7d). These M-preheads I had the same

morphology as the preheadsaccumulated after infection of mutantsin gene 5, 8, 15, and 16of the normal host (29). The M-preheadsII

repre-sented the bulk ofthe isolated preheads; they

had thin shells, were often collapsed, and also

contained a core(Fig. 7e). We were not ableto separateM-preheads I andIIbysedimentation.

In the following experiments, the mixture of

these two types of particles had to be used.

Preheads II were not produced in the normal

host after infection withgene 5 mutants(datanot


InTable 1 we give theresults ofourin vitro packaging experiment. Both gp5- and M-pre-heads werecompetent tobecomepackaged.The mostdelicatestepoftheprocedurewasfoundto

bethe quantitative lysis ofthe cell suspension.

Invitro maturation of the "gp5- preheads" has

already been demonstrated by several authors (17, 25). In our experiments the isolated

M-preheads werematurable into infectious phages with an efficiency (resulting PFU/input

pre-heads) of1%. This in vitro maturation efficiency

was about the same aswithgp5- preheads. We

donotthinkthatthedifference ofafactor of2is significant. The factthat theefficiencyof in vitro packaging was the same for preheads ofthese two origins argues strongly


the first hypothesis, that M-preheads areabortive struc-tures.

Inaddition to theM-preheads I and II, we also

discoveredsomeparticles whichappearedtobe

partially filled (Fig. 7c). Weinvestigated wheth-er they might represent intermediates of the DNA-packaging process or whether they were

breakdownproducts of already packaged


Isolation ofputative DNA-packaging intermedi-ates. To isolate putative


inter-mediates, we followed the isolation procedure already described (27). T7-infected E. coli


and K-12-M cells were labeled with 14C-amino acids and


at 5 min


At the

indicated times the cells were harvested and


and the extracts were analyzed on a

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FIG. 6. Thin-sectioned BE cells after 17 infection. T7-infected E. coli BE cells were processed for thin sectioningasdescribed in Material and Methods.(a)10minafter infectionat37°C; (b)14minafterinfectionat


sucrosegradient inheavy water (for details, see

Materials and Methods). Figure 8a shows the

analysis of radioactivity inthe gradient of a



culture 9 min p.i. The fractions

obtainedwereanalyzed by electron microscopy. Phage particles sedimented atfraction 16;

pre-heads I and II sedimented atfraction 35.



inter-VOL. 41,1982 665

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FIG. 7. Electronmicrographs of preheads accumulated in infectedK-12-Mcells.(a)Thin-sectioned K-12-M cells14minafterT7infection. The barrepresents500 nm.(b) Thin-sectioned M-preheads.(c) Isolated prehead fraction ofasucrosegradientof alysate ofT7-infectedK-12-Mcells.Wedistinguish "immature" preheadI(1),

"mature" prehead 11(2), and suspectedpackaging intermediates (3). The sampleswere dialyzed againstthe

isolation bufferandnegatively stained with1%uranylacetate.(d) IsolatedpreheadIfrominfectedK-12-M cells

negatively stained with 1% uranylacetate.(e) Isolated preheadIIfrominfected K-12-Mcellsnegatively stained with1%uranyl acetate. Thebars in panelsbthrough e represent 100 nm.

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TABLE 1. Invitro packagingof isolated preheads

Group In vitro components PFU x 106/ml

Controls gplO- extract 2.5

gp5-preheads 0.001

Mpreheads 0.05

Experiments gplO- extract+gp5- 170


gplO- extract + M 75


mediates but also preheads II. The upper

fractions containedDNAaggregates,freeDNA, and cell components, but no capsid-like


In the corresponding experiment with a

T7-infected K-12-M culture the cellswereharvested

14 min after infection and assayed for radioac-tivity (Fig. 8b). It is striking that almost no

labeled material was found in fraction 16,

con-firming that very fewphage particles were

pro-duced.By electron microscopyof material from fractions 31 to 34, we detected some particles

with an apparently incomplete DNA content

(Fig. 9). These particles werealways

accompa-nied by random coils of DNA. For the sake of simplicity, wewillcall these particles packaging

intermediates, although we can obviously not

rule outthat the observed particles had simply lost some of their DNA during isolation or

specimen preparation for electron microscopy. Acommonfeatureof all of these particleswas

that they were apparently attached to DNA in

some way. We thus prepared the fractions of

interestby the method of Dubochetetal.(5), in

current use for observing DNA and DNA-pro-tein complexes by electron microscopy, as de-scribed inMaterialsand Methods(Table 2).

In fractions 31 and 34 we observed a large

amount of capsid-DNA complexes, but not in theupperfractions(Table 2). Stretches of DNA

were found to emerge from single capsids; in

othercasesuptofourcapsidswerealigned with

DNA to form a linear stretch (Fig. 10c). We

measured the total length of DNA extruding from the associated particles and found that it

waslonger than T7 unit length (15.5to36.2


Thestretches of DNAbetween associated parti-cles did notbelong todistinct length classes,as

judged from the 12 moleculesmeasuredon

elec-tron micrographs (data not shown). In most cases observed, two DNA filaments protruded fromoneparticle (Fig. 10a,b). This canbe

inter-pretedas asingle filament entering and leavinga

particle and forming a loop inside or as ends of twoindividualDNA moleculesenteringthesame

particle. To distinguish between thesetwo possi-bilities, we analyzed the DNA length of the

complexes on sedimentation gradients after

di-gesting the capsids by pronase as described below.

Analysis of the DNA of possible packaging intermediates. Thefractions ofinterestof BE and

K-12-M extracts (Fig. 8)were treated with pro-nase and analyzed on 5 to 20% neutral and

alkaline sucrose gradients. Figure 11 shows re-sults obtained forfractions 30, 31, and 34. In

fraction 30 of the BE extract, we observed, besides T7 unit-length DNA, some DNA in its concatemeric form(Fig. lla). Practicallynounit length DNA wasobserved in T7-infected K-12-M cells. The DNA of fractions 31 and 34

sedi-mented veryfast andwastherefore in its

concat-emeric form(Fig. llb, c). Alkalinetreatmentof

these concatemers showed that they all had

nicks at a distance corresponding to about one T7 genomelength(Fig. lld). With regardtothe

other fractions of the K-12-M extract, we ob-served mainlyunnickedconcatemersinfraction

45 and some T7 unit-length DNA, but mainly pieces of smaller sizes in fraction 50 (data not



Multiplication of bacteriophages depends on many host functions. A mutation of a gene

coding for one of these functions may prevent

the production of phage progeny, but not, at

least under laboratory conditions, measurably affect growth ofthe uninfected bacterium. Itis

therefore conceivable that suchamutantisonly detectable because ofan incompatibility regard-ing virus host interactions. Many such host

mutants affecting the growth of several coli-phages have been characterized: mutations in the groE gene of E. coli B affect the head assembly of T4 and lambda (9, 15); those in

groN prevent the correct interaction of the

lambda gene N product with the host

RNA-polymerase and block the transcription of the

early genes (8). In the case of phage T7, the


thioredoxin has been found to be an

obligatory componentofthe T7 DNA polymer-ase(24); inanothermutant, E. coli Y49, the T7

head assembly seems tobe abnormal (40).

Be-sides host-dependent DNA restriction, various functions interfere with early or late events of phage multiplication. Accordingly, they might


transcription, translation, or replication of

phageDNA, ortheymightblock DNA

matura-tion,head assembly, or DNApackaging. Ourattempt was toelucidatethemechanisms ofthe T7abortion factor, groM. In afirst step, we investigated the early events of T7 phage

multiplication.Host DNAdegradationaswellas

phage-directedDNAandprotein synthesis indi-cated that none of these processes was qualita-tivelychanged. In comparison with the normal

T7-infected K-12 strain, the rateofphage DNA

VOL.41,1982 667

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10 8
















BE 20

15 10




0 10 16





















...r / \/k~~~

0 10 16 20 30 31 34 40


FIG. 8. Fractionation ofextractsfrom T7-infectedBE (a)and K-12-M(b) cells aftercentrifugation through

discontinuoussucrosegradients inheavywater.Thecultures(4x108cells/ml)wereinfectedat anMOI of fivephage per celland4minlaterdoubly labeled with14C-aminoacids(0)and[3H]thymidine


BE cellswereharvested

at9minp.i. (a), andK-12-Mcellswereharvestedat14minp.i.(b).Theextractswereanalyzedondiscontinuous

sucrosegradientsinheavywater asdescribed in Materials and Methods(27),and theresulting0.6-ml fractions

wereassayed forradioactivity. Centrifugation wasfromrighttoleft.

andprotein synthesiswas reducedto20to30% We then


the maturation of

phage-in T7-infected K-12-M cells. We do not think directed DNA. The intracellular maturation of thatthis reductioncanexplaintheblocked mul- T7 DNA


startswith the



tiplication, since only 0.5 phage was produced T7unit-sizedmolecules,asproposed





These molecules are then connected to



on November 10, 2019 by guest




4!,. ,,

FIG. 9. T7packagingintermediates isolatedfrom K-12-M cells. The fractions obtainedaftercentrifugationon adiscontinuous sucrosegradient (Fig. 8)weredialyzed against20 mMTris-hydrochloride, pH7.9,containing

200 mMNaCl and 1 mMEDTA,fixedby2%glutaraldehydefor 10min,andnegativelystainedwith 1% uranyl

acetate.Particles from fractions 31(aandb)and 34(c)in which the T7 DNA sedimented in its concatemeric form

(Fig. 11). The barsrepresent100nm.

formconcatemersof variouslengths (30).

Short-ly before or during the packaging process, the

concatemers are cutagainto unit length bythe

products of the T7genes3, 6, and 19 (14, 33).

Thesucrosegradient sedimentation profiles of

extracts of T7-infected K-12-M cells showed that thenewly synthesized phage DNA

accumu-lated inafast-sedimenting form (Fig. 3b and 4b).

The heterogeneity of the profile of this

fast-sedimenting DNA showed that theconcatemers

had various lengths. Alkaline treatment of the DNA(Fig. 5) showed that nicking of the

concat-emers was not prevented, as the DNA was in

partnickedatintervals of about unit-sizelength. Infectionof the normal host by a T7 mutant

defectivefor head assembly (gene 10) led alsoto an accumulation of concatemeric forms (Fig.

3d). The maturation of T7 DNA from its

concat-TABLE 2. Content of fractions treatedasdescribed in thelegend of Fig.9and10andanalyzed byelectron


Infectedhost Fraction Observed structures

BE 16 Phages

30 DNAcapsid complexes, preheadII,free DNA

35 PreheadsIand II

50 Ribosomes andproteinaggregates, nocapsids

K-12-M 16 Fewphages

31, 34 DNAcapsid complexes, freeDNAof distinctlengthclasses 45 Ribosomes,DNAof variablelength,nocapsids

50 Vesicles, proteinaggregates, fewcapsids

54 DNApieces shorter than T7unitlength, nocapsids

VOL.41, 1982 669

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on November 10, 2019 by guest





E 100


500 r



0 21 0 22

C K-12-M


- 10


150 100 50

I I I 1




d K-12-M






FIG. 11. DNAsedimentationpatternsofthefractions containingpackaging intermediates in neutral (atoc) and alkaline (d)sucrosegradients. Equal portions of the pooled fractions showninFig. 8werepronasetreated

andlayeredon5to20% linearsucrosegradients. T7DNABEextracts(9min p.i.)infraction 35(a); T7 DNAof

K-12-M extracts(14 min p.i.) in fractions 34 (b) and 31 (c, d). [14C]thymidine-labeled T7 DNAwasusedas a


emericform into its unitlength obviously needs thepresenceofpreheads. Thestateof the phage DNA accumulated in the K-12-M cells points, therefore, to a possible block in the prehead

assembly or a block in the DNA-packaging process. In thin sections of infected K-12-M

cells, wedetected preheads. They appeared at

theperiphery ofanucleoid-like structure,which presumably contained vegetative T7 DNA (since host DNA isalreadydegradedatthat time after infection). Blackparticles were notfound (Fig. 7). This doesnotexclude thehypothesis that the M-preheads might have contained DNA and

wereartifactually emptied by the fixation

proce-dure. Suchaninduced loss of alreadypackaged

DNAhasbeenobservedfor the T4 gp49- parti-cles (10, 20, 22, 23) and for some mutants of phage P22 (21).

Consequently, two possibilities still remain

forexplainingmorepreciselythe T7 maturation block: either DNAorthepreheadsare

incompe-tent for packaging, or the packaging process

itself isblocked. Todecide between these possi-bilities, we compared normal and M-preheads

by in vitropackaging experiments. In both cas-es,the sameorder ofmagnitude of the

accumu-lated particles was maturable (Table 1). Thus,

thepreheads which accumulated in K-12-M and those of the usualhostwere functionally

indis-tinguishable. We conclude that groM does not

affect assembly ofpreheads but interferes with their DNA packaging. The isolation of

phage-related particles from the T7-infected K-12-M cells revealed at least three morphologically distinguishable particles (Fig. 7C). The

preva-lenttypeofparticleswereM-preheads II, which

contained a core and a thin shell (Fig. 7e). Accordingtotheusual nomenclature, these

par-ticles represent mature preheads (7). Besides these, wefound M-preheadsI,whichalso hada corebut amuch thicker shell (Fig. 7d). The

M-preheads I were morphologically identical to

thosepreheads which accumulate during DNA

replication-defectiveT7infections (29). Matura-tion of preheads I to preheads II might be

coupledto aninteraction with DNA (7). This is

furthercorroborated by theobservation thatno

preheads oftypeIIwerefound in the lysate ofa

T7mutantdefective in DNA synthesis (gene 5). The appearance of prehead II might thus be

induced byphage DNA.

The following observations support the hy-pothesis that K-12-M preheads undergo a

first-step interaction with phage DNA. Among the

phage-relatedparticles isolated from the K-12-M cells, we observed some which appeared to be halffilledwith DNA (Fig. 7c). From work with T4phage,wehave becomeawareof

experimen-tal difficulties in demonstrating true packaging intermediates. Invivo-formed DNA-capsid (pre-head II) complexes of T7 were isolated by

Serwer and Watson (31, 32). It could not be

proven whether these complexes represented

true packaging intermediates or whether they 150



CL u 50




on November 10, 2019 by guest



were breakdown products of fragile particles which contained a complete complement of DNA. Inour caseofK-12-M,it ismorerelevant to ask whether packaging was initiated at all. Ouranalysis by means of lengthmeasurements

anddetermination of sedimentation behavior of

DNAattached topreheads showedDNA exclu-sively in concatemeric states (Fig. 11). This

suggeststhat thesestructures are truepackaging intermediates. From theseresults,we tentative-ly conclude that binding of preheads to phage

DNAin its concatemeric form is the firststepof the packagingprocess (and not the cuttinginto unit length). Similar structures have been de-scribed in othersystems,for example, forphage

A (41)and for adenovirustype 5(27). Apossible pathway proposed by Serwer and Watson (32) is in accordance withthe structures weobserved. The rather smallamountof suchDNA-capsid complexes inextractsof the normalT7infection (Fig. 8a)suggeststhat either the time needed for packaging and cutting of the packaged DNA is

very short or that suchintermediate structures arenotstable under theextraction conditionswe

used. According to this, we propose that the

block ofT7 maturation in the K-12-Mcells isa

late step in the DNA packaging reaction, i.e.,

the completion or the stabilization of the

pack-age of DNA or the final cutting of the DNA

(terminase). Three observations favor this hy-pothesis: thepresenceofDNA-capsid

complex-esfound by electron microscopy, the maturation ofpreheadsIintopreheads II, and the

accumu-lation ofconcatemericT7 DNA.

Ifthe terminalcutting of the DNAisblocked


our results could be



follows. The




intermedi-ate allows us to observe


breakdown products. Second, if the


proc-ess itself is slowed down, only a few




filled with DNA and will accumulate. Several


can be

pro-posed for such a


block of the

DNA-packagingprocess. Ahost-codedenzyme

neces-saryfor the





defective or missing. Another


is a lowered intracellular ATP level at this stage,

since it has been shown that ATP is necessary

for the




Further-more, abnormal internal conditions in the host

mightinhibit a stablecondensation ofDNA. Itis also knownthat T7 in vitro


systems show an absolute requirement for


ions (17). Changed ionic conditions could have an

effect onthe condensation ofphageDNA, e.g.,

by affecting its folding. As a consequence, the DNA, although in functional form, could not

enter the prehead


In a


paper(Kuhnetal., inpreparation) wewillshow a lower level ofintracellular K, Mg, and

poly-amines inT7-infected K-12-Mcomparedwith K-12and BE.By compensating fortheleakageby Mg2+ ions, we were able to show that the

multiplication blockcanbeovercome. This fact strongly suggests that the decreased internal

level ofKand Mg concentration is the common cause of the morphogenetic block, the reduced syntheses, and lysis inhibition. Cumulative ef-fectsof several separately contributing host

fac-tors aretherefore at least no longer to be consid-eredas afirstpriority.


We thank A. P. Pugsley and T.A.Bickle for theircritical reading of the manuscript. We are most indebted to Elvira Amstutz andBeatrice Meier for patient typing of this manu-script.

This investigation was supported by grants from the Deut-scheForschungsgesellschaft (Ha 709) to R.H., from the Swiss National Science Foundation to E.K., and through a Europe-anMolecular Biology Organization fellowshiptoM.L.J.M.


1. Adams, M. A. 1959.Thebacteriophages. Wiley Intersci-ence, New York.

2. Arber, W., and D. Dussoix. 1962. Host specificity of DNA produced by Escherichia coli. I. Host controlled modifica-tion of bacteriophage K. J. Mol. Biol. 5:18-36.

3. Center,M. S.1975. Roleof gene2inbacteriophage T7 DNAsynthesis. J. Virol. 16:94-100.

4. Clark,R.W.,G. H.Wever,andJ. S. Wiberg. 1980. High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on T7 bacteriophage and two novel formsof T4 bacteriophage. J. Virol. 33:438-448. 5. Dubochet, J., M. Ducommun, M. Zollinger, and E.

Kellen-berger. 1971. A new preparation method for dark field electron microscopy of biomacromolecules. J. Ultra-struct.Res. 35:147-167.

6. Duckworth, D. H., J. Glenn, and D. J. McCorquodale. 1981. Inhibition ofbacteriophage replication by extra-chromosomalgenetic elements. Microbiol. Rev. 45:52-71. 7. Earnshaw,W.C., and S. R. Casjens. 1980. DNA packag-ing by the double-stranded bacteriophages. Cell 21:319-331.

8. Georgopoulos,C. P.1971.Bacterial mutants in which the geneNfunction of bacteriophage lambda is blocked have an altered RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 68:2977-2981.

9. Georgopoulos, C. P.,R.W.Hendrix, S.R.Casjens, andA. D.Kaiser. 1973. Hostparticipation in bacteriophage lamb-da headassembly. J. Mol. Biol. 76:45-60.

10. Hamilton,D.L., and R. B. Luftig. 1976.Bacteriophage T4 headmorphogenesis.VII.Terminalstages of head matu-ration. J. Virol.17:550-567.

11. Hausmann, R. 1973. Host-dependent geneexpression of

bacteriophagesT3 andT7,p. 263-276.In B. A.Hamkalo and J.Papaconstatinou, Molecular cytogenetics. Plenum Publishing Corp.,NewYork.

12. Hausmann, R. 1976. Bacteriophage T7 genetics. Curr. Top. Microbiol. Immunol. 75:77-110.

13. Hausmann, R., and B. Gomez. 1967. Amber mutants of bacteriophage T3 and T7 defective in phage-directed deoxyribonucleic acid synthesis. J. Virol. 1:779-792. 14. Hausmann, R., and K. LaRue.1969. Variations in

sedi-mentationpatterns among deoxyribonucleic acids synthe-sized after infection of Escherichia coliby different amber mutantsofbacteriophage T7. J. Virol. 3:278-281. 15. Hendrix, R. W., and L. Tsui. 1978. Roleof the host in

virus assembly: cloning of the Escherichia coli groE gene andidentificationof itsprotein product. Proc. Natl. Acad. Sci. U.S.A.75:136-139.

on November 10, 2019 by guest




16. Kelly,T.J., Jr., andC. A. Thomas, Jr.1969. An interme-diate in the replication ofbacteriophage T7 DNA mole-cules. J.Mol.Biol. 44:459-475.

17. Kerr, C., and P. D. Sadowski. 1974. Packaging and maturation of DNA of bacteriophage T7 in vitro. Proc. Natl. Acad.Sci. U.S.A. 71:3545-3549.

18. Korsten, K. H., C. Tomkiewicz, and R. Hausmann. 1979. The strategyof infection as a criterion for phylogenetic relationship of non-coli phagesmorphologically similar to phage T7. J. Gen.Virol. 43:57-73.

19. Kruger, D. H., and C. Schroder. 1981. Bacteriophage T3 andbacteriophage T7 virus-host cell interactions. Micro-biol. Rev. 45:9-51.

20. Limmli,U.K.,N.Teaff,andJ.D'Ambrosia.1974. Matu-rationofthe head of bacteriophage T4. III. DNA

packag-ing into preformed heads. J. Mol. Biol. 88:749-765. 21. Lenk, E., S. Casjens, J. Weeks, and J. King. 1975.

Intracellularvisualization of precursorcapsids in phage P22 mutantinfected cells.Virology68:182-199. 22. Luftig, R. B., and C. Ganz. 1972.Bacteriophage T4 head

morphogenesisII. Studies on the maturation of gene 49-defective head intermediates. J. Virol. 9:377-389. 23. Luftig, R. B., W. B. Wood, and R. Okinaka. 1971.

Bacteriophage T4 head morphogenesis. On the nature of gene49-defective heads and their roleasintermediates. J. Mol.Biol. 57:555-573.

24. Mark, D. F., and C. C. Richardson. 1976. Escherichia coli thioredoxin: asubunit ofbacteriophageT7 DNA polymer-ase.Proc. Natl.Acad. Sci. U.S.A. 73:780-784. 25. Masker, W. E., N.B.Kuemmerle,andD. P.Allison. 1978.

In vitropackaging ofbacteriophage T7 DNAsynthesized

in vitro. J. Virol. 27:149-163.

26. Miyazaki, J. 1., H. Fujisawa, and T. Minagawa. 1979. Biological activity of purifiedbacteriophage T3 prohead andprohead-like structuresasprecursorsforin vitrohead assembly.Virology 91:283-290.

27. Moncany, M. L. J., B. Revet, and M. Girard. 1980. Characterization ofa new adenovirus type 5 assembly

intermediate. J.Gen. Virol. 50:33-47.

28. Reynolds,E.S.1963.The use of lead citrateathigh pHas anelectron-opaque stain in electronmicroscopy. J. Cell.

Biol. 17:208-225.

29. Roeder, S., andP.D.Sadowski.1977. Bacteriophage T7 morphogenesis. Phage-related particles in cellsinfected with wild-type and mutant T7 phage. Virology 76:263-285. 30. Schlegel,R.A.,andC. A.Thomas, Jr.1972.Somespecial

features of intracellularbacteriophage T7 concatemers. J. Mol. Biol. 68:319-345.

31. Serwer, P. 1974. Complexes between bacteriophage T7

capsidsandT7 DNA.Virology59.

32. Serwer,P., and R. H. Watson. 1981.Capsid-DNA com-plexes in the DNA packaging pathway ofbacteriophage

T7:characterization of thecapsids bound to monomeric and concatemeric DNA.Virology 108:164-176. 33. Stratling, W., E. Krause, and R. Knippers. 1973. Fast

sedimenting deoxyribonucleic acid in bacteriophage T7-infected cells. Virology 51:109-119.

34. Studier, F. W. 1973. Analysis of bacteriophage T7 early RNAs andproteins on slab gels. J. Mol. Biol. 79:237-248. 35. Studier, F. W. 1976. Bacteriophage T7.Science


36. Studier, F. W. 1979. Relationships among different strains of T7 and among T7-related bacteriophages. Virology 95:70-84.

37. VanDriel, R., F. Traub, and M. K. Showe.1980.Probable localization of thebacteriophage T4 prehead proteinase zymogen in the center of the prehead core. J. Virol. 36:220-223.

38. Watson, J. D. 1972. Origin ofconcatemeric DNA. Nature (London) New Biol. 239:197-201.

39. Wunderli, H., J. van den Broek, and E. Kellenberger. 1977. Studies related to the head maturation pathway of bacteriophages T4 and T2. I. Morphology and kinetics of intracellular particles produced by mutants in the matura-tion genes. J.Supramol. Struct. 7:135-161.

40. Yamada, Y., J. Silnutzer, and D. Nakada. 1979. Accumu-lation of bacteriophage T7 head-related particles in an Escherichia coli mutant. J. Virol. 31:209-219.

41. Yamaguishi, H., and M. Okamoto. 1978. Visualization of the intracellular development of bacteriophage X, with special reference to DNA packaging. Proc. Natl. Acad. Sci. U.S.A. 75:3206-3210.

VOL.41, 1982 673

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FIG. 1.After(MOIprecipitablevariousBE,K-12,12, T7-induced degradation of host DNA and phage-directed DNA synthesis
FIG. 2.phage-encodedinfectionnumbers SDS-polyacrylamide gel electrophoresis. (a) Time course of synthesis of phage-coded proteins after of K-12-M
FIG. 3.fractionscoliactualandlabeledwild-typemarkersynthesized Analysis of phage-directed DNA on neutral sucrose gradients
FIG. 4.0.2chase.wasculturegradientactivetakenculturewere(open[14C]thymidine-labeled Maturation of phage DNA studied by pulse [3H]thymidine incorporation into phage DNA performed as follows


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