Isolation and Characterization of Bacteriophage T4 Base Plates

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Vol.10,No. 4 PrilztediltU.S.A.

Isolation and Characterization of

Bacteriophage T4

Base

Plates

B. F. POGLAZOV, L. P. RODIKOVA, AND R. A. SULTANOVA LaboratoryofBioorgainic Clhemistry, Moscow State Unliversity, Moscow, U.S.S.R.

Received forpublication20July1972

Amethod for isolating bacteriophage T4 base platesfromlysates of Escherichia

coli B cells infected with thets mutant ingene 19, tsB31 has been developed. By

electrophoresis in polyacrylamide gel with sodium dodecyl sulfate the base plates

have been showntocontain fivetosevenproteincomponentswith molecularweights

of 36,000, 53,000, 66,000, 81,000, 87,000, and probably about 100,000. Electron

microscope studies have demonstrated that baseplatesmayoccur intwostructural

states:in the form ofhexagonsorstars.Starraysand short fibrilsarenotradialor

elongated and are turned sideways at an angle to the radius. Base plates do not

complement in vitro with free tail coresisolatedafter disintegration of particles of

thewild-type bacteriophage.

Base plates are the central tail element of a

particle of T-even bacteriophages to which long

fibrils,tail cores, sheaths, and short fibrils attach. They occupy the central position and, therefore,

play a leading part in bacteriophage

morpho-genesis (6). Due to this, base plates have long

been attracting the attention of investigators.

However, because of technical difficulties

in-volved in their isolation, extensive studies of base

plateshavecommenced only recently (2,8).Some

electronmicroscope investigationshaveprovided

evidencethat base plates mayexist in two states (H. Fernandez-Morgan, 1962, Symp. Int. Soc. Cell Biol., vol. 1, p. 411; references 1 and 9). As aconstituent ofanintact phage particlewith

anextendedtail sheaththebaseplatesformathin

hexagonal plate of 310 to 400 nm in maximal

diameter. In the center of the plate there is a

pluggedholesurrounded byahexagonaldisc with

a diameter of 180 nm situated on the inner side

of theplate (2,6,9).Inresponse tosheath

shorten-ing, base plates transform into their second state

and change their form from hexagons to

hex-agonal stars with a maximal diameter of 500 to

600nm.

In their recent work Cummings et al. (2) have

shown, by using separation on the agarose column, that base plates are composed of three proteins with molecular weights of 17,000, 31,000,

and 53,000. These authors have also advanced a

model of a base plate built of tetrade complexes of protein subunits. Also, recently Kozloff and

co-workers (8) have isolated T4 base plates and

have shownthat the phage-induced dihydrofolate

reductase of molecular weight 30,000 is a tail

plate component.

The present study deals with isolation of base

plates from the ts mutant of a T4 phage damaged

in gene 19 and theinvestigation of the base plates

by electron microscopy and physicochemical methods.

MATERIALS AND METHODS

Baseplates were produced with the aid ofa tem-perature-sensitive mutant T4 B31 damaged in gene

19, kindly provided by King (6). The mutant was

grownonEscherichiacoliBat 42 C,usingasynthetic medium and intensive aeration in a 100-liter fer-mentor. E. coli B was grown to a concentration of 2.108 cells/ml and then infected with phage with a

multiplicity of 5, superinfected 15 min laterwithan

equivalent amount of bacteriophage, and incubated

for another 40 min. The bacterial mass was

centri-fugedand resuspended inasmallamountof the

cul-tureliquidtothefinalvolume of1.5liters. Cellswere

lysed with chloroform and kept overnightat4C.The resultant lysate was supplemented with deoxyribo-nuclease (40 Ag/ml) in the presence of Mg2+ and ribonuclease (100

jig/ml)

and keptfor 2 hr at 37 C with continuous stirring. The bacterial cell debris wereseparated by centrifugationat6,000 X gfor 30 min.Elementsofphageparticlesthatremainedinthe supernatantfluid were precipitated by further centri-fugation at 78,000 X g for 7 hr. The residue was suspended in 0.01 M tris(hydroxymethyl)amino-methane (Tris)-hydrochloride buffer, pH 7.4, in the presence of 0.001 M ethylenediaminetetraacetic acid (EDTA). The resulting suspension contained, in addition to minorcell structures, amixture of struc-tural elements ofaphageparticle (heads,polysheaths,

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BACTERIOPHAGE T4 BASE PLATES

base plates, base plates plus cores, fibrils) and was usedtoisolate and purify base plates by means of step-wise centrifugation in the sucrose gradient. In the first stage, heads and polysheaths were roughly separatedfrom base plates. For this purpose 5 ml of 60% sucrose was poured into a centrifuge tube, and then 10ml of20%sucrose was added and20 to 30 ml of a suspension of bacteriophage structuralelements

waslayeredontopof the sucrosesolution. Asaresult

ofcentrifugation at40,000 X g for 1 hr, two layers wereformed. The upperlayer contained mainly base plates, empty discs with a diameter that equaled the corediameter, andasmallamountof heads and

poly-sheaths. Thelower layerincluded a large accumulation of heads,polysheaths, and intact phage particles. Both layers werecollected with a syringe and centrifuged at 165,000 X g for5 hr. The residues were suspended

with 0.01 M Tris-hydrochloride buffer, pH 7.4,

con-taining 0.001 M EDTA. The residue of the upperlayer was then centrifuged at 40,000 X g for 4 hrin the linear sucrose gradientat aconcentration of5to 20% withabottomcushion of60% sucrose. This resulted chiefly in the formation ofthreeopalescent layers. The lowerlayer thatwas onthe cushioncontaineda mix-ture ofpolysheaths and heads; the middle layer in-cluded baseplates and baseplates plus cores (forks); the upperlayer showed empty discs that were probably core fragments or connectors. Base plates from the middle layer were precipitated by centrifugation at 165,000 X gfor7hr andresuspended indistilledwater atpH 6.0. Theresultantpreparation of baseplates was occasionally passed through the linear sucrose gradient.

Tail cores ofbacteriophages T2 and T4 were ob-tained according to the previously developed pro-cedure (PoglazovandNikolskaya, 1969). Inthe case ofbacteriophage T4, cores emerged attached to the shortened sheaths and connectors (socalled grenades). Thecomplementation experimentswerecarriedout inaccordancewith the method of Edgar and Wood (4). The preparation of bacteriophage T4 ts B31 base plates was mixed with that of bacteriophageT2Lcores

orbacteriophageT4D inequalvolumes. The mixture

was addedto the lysateofphage T4D-infected bac-teria, incubated for 3hrat30C, and then examined in theelectronmicroscope.Thelysatewaspreparedin

thefollowingway.BacteriophageT4D wasgrownin

200ml of Hottinger broth, thelysis beingdelayedfor 40min. Without lysingthe bacteria, they were pre-cipitated at 6,000 X gfor 1 hr. The residuewas re-suspended in 25 ml of phosphate buffer (0.039 M

Na2HPO4+ 0.022MKH2PO4+ 0.07MNaCl + 0.02 M

MgSO4) and lysed by freezing and thawing. Cell debris and intact phage particles were removed by centrifugationat30,000 Xg for1hr.Thesupernatant fluid was used as lysatein complementation experi-ments.

Electronmicroscope examinations were performed in a Hitachi 12 microscope with a magnification of

X50,000.Thespecimenswerestainedwith2% sodium

phosphotungstate and placed on the Formvar film support.

Thesedimentation analysis was conducted in the

Spinco Ecentrifugeequippedwith ascanningsystem

adjusted for 230 nm. For sedimentation experiments, base plates were placed in 0.01 M Tris-hydrochloride buffer, pH 7.4, to bring the solutionEm0density to 1. Thecentrifugationvelocity was 26,000 rev/min.

Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS) was carried out according to Maizel (Maizel, 1969; Laemmli, 1970). The protein solution with a concentration of 100 to 200

mg/ml

was placed in 0.031 M Tris-hydrodiloride buffer, pH 6.8, containing 1% SDS, 5% glycerol, 2.5% mercapto-ethanol, and 0.0025%c bromophenol blue. The mixture was kept for 1.5 min in aboiling bath, cooled, and placed on the gel. The separating polyacrylamide gel was used in the 10%c concentration. The separation was continued for5 to7 hrusing a current of 3 amps.

RESULTS

Base plates wereisolated using the above

pro-cedure with constant electron microscopy and

sedimentation analysis to control their

purifica-tion. Theonlyadmixture that occurred

occasion-ally with base plates was core material; in this

case we saw not only base plates but also base

plates connected with cores. Figure 1 shows a

typical electron micrograph of a base plate frac-tion.

The sedimentation analysis confirmed

homo-geneity of the base platepreparation yielding the

only peak that corresponded to a sedimentation

coefficient of69S.

A thorough study of electron micrographs

warranted the following major conclusions

con-cerningthepreparationand the structure of base

plates. In addition to hexagonal base plates, the preparation also contained a small number of

stars, which varied from experiment to

experi-ment, apparentlydepending ontheeffect of

cer-tain damaging factors attending the isolation of

base plates and their preparation for electron

microscopy.It wasnoted thatasmall increase of

alkalinity favored an irreversible transformation ofhexagonal base platesinto stars. Thisprocess

occurred most intensively ina narrow pH zone

of 10.5 to 11.0. At early stages of alkalization,

beginning from 10.0, a small number of tetrades

was seen toappearinthemedium,aphenomenon already referred to by Cummings et al. (2). An

increase in pH above 11.0brought about a

dis-sociation of base plates and disappearance of

tetrades. Tostudythereversibilityof dissociation

of baseplates,analkalinizedsolutionwas

neutra-lizedbyaddingHCIorbydialysis againstneutral

buffer. In either case we failed to find

recon-structedbase plates, and amorphous aggregates of

subunitmaterialwere seentoform in thesolution

instead.

A statistical study shows that the largest

diameter of hexagonal base plates is

approxi-mately 360 nm and of star base plates about

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POGLAZOV, RODIKOVA, AND SULTANOVA

FIG.1.Electron micrograhoffraction.X51a base platet6 O

FIG. 1.Electronmicrograph ofabaseplate fractioni. X565,000.

480 nm. In the center ofhexagons we discrimi- withcores wereturnedsideways

(Fig.

2).

Besides,

nated a plug and a hexagonal disc of about hexagonal baseplates (Fig. 3a) displayeda sub-180nmin diameter.Thediscwasveryclear when structure situated along the

edge

of the plate

thebase plates that formed dimersor combined which looked like tilted gear teeth. A schematic

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..A

FIG.2.Electronmicrograph of base plates

conijugated

withcores. X825,(00.

representationofahexagonal base plate isgiven

inFig. 4a. Sofarwehave accumulatednodirect

evidence butwe cansupposethat these teethare

none other than a projection of short fibrils. If

this is true,then shortfibrilscannot run

radially

inrelationtothecenterofabaseplatebutshould

be

disposed

at acertainangle.It shouldbenoted thatstars

(Fig.

3b and

c)

alsoexhibitnot

straight

radialraysbutslightly inclinedraysrunningat an

angletotheradius.

Accordingly,

the

long

fibrils thatare attachedtothemarealso turned in one

direction.Aschematicdiagram of thestaroutline

ispresentedinFig. 4b.

Simple

electron

microscope

examination of

base platesdemonstrate that

they

are

composed

ofatleastfour different elements

(short

fibrils,

a

plug, the plate

itself,

and a disc located in the

inner partofthe baseplate).Theprotein

composi-tion was verified

through

studying

dissociated base plates with the aid of

electrophoresis

in

polyacrylamide gel

with SDS. In most

experi-ments we clearly

distinguished

five bands that

corresponded

to

proteins

withmolecular

weights

of36,000,53,000, 66,000,

81,000,

and

87,000.

We

also hadanimpressionthat theband of53,000is

a double one. This idea needs, however, further

investigation. Furthermore, we often observed

protein material with a molecular weight of

slightly more than 100,000. Thus, according to

ourresults,thebaseplatesarecomposed of5to 7

proteincomponents.Nevertheless, we cannot rule

outthe

possibility

that someproteincomponents

occurring in minor amounts such as the phage dihydrofolate reductasearebeyond the sensitivity ofourmethod.

Owing to the fact that the first product with whichthe base plateinteracts during the process

of bacteriophage

particle assembling

is a core

protein and also because the mechanism of its

polymerization on a baseplateareunknown, we

exploredthepossibility ofabase platecombining

in vitro with free tail core in the presence of

extracts from phage T4D-infected cells. These

experimentsweredesignedtoverifythehypothesis

thata coremaybegintogetassembledonlyona

baseplate rather thangetting attachedtoit in a

freeform(6). In theseexperiments, purifiedbase

platesweremixed with isolatedcoresof

bacterio-813

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POGLAZOV, RODIKOVA, AND SULTANOVA

FIG. 3. Electroni micrographofabaseplateintwostates. X825,000. a,Hexagonal form;b anzdc, starforms.

360A 460i

A B

FIG. 4. Schematic representation ofa base plate

projectionl in two forms. a, Hexagonal projection

(spinnzedshortfibrilsareseen); b,starprojection (some

turni of rays in one directiont can be distiniguished).

phagesT2 orT4,the mixturewascombined with

thelysate, incubated for 3 hr at 30C, and then

examined under the electron microscope.

Con-jugations of base plates with cores were not

ob-served in anyof the experiments.

DISCUSSION

Abase plate is themost

complicated

structural

element of the tail of T-even

bacteriophages.

Fifteen genesareinvolvedinitsformation (3,5).

Some of them produce structural

proteins

and

others play a regulatory,

catalytic

part. Our

electrophoretic experiments haveclearlyrevealed

5 to 7proteinfractions,althoughwecansuppose

the existence of additionalminor

proteins

which

wehave failed todiscriminate.

It isobvious that theelectrophoretic separation

we have employed is more sensitive than the

separation on the agarose column used by

Cummings et al. (2). Apparently,thishasenabled

ustodiscriminate additionalproteincomponents

ofa baseplate-proteinswith molecular

weights

of 36,000 and 53,000. More detailed studies on

theproteins ofthebaseplatewill bereported

by

Kingand Lacmmli(personalcommunication).

In ouropinion, the component withamolecular

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weight of 17,000 (2) should bereferredtothecore

protein because we also havedetected it

(molec-ular weight = 19,000) mainly in the fraction of

base plates plus cores (forks).

Asmentioned above, the electron microscopy

data suggest the presence of at least four

struc-tural elements in a base plate (short fibrils, a

centraldisc, the plateitself,andaplug).Theplug

protein occursundoubtedly inthe least amount.

Thebase plateitself contains certainspecial

sub-units whichareincontact with the discandwith

short and long fibrils. The central disc can

cer-tainly be considered an

integral

part of base

base plates, as follows from the recent

experi-mentswithdoublemutants (48-, 18-)carriedout

byKing (6).

We have failed to reconstruct base plates in

vitro aftertheir dissociation. The results of these

experimentsservetoemphasizeacomplex organi-zation ofa base plate and point to the need to

implicate someadditionalregulatory factors. We

donotknow asyethowto explainthefact that baseplates cannot complement with free cores.

Considering that we have experimented with

highlypurifiedbaseplatesandaddedall necessary

cofactors into the reaction mixture with the

lysate,it remainstobeassumed thatduring

isola-tion the

artificially produced

cores may lose a

componentwhich should lieatthe end ofa core

and contactwith the base plate.

Of great interest is the structural

transforma-tion of a hexagonal base

plate

into a star base

plate, although the mechanism and function of

the processremain unknown. It may be supposed

that the conversion is accompanied by a

con-formational transformation of base plate

pro-teins and their transfer to an active state necessary

for an interaction with the cell envelope and a

liberation of the core tail. The spinning of star

rays and nonradial arrangement of short fibrils

we have observed correlate to a certain extent

with the shape of an extended tail sheath in the

crosssection found by Klug and DeRosier (7).

LITERATURE CITED

l. Anderson, T. E.,andR.Stephens. 1964.Decomposition of T6 bacteriophage inalkaline solutions.Virology 23:113-116. 2. Cummings, D. J., V. A.Chapman,S.S. DeLong, A. R. Kusy,

and K. R.Stone. 1970.Characterization of T-even bacterio-phagesubstructures. II.Tailplates.J. Virol.6:545-55. 3. Edgar, R. S., and I. Lielausis. 1968.Some steps in the

as-semblyofbacteriophageT4.J. Mol.Biol. 32:263-276. 4. Edgar, R. S., and W.B. Wood. 1966.Morphogenesis of

bac-teriophage T4 in extracts of mutant-infected cells. Proc. Nat. Acad. Sci.U.S.A. 55:498-505.

5. King, J. 1968. Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32:231-262.

6. King, J. 1971.Bacteriophage T4 tail assembly: four steps in coreformation.J.Mol.Biol. 58:693-709.

7. Klug, A.,and D. J.DeRosier.1966.Optical filteringof electron micrographs: reconstruction ofone-sided images. Nature (London)212:29-32.

8. Kozloff, L. M., C. Verses, M. Lute, and L. K. Crosby. 1970. Bacteriophage tailcomponents. II. Dihydrofolate reductase in T4D bacteriophage. J. Virol. 5:740-753.

9. Simon, L.D., and T F. Anderson. 1967. The infection of Escherichia coliby T2and T4bacteriophagesas seenin the electronmicroscope.II.Structureandfunctionofthe base-plate. Virology 32:298-305.

VOL. 10, 1972 815

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ERRATA

Isolation and Characterization of Bacteriophage T4

Base Plates

B. F. POGLAZOV, P. P. RODIKOVA, AND R. A. SULTANOVA

Laboratoryof Bioorganic Chemistry, Moscow State University, Moscow, U.S.S.R.

Volume 10, no. 4, p. 810, abstract: Change thesecond sentence, "By electrophoresis in polyacryla-mide gel with sodiumdodecyl sulfate the base plates have been shown to contain five to seven protein components with molecular weights of 36,000, 53,000, 66,000, 81,000, 87,000, and probably about 100,000." to read "By electrophoresis in polyacrylamide gel with sodium dodecyl sulfate the base plates havebeen shown to contain seven protein components with molecular weights of 30,000, 36,000, 46,000, 60,000, 86,000,

and

about 100,000 and 115,000."

P. 813,left column, beginning with line 21: Change "In most experiments we clearly distinguished

five

bands

that

corresponded

toproteins with molecularweights of 36,000, 53,000, 66,000, 81,000, and 87,000. We also hadan

impression

that the band of53,000 isadoubleone.This idea

needs, however,

further investigation.

Furthermore,

we often

observed

protein material with a molecular weight of slightlymore than 100,000. Thus,

according

to ourresults, the base plates are

composed

of 5 to 7 protein components. Nevertheless, we cannot rule out the

possibility

that some protein components occurring in minoramounts such asthephage

dihydrofolate reductase

are

beyond

the sensitivity of our

method."

to

read

"In mostexperiments we

clearly

distinguished five bands that

corresponded

to proteins withmolecular weights of 30,000, 36,000, 46,000, 60,000, and 86,000. We also had an im-pression that the band of 46,000 is a double one. This idea needs, however, further investigation. Furthermore, we

often

observed

proteinmaterial with a molecular weight of about 100,000 and 115,000. Thus,

according

to our

results,

the base plates are composed of 7 protein components. Nevertheless, we cannot

rule

out the

possibility

that some protein components occurring in minor amounts are

beyond

thesensitivity ofour

method."

P. 814, right column, line 16: Change "of 36,000 and 53,000." to read "of 31,000 and53,000."

Latency of

Human Measles

Virus

in

Hamster

Cells

PAUL KNIGHT, RONALD DUFF, AND FRED RAPP

Department ofMicrobiology, CollegeofMedicine, TheMilton S. Hershey Medical Center,

ThePennsylvania State University, Hershey, Pennsylvania 17083

Volumiie

10, no. 5, p. 995, column

2,

line 7:

Change

"The Schwarz measles

virus,

vaccine lot no 94217, was

obtained

from

Merck, Sharp

& Dohme. This virus was

passaged

eight

times in

BSC-1

cells."

to

read

"The virus

employed

was obtained

commercially

fromThe Dow Chemical Co. Itwas

derived

from the Schwarz strain but had been

passed eight

times in13SC-1 cells at37 C

prior

toits use inthis

study."

Ribonuclease H: a Ubiquitous

Activity

in

Virions

of

Ribonucleic

Acid

Tumor

Viruses

DUANE P. GRANDGENETI, GARY F. GERARD, AND MAURICE GREEN

InstituteforMolecular Virology,Saint LouisUniversity SchoolofMedicine,St.Louis,Missouri63110