Characterization of T-Even Bacteriophage Substructures

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JOURNAL OFVIROLOGY, Oct. 1970, p. 534-544 Copyright©1970 American Society for Microbiology

Vol. 6, No. 4 Printedin U.S.A.

Characterization of T-Even Bacteriophage

Substructures

I.

Tail Fibers and Tail Tubes

DONALD J. CUMMINGS, A. R. KUSY, V. A. CHAPMAN, S. S. DELONG, AND K. R. STONE

Departmenitof Microbiology, University of ColoradoMedicalCenter, Denver, Colorado 80220

Received for publication 21 May 1970

T-even bacteriophages were grown and purified in bulk quantities. The

pro-tein coats were disrupted into their component substructures by treatment with

67% dimethyl sulfoxide (DMSO). Tail fibers and tubeswere purifiedon

glycerol-CsCI-D20 gradients and examined with respect to sedimentation properties,

sub-unit molecular weights, amino acid composition, isoelectric points, and

morphol-ogy.It wasfoundthat intact tailfibershadasedimentationcoefficient of 12to 13S

and that dissociated fibers consisted of three classes ofproteins having molecular

weights of 150 K ti 10, 42 K 4 4, and 28 K i 3 daltons. A model was

con-structed in which the 150-K subunit folded back on itself twice to give a

three-stranded rope. Each 150-K subunit then represented a half-fiber and it was

pro-posed thatthe role of the 42-and28-Ksubunitswastohold each half-fiber together

aswellasserve asapossiblelinkwith other substructures. Isoelectric pointstudies

also indicated that there were three different proteins with pl values of 3.5, 5.7,

and 8.0. Amino acidanalyses indicated that fibers hada compositiondistinct from

other phage substructures. In addition, a striking difference was noted in the

con-tentoftryptophanamongthephagesexamined. T4B had threeto five timesmore

tryptophan thandid T2L, T2H, T4D, andT6. Intact tail tubes had anS20o, of 31

to 38S and dissociated tubes consisted of three proteins of molecular weights 57

K + 5, 38 K + 4, and 25 K i 3 daltons. Based on degradation studies with

DMSO, it was proposed that these three proteinswere arranged inahelicalarray

yielding the tube structure. Isoelectric point studies indicated that therewere three

major proteins in the tube whose pl values were 5.1, 5.7, and 8.5. No significant

differenceswereobserved inthe amino acidcontent of tubes obtained from all the

T-even bacteriophages.

T-evenbacteriophagearecomplexviruses

com-posedof distinct substructural elements. The head substructure (4, 16;unpublished data) and the tail sheath (3, 21, 25) have been well characterized, but the physical and chemical properties of the neck (6, 21), collar, tail tube, tailplate, and tail fibers havenotbeenreported. Thishas beendue

to primarily the lack of methods for separating intact substructures and alsotothesmall relative

amounts of these substructures. Recently,

methods havebeenreported (6, 28)which doyield intact substructures and isolation procedures for handling large quantities of bacteriophage have

beendeveloped (1, 16).

Inthis paper, wewill present the resultsonthe physicaland chemicalpropertiesof tail tubes and tail fibers, and, in thecompanion paper (8), we

will present theresultsontailplates.

MATERIALS AND METHODS

Bacteriophage growth conditions and purification.

Bacteriophages T2L, T2H, T4B, T4D, and T6 were

grownin70-liter quantities under standard conditions

(4, 16). Toinsure maximum yields, thebacteriawere

infected with a multiplicity of infection of 3:1 with

phageprepared2daysbeforeuse;also, 30 ,ugof

thy-minepermlwasaddedatthe time of infection.

Con-centration of these bulk lysateswasachieved byusing

atwo-phasepolymermethod (1,16;S.Ward, personal

communication). At the end of the growthperiod, 1.0

ml ofchloroformperliterand1mgeachof

deoxyribo-nuclease and ribonuclease wereadded. Withmixing,

thefollowing materialswereadded, intheorderlisted,

per liter: 17 g of sodium chloride, 2.3 g of sodium

dextran sulfate (Pharmacia, Uppsala, Sweden), and

71gofpolyethylene glycol6000 (Chemical Sales Co.,

Denver, Colo.). Eachcompound was dissolved

com-pletelybefore addingthe next. The lysatewas placed

inacoldroomat4Cfor24to 30 hr. Mostof theupper

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T-EVEN PHAGE SUBSTRUCTURES. I

phasecontainingthepolyethylene glycolwasremoved

anddiscarded, and theremaining mixturewasplaced

inasmaller containerandwasagainallowedtosettle

inthecold overnight. Then theresidual polyethylene

glycol intheupperlayerwasremovedagain, and the

whitishdextranlayerwasdiluted withtwovolumes of

saline stock solution (0.15 MNaCl, 1 mM MgSO4, 1

mM phosphate buffer, pH 7.3, designated 1X SSS).

This final solutionwas mixed, and,toprecipitatethe

dextransulfate,0.22volume of3MKCIwasadded, in

the cold,slowly with mixing. This solutionwasplaced

inacoldroomovernightand thesupernatant,which

contained the phage, wascollectedandcentrifugedat

4,500 X gfor 10mintoremove debris and residual

dextran sulfate. The supernatant solution containing

the phagewascentrifugedat18,000X gfor 2 hr.The

phage pelletswereresuspendedinsalinestock solution

andrepurifiedtwotimesbycentrifugation asbefore.

Capsid preparationandpurification. The highly

con-centrated phage (25to50ml of phageat4 X 10's to

6 X 1013particles/ml) wereosmotically shocked(17),

and the released DNA was removed by

deoxyribo-nuclease digestion for 2 hratroomtemperature (4).

Theemptycapsids wereconcentrated by

centrifuga-tion at40,000 X gfor 3 hr. The pellets were

resus-pended insalinestock solution and purified by

sedi-mentationthrougha1.33g/ml density CsCl-D20step

gradient [5to 10 mlof concentratedcapsids layered

over 2 molal CsCl (Gallard-Schlesinger Chemical

Mfg.Corp., CarlePlace, N.Y.),2mmMgSO4, 2mM

phosphatebuffer(pH 7.7),and100%deuteriumoxide

(Bio-Rad Laboratories, Richmond, Calif.)] for 1 hr

at35,000 X gin aSpinco SW25rotor. Thecapsids

sedimentedto theirflotation density and were

with-drawn and diluted to 28 ml, for convenience, with

2X SSS.Thisprocedure yielded between 100 and 300

mgofpurified capsids.It should bepointedout that

this procedure of purifying the intact bacteriophage

and thenpurifyingtheemptycapsids effectively

elimi-natesthepossibilitylaterof contamination ofthe sub-structuresobtainedfromtheemptycapsidswith

bac-terialstructuressuchaspili of flagella.

Disruption ofcapsids and purification of

substruc-tures.The 28 ml ofpurified capsids was placed in a

100-ml beakerandstirred, withamagnetic bar, slowly

inanice-water bath. Twovolumes of dimethyl

sulf-oxide (DMSO; Mallinckrodt Chemical Works, St.

Louis,Mo.)wasaddedveryslowly;itwasessential(6)

tomaintain the temperatureat0C.Consequently,the

DMSO wasaddedbyusinga25-mlburet througha

Teflon needle valve (Manostat Corp.) dropwise at a

ratewhich didnotexceed 1 ml/min.Themixturewas

then transferred, in the cold, to dialysis bags and

dialyzed against twochanges of 4 liters of2X SSS.

Onthenextday,the substructuremixturewas

centri-fuged at 30,000 X gfor 10min to remove a slight

precipitate.Most of themasscontained in the mixture

represented heads. It was necessary to remove the

majority ofthese headsbeforefractionation. Thiswas

accomplished byuseofapreliminarygradient

consist-ingofabottomlayer (10 ml, 1.30g/ml density

CsCl-D20), anintermediate layer (2 ml, 1.20g/ml density

CsCl-D20),and 10to20mlofthephagesubstructure

mix. This was centrifuged for 45 minin an

Interna-tional B20centrifuge at30,000 X g. The heads

sedi-mented totheir density, and theslower sedimenting

substructures remained primarily at their original

position.Thesamevolumeappliedtothegradientwas

withdrawn from the top andcentrifugedfor 5 hr ina

Ti-50Beckman rotor at 125,000 X g. These pellets

wereresuspended in 3 to 4 mlof 2X SSS (about 0.3

ml/tube), and these phage parts were fractionated

on aCsCl-D20-glycerolstepgradient [master solution:

4.5gof CsCl, 2mmphosphatebuffer (pH7.7), 2mm

MgSO4, 13 ml of D20; bottom layer: 2.65 ml of

mastersolution,0.45mlofglycerol;secondlayer: 4.3 ml ofmaster solution, 0.6 ml ofglycerol, 1.2 ml of

D20;thirdlayer: 3.1 mlofmastersolution,0.4mlof

glycerol, 2.6 ml of D20; fourth layer: 1.9 ml of

mastersolution,0.2mlofglycerol,5.0mlof D20; top

layer: 3 to 4 ml ofthe substructure mixture.] This

gradientwascentrifuged for3 hrat 35,000X gina

Beckman SW25 rotor.Figure1displaystheseparation

achieved with thisgradient.Thefiberssedimented the

least followed by tubes, etc.Ascan benoted, intact

plates were not observed; plates tended to be

disso-ciated bythe DMS0 and this will be exploited and

discussed in theaccompanying paper.Thefibers and

the tubes were reconcentrated by centrifugation and

re-run onseparategradientstoachievebetter purity.

Studies of their sedimentation properties, electro-phoresisbehavior, anddirectexaminationin the

elec-tron microscope indicated that each preparation of

fiberswascontaminated byno morethan 5%

extra-neous material and the tubes were contaminated by

no more than15%.

Dependingonthephageyield (T6gavethe poorest

yields and was somewhat susceptible to degradation

by theDMSO),weobtainedbetween0.5and2mgof

both fibers and tubes. Basedontheirdimensions (see

below) and byusingapartial specific volume of0.75

g/cc,six fiberswould have amass of about2 X 106

daltons, and tubeswould havea mass of about 3 X 106daltons,or 2 and3%,respectively, of themassof the completecapsid. Itcan be estimated, therefore,

thatwerecoveredin pure form about 15to25%of the

desiredsubstructures from theoriginal capsids.

Con-sideringlosses inpurificationand thatnotall

substruc-tures were separated from one another, witness the

tridentsobserved,thiswasquite acceptable.

Amino acidanalysis.Theaminoacidcompositionof

fibers and tubeswasdeterminedinaBeckman/Spinco

amino acidanalyzer (19) equipped withapeak

inte-grator. A 0.2-to0.4-mgamountofproteinwas

hydro-lyzed for 22 to96 hr in2ml of 6N HClcontaining

B-mercaptoethanol and phenol (10ml of threetimes

distilled 6NHClplus5,ulitersofphenoland5,lAiters

of ,3-mercaptoethanol). The 3-mercaptoethanol and

phenol protectedtryptophanresiduesfrom acid

hydrol-ysis destruction (J. Stewart, personal communication).

Byusinglysozymeorbovineserumalbuminas

stand-ards, we wereableto account for 75 to85% of the

expected tryptophancontent.Half-cystine was

deter-mined after performic oxidation (18); with

ribonu-clease as a standard, we recovered all of the

half-cystine content ofthe proteinsexamined. Itwasnot

possibletoobtainprecise dryweightsonthe

substruc-tureproteins.Consequently,theaminoacid composi-535

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SEPARATION

OF

T-EVEN

PHAGE

PARTS

F

I

BE RS

T

U B E

S

/

TRIDENTS

/

S

H

E

AT

HS

H

E

ADS

FIG. 1. CsCI-D20gradient for thefractionation of phagesubstructures. The bar indicates 10 nm. It is clear

from this gradient thatnotallof the substructures detach one from the other. Tridents, i.e., asubstructure

con-sistingoftailplate,tailtube, andtailfiber, wereobserved in various degrees in allpreparations.This was a limiting

factor in the ultimate yield of isolatedsubstructures.

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T-EVEN PHAGE SUBSTRUCTURES. I

tion was calculated on a micromole per cent basis,

with100% set as the total for 18 aminoacids.

Sedimentation analysis. We did not have sufficient

material todetermine the sedimentation coefficient by

zonecentrifugation.Consequently, we used the band-forming centerpiece technique (27). This has the

ad-vantage ofbeing more precise than zone

sedimenta-tion both in measuring the S2,, and in detecting

impurities and also required very little material. For

both fibers andtubes, welayered20,lAitersof about a

0.5% solution ontoD20 phosphate buffer (pH 5.8).

Thefiberswerecentrifugedatl20,000Xgand the tubes

at80,000 X g inaSpinco modelE, and pictures were

taken by using monochromatic light at 275 nm (9).

Thesedimentationconstant wascalculated by using a

Nikon two-dimensional comparator, and the values

werecorrected to awater basis at 20 C (S2o w). For

both fibers and tubes, a single band was observed

throughout thesedimentation;however,wewould not

detectless thana10%contaminating band.

Determination of isoelectric points. The phage

sub-structures were disrupted into their component

pro-teins byusing 10 M ureaand were separated bytheir

isoelectric points by usingamodification (Stone and

Cummings, unpublisheddata) of the acrylamide

gel-ampholine technique developed byDale and Latner

(10). The details ofthis technique will be reported

elsewhere.

Determination ofthemolecularweights of

substruc-tural proteins. The molecular weight profile of the

protein composing both fibers and tubes (and also

plates) was obtained by using guanidine-agarose

columns(11).Thedetailsof this method,includingthe

molecular weight calibration, have already been

de-scribed (16). We labeled the isolated substructures

with 1261 by the method of McConahey and Dixon

(23). Approximately 0.10 mg of substructure was

placed in 2 ml of phosphate buffer(0.10 ionicstrength,

pH7.0). Then 50to100,uCi of 1261 in 20

,liters

was

added and the mixture was stirred gently in an ice

bath.Theiodination reactionwasstartedby the

addi-tionof 0.04to0.08 ml ofchloramine-T (1 mg/ml in

the same phosphate buffer) and allowed to proceed

for 10 min. Thereactionwasstopped by theaddition

of 0.04to0.08mlof sodiummetabisuffite (1 mg/mlin

thesame phosphatebuffer).Themixturewas diluted

to 10 ml with 2X SSS, the substructure was

centri-fugedasbefore,and thepelletwasdissolved in 1 ml of 6 M guanidine hydrochloride, containing 3 mm

Cle-lands reagent, 0.05 M LiCl, and 0.01 M disodium

(ethylenedinitrilo) tetraacetate, pH 6.8. Theonly

sub-structurewhichresisteddissolutioninthis solventwas

sheath, in agreement withprevious studies (28). The

dissociatedsubstructureswerethenplacedoneithera

6 or 4% agarose (BioRad Laboratories, Richmond,

Calif.) gel column and eluted with 5 M guanidine hydrochloride, containing the other reagents listed

above (16). Sampleswerecollected and counted ina

Nuclear-Chicagogammascintillation-well counter.

Electron microscopy. Electron micrographs of the

phagepreparationsweremadebyuseof the

phospho-tungstic acid-negative staining method (2) with an

RCA-EMU4electronmicroscope asbefore (6, 7).It

wasdifficulttodepositthetubesontheelectron

micro-scropegrids without first wetting the grids with 0.5%

bovineserumalbumin.Infact, the more pure the tube preparation, the more difficult it was to have them

deposit uniformly. Final results were recorded on

DupontCronar Ortho 708LithoAfilmat a

magnifi-cation of about 15,000 times and were

photographi-callyenlarged four to five times.

RESULTS

Sedimentation analysis. In our laboratory as well asin others (22, 24), it was estimated from gradients thattail fibershad a sedimentation co-efficient ofabout 12S and tail tubes had a sedi-mentation coefficient of about 30S. This was verifiedanalytically(Table 1); it can be noted that tailfibers hadanaverage

S20,,

of 12.8Sand tail tubes hadanaverageS20,w of 35S.The sedimenta-tion coefficient oftubes was morevariable than thatof fibers due,webelieve,tothe fact thattubes were more susceptible to degradation by DMSO and also tended to aggregate end to end (see below).Itshouldbepointedout,aswaspreviously reported (24),that theS20,W observedhereis quite different fromthatfound by Sarkar, Sarkar, and Kozloff (26).Intheirpaper, avalueof11.6S was reported. It is not clear why this discrepancy exists, but it is likely that these workers (26) studieddegraded tubes

possibly

mixedwith other degraded substructures.Itwill be shown later that theamino acidcompositionof tubes reported by Sarkar etal. (26) differed from that found here, particularly with regard to proline and half-cystine.

Molecular weight analysis. The molecular weightsof theprotein subunitsweremeasured by

1251I

labeling, and the profiles are illustrated in Fig. 2for fibers and in Fig. 3 fortubes.

Qualita-tively, about 80% of the fiber

protein

was con-tained in a subunit of 150,000

(150

K) daltons,

andtheremaining20%wascontainedintwo pro-teinshaving molecular

weights

of42Kand28 K daltons. Theseestimationsassumeuniform label-ingoftheproteinsinvolved andmaybe

quantita-tivelyinerror.It shouldbe

pointed

outhere that repeated analyses

yielded

the molecular

weight

valuesreported in bothFig.2and 3. However,in thecaseoffibers,themolecular

weight

of150 K

for the

major

subunit, although

precise,

was not

accurate; this value was obtained

by

extrapola-tionofourstandardcurveandcould range from 110to170 K.Thewidth of thecurve

might

sug-gest that the molecular

weight

distribution ob-tainedwas notuniform and couldindicate more

than one protein. S. Ward

(personal

communica-tion)

has

recently

shown thatonehalf-fiberhasa

subunit

having

a molecular

weight

of 114 K whereas the otherhalf-fiber hasa subunit

having

a mass of 160 K. A

single

polypeptide

chain of

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CUMMINGS ET AL.

TABLE 1. S20w ofT-evenphage tail parts

Phage Fibers Tubes

T2L 12.8S 315

T2H 13.4S 36S

T4B 12.2S 38S

z

z

D

0 U)

150 K daltons would have dimensions of 1 by 250 nm and could represent themass ofa

half-fiber. We willusethisapproach later in

construct-ing a model simulating the structure ofa fiber,

utilizing the 42- and 28-K proteins as linkers.

Tubes hadadifferent molecularweight profileand

appeared to consist of three proteins having

FRACTION NUMBER FRACTION NUMBER

FIG. 2. 12Sf labeling oftailfibers. The voidvolume (arrow) contains materialofmassgreater than theupper rangeoftheagarose.In the4%agarose,this material contained less than5% ofthecountsin theprotein peaks.

Free 125I indicates the unbound 1251 whichelutedlast.

z

z

0

U

FRACTION NUMBER

FIG. 3. 12SIlabeling oftailtubes. Thepercentagevalues under each peak representthepercentageof the total protein counts within each peak. Thearrows have thesame meaningasin Fig. 2. Somemass (less than15%)

eluted in the voidvolume. This wastypical inall the tubepatterns, andwe werenotabletoeliminateitby

in-creasingtheguanidinecontentorbyadding guanidine thiocyanate. Itmayrepresentsheathmaterial which is

re-markably resistant todissolution orit may be undissociated tubes.

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T-EVEN PHAGE SUBSTRUCTURES. I

molecular weights of 57,000, 38,000, and 25,000 daltons. Wecalculated that about half the mass of thetubes wascomposed of 57 K-dalton subunits and the other half comprised the remaining two subunits. Inconstructing a model for tube struc-ture,itshould be considered that the tube may be composedof two physical subunits each having a mass of about 60 K daltons, with one being a single polypeptidechainand the other consisting oftwochains, i.e.,the38 K-and 25K-dalton sub-units.

Amino acid composition.Multiple hydrolyses of different batches of fibers and tubes were per-formed for 22to96 hr (19.) In addition, the per-formic oxidation determinations for half-cystine also yielded analyses for all amino acids, except tryptophan, lysine, histidine, and arginine. Com-paring all of these analyses indicated that the amount of destruction of some residues and

in-creasesin other residueswere somewhatvariable

(-

10%).

Consequently, we felt that the com-position obtained at a 24-hr hydrolysis repre-sented the least variation on the average, and those values

(duplicate

determinations on two

different phage preparations) are reported here andinthe

accompanying

paperontailplates

(8).

Table 2 lists the percentage composition of tail fibers for the T-even bacteriophages examined. Comparedwith otherphagesubstructures(heads; 16,unpublished data; sheath, 3, 25;andplates,8), themost striking difference in fiber composition

was in the high glycine content. Compared with tubesand plates

(see

below)

fibers were alsolow

in half-cystine. This high glycine content is some-what similar to that of collagen (14) and may suggestthatfibers have a structure held intact by cross-linking of adjacent strands. Comparing the composition of the different phage fibers, the only difference noted was in the tryptophan content. T4B had three to five times as much tryptophan

asdid any of the other phages examined. It iswell known (5, 7, 20) that the orientation of the tail fibers on T4B is affected by the presence of L-tryptophan, whereas the fiber orientation on the other phages is unaffected. Whether the high content of tryptophan in T4B tail fibers is con-nectedwiththisconformational effect is not clear, but it is highly suggestive. It could well be that exogenoustryptophan interferes with the binding of specific endogenous tryptophan residues and ultimatelyaffects fiber orientation.

Tail tubes had an amino acid composition similar in some respects to fibers but, as was noted in Table 3,different in some residues. No

signifi-cantdifferenceswereobserved in the composition of tubes isolated fromT2L, T2H, T4B, T4D, or T6,and thecompositionreported is an average of 12different analyses. Again, itshould be pointed out that the tail tubes had

3.9%/

proline and

0.55%NO

half-cystine, whereas Sarkar et al. (26) found

7.0%/

proline and

0.10%/'

half-cystine for material thought to be tail tubes. Another indica-tion that these workers were studying degraded material was that they reported a molecular weight of about 500,000 daltons for intact tail tubes. Based on the dimensions of tail tubes, the

TABLE 2. Amino acidcompositioni oftailfibers

Amino acid

Half-cystine ...

Aspartic plus asparagine...

Threonine... Serine...

Glutamic plus glutamine Proline... Glycine ... Alanine ... Valine .... Methionine... Isoleucine ... Leucine ... Tyrosine... Phenylalanine ... Tryptophan... Lysine... Histidine... Arginine... Bacteriophage T2L 0.25a 12.8 8.5 7.6 8.1 3.3 13.4 7.6 7.3 1.2 6.8 6.1 3.0 3.3 0.30 4.6 0.80 5.0 T2H 0.36 13.3 8.9 8.0 9.2 3.2 11.3 7.8 6.4 1.3 6.7 5.8 3.2 3.2 0.48 4.7 1.0 5.0 T4B T4D 0.30 0.26 12.8 12.5 9.0 8.9 7.7 7.7 8.7 9.2 3.5 3.5 11.5 11.7 8.1 8.0 7.0 7.2 1.5 1.0 6.6 6.8 5.5 5.6 2.7 3.3 3.4 3.6 1.50 0.60 4.7 4.7 1.2 1.0 4.3 4.3

aValues expressed asmicromole percent.

T6 0.30 11.6 8.0 7.2 10.0 3.9 15.2 7.8 6.0 1.3 6.5 6.0 3.0 2.9 0.41 4.5 1.0 4.4

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CUMMINGS ET AL.

TABLE 3. Amino acid composition ofT-evenz phage

tail tubes

Amino acid Micromolepercenta

Half-cystine ... 0.55 4 0.10

Aspartic plus asparagine 13.1 ± 0.4

Threonine 7.5 ± 0.5

Serine... 7.0 i 0.5

Glutamic plus glutamine... 11.1 i0.2

Proline 3.9 ± 0.5

Glycine... 12.5 i 1.2

Alanine 8.5 i0.7

Valine...

5.6 ± 0.4

Methionine 0.90 i 0.1

Isoleucine 6.3 i0.4

Leucine 6.3 i0.4

Tyrosine 3.0 ± 0.2

Phenylalanine ... 3.1 ± 0.1

Tryptophan ... 0.65 i 0.20

Lysine... 5.0 4 0.4

Histidine ... 1.0 ± 0.1

Arginine... 4.0 + 0.40

aTail tubes contained less threonine, more

glutamic plus glutamine, less valine, and more

half-cystine than did tail fibers.

molecular weight should be about 3 X 106

dal-tons.

Isoelectric points of fiber and tail protein sub-units. Theisoelectricpoints (pl) of thedenatured proteins weredetermined by using the technique ofelectrofocusinginacrylamide gels (10) followed by stainingwith bromophenolblue. Figure4is a schematicdiagram depictingtheisoelectric points for both fibers and tubes obtained from all of the

T-evenphageexamined. Inthecase offibers, the

majorbandwasactuallyaprecipitated proteinat pl 3.5 i 0.2. This band is illustrated as abroad, stripedareabecauseit did notstain, possiblydue

toitsprecipitationortothe pH of thatregionof thegradient.Presumably,thiswasthemajorfiber subunit of 150 K daltons molecular weight. Al-though it was difficult to reisolate this band, aminoacidanalysis indicatedthatthisproteinhad the same composition as did intact fibers. The acidity ofthepl ofthis protein suggests that the

high content of aspartic plus asparagine and

glutamic plus glutamine in fibers was largely asparticacid andglutamic acid. The pl values of

the other two proteins were found to be 5.7 +

0.2and 8.0 4 0.3.One of theseproteins, at pl 5.7,

occasionally had a satellite band of much less intensity, indicated by the dotted line. We could

notascertain whether this was another protein or

was due to subunit interaction on the gel. For example,themajorheadprotein(16) which hada

molecular weight of 40 K daltons never gave a single pl value (unpublished data) but rather a

minimum of three bands, and at higher

concen-ISOELECTRIC

PATTERNS

TUBES

pI

5.1

5.7

8.5

F I

B E R S

pI

3.5

5.7

8.0

FIG.4. Isoelectric pointsfor tailfibers and tubes.

The values werecalculatedby using known values for

lysozyme,trypsini,andbovineserumalbumin.

trations of protein additional bands were ob-served. Thesame wasfound for tubes andplates (8). Fortubes, threemajorbandswerenoted with pIvalues of5.1 i 0.2, 5.7 i0.2, and 8.5 + 0.3. Thissuggested thatabout half theproteinwasin the pI 5.1 bandwith lesseramountsin the5.7and 8.5bands. Thiscorrelates well with themolecular weight profile. However, several minor bands also occurred (dotted lines), and again we cannot be certain that subunit interaction didnotaffectthe patterns. In any case, the patterns clearly indi-cated that fiber and tube proteins were unique with respect to one another and that both were

different fromplates(8). DISCUSSION

We have reported a straightforward physico-chemical study of T-even phage tail fibers and tubes. Based on these studies, models for the

structure of these substructureswereconstructed

andarepresentedinFig.5and6.

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T-EVEN PHAGE SUBSTRUCTURES. I

0

20 A

0

IA

GUANIDI N E

650 A

0

1OA

+

150 K

T

0

2500 A

+ 9

+

o

42K 28 K

150 K

FIG. 5. Tailfibermodel. Thedimensionsgivenforintactfibers weredeterminedfromamicrograph suchasA.

(A) Thedimensionsgivenforthe 150-Kdenaturedsubunitswerecalculatedandthe42-and 28-Kwereidealized

asellipsoidalparticles. (B)Atailplatewhich has whatcouldbeconsideredsplit fibers. (C) Representativeelectron

microscopicfieldofthe 150-Ksubutnit; thepuddledmass on the right was observedas well astheamorphous

materialon the left. The bar indicates 10nm.

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542 CUMMINGS ET AL. J. V IROL.

35A

0C

0

PROTEINS ~~~1 00A

0

FIG.6. Tailtube model. Thetailtube isdepictedas ahelical substructurecomposedofthe 57-, 38-, and25-K

proteins. In this model, the diameters ofthe tube were obtained from measuringatleast 20 separate tubes,and

the length was obtainedfrom the length ofthe tube while still attached to the phageparticle. InAandB,the

arrowsinzdicatepossibletubesubuntits;similar subunits wererecentlyindicated by To et al. (28). Thebluntarrow

in A indicatesapossible neck substructure (6, 21) which iswider than tubes (6) andwas observedattachedto

either the head or tube (6).

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T-EVEN PHAGE SUBSTRUCTURES. I

The tail fiber (Fig. 5) isdepicted as two half-fibers (22) held together byone of thetwo low-molecular-weight protein subunits, with the

re-maining low-molecular-weight protein serving as

alinkwith the baseplate (not shown).Each half-fiberwasconstructed as athree-stranded ropeby using the 150-K subunit; this subunit would be sufficiently long (250 nm) tocurl back onitself twice, asshown,andthentwisttoformthe rope. This type of model has the advantageof specify-ingthelength ofeachhalf-fiber. Thehigh glycine

contentsuggeststhat this residue may beinvolved

in thefree rotationrequired for athree-stranded rope. InFig. SB,abaseplate isshownwith fibers attached which appear to be split; a three-stranded rope could besplitinjustsucha manner.

However,it isdifficulttobecertainthat the"split fiber" isnot another free fiberlying in the field. Figure SC isanelectronmicrographof the 150-K subunit; this was obtained by centrifuging the 150-K subunit isolated from the guanidine agarosecolumn (Fig. 2) under conditionswhich would sedimentahalf-fiber.Allthatwasvisiblein thefield was anamorphous substance which ap-pearedtobe aggregatesofasymmetricmolecules. The evidence for this model is indirect but the model does accountforthefibermorphology. In

terms ofreconciling this model with the genetics

of tailfibers,it is known that therearethreefiber antigens, A, B, and C (12), controlled by six genes (13). In addition, antigen B and C appear

tobegeneticallylinked(12).

Lessinformationisavailable forthetubes. The only information we had concerning their

struc-ture was that tubes appeared to be somewhat

labiletoDMSO. Occasionally breakage products appeared and also

polytubes

(Fig. 6A and

B),

suggesting that the tube had helical symmetry. Therefore, the model was considered to be a

helical structure composed of the three subunits with molecularweights of57K, 38K, and 25 K daltons. No distinction was made in subunit size; it ispossible,assuggested earlier,thattwo physi-cal units exist: a 57-K single polypeptide chain and a 63-K double polypeptide chain. Alterna-tively,it maybe that

only

oneof theseproteinsis theactualsubunitused in the helix and the other

two arenecessaryforholdingthetubetotheneck

and plate. As was

pointed

out, the molecular weight profileswere obtained after iodination of the intact tube and this has its limitations. Preli-minary experiments have indicated that, when iodination was carried out ondissociated tubes,

more of the low-molecular-weight subunit was

found. Unfortunately, tubes are difficult to ob-tain in convenient

quantities

and these

experi-ments require additional work. Genetically, very

little is known about the tail tube.About 15 genes

(13) have been implicated with the tail plate and tube (trident)substructure, but only one, gene 54 (13), has been found to be associated with tube assembly alone. Recently, J. King and U. K. Laemmli (personal communication) showed that gene19 directs thesynthesis of a tube polypeptide chain having a molecular weight of20 K. Ob-viously, with three proteins more than one gene would berequired, andmoregeneticinformation mayclarify tail tube structure and assembly. Our model represents a first approach to understand-ingthe structure of this interesting bacteriophage substructure.

ACKNOWLEDGMMENTS

This investigation was supported by Public Health Service grants Al 06472 and Al08265 from the National Institute of Allergyand Infections Diseases.

We alsothankB.F. Pollock forassistancein the aminoacid analyses.

LITERATURE CITED

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fromT-evenbacteriophage.I.Molecularweight characteri-zation. J. Virol. 5:398-405.

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I.Bendet, D. Goldstein, and M. A. Lauffer. 1965. Functions

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22. King, J., and W. B. Wood. 1969. Assembly of bacteriophage T4tail fibers: the sequence ofgene product interaction. J. Mol. Biol. 39:583-601.

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iodination of proteins for immunologic studies. Int. Arch. Allergy29:185-189.

24. Poglazov, B. F., and T. I.Nikolshaya. 1969. Self-assembly

of theprotein of bacteriophage T2 tailcores.J. Mol. Biol. 43:231-233.

25. Sarkar, N., S. Sarkar, and L. M. Kozloff. 1964. Tail

compo-nents of T2 bacteriophage. I. Properties of the isolated contractile tail sheath. Biochemistry 3:511-517. 26. Sarkar, S., N. Sarkar, and L. M. Kozloff. 1964. Tail

compo-nents of T2bacteriophage. II.Properties of the isolated

tailcores.Biochemistry 3:517-521.

27. Studier, F. W. 1965. Sedimentation studiesonthe size and shape of DNA. J. Mol. Biol. 11:373-390.

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Figure

FIG. 1.fromfactorsisting CsCI-D20 gradient for the fractionation ofphage substructures
FIG. 1.fromfactorsisting CsCI-D20 gradient for the fractionation ofphage substructures p.3
TABLE 1. S20w of T-even phage tail parts

TABLE 1.

S20w of T-even phage tail parts p.5
FIG. 2.Freerange12Sf labeling of tail fibers. The void volume (arrow) contains material of mass greater than the upper of the agarose
FIG. 2.Freerange12Sf labeling of tail fibers. The void volume (arrow) contains material of mass greater than the upper of the agarose p.5
Table 2 lists the percentage composition of tailfibers for the T-even bacteriophages examined.Compared with other phage substructures (heads;

Table 2

lists the percentage composition of tailfibers for the T-even bacteriophages examined.Compared with other phage substructures (heads; p.6
TABLE 2. Amino acid compositioni of tail fibers

TABLE 2.

Amino acid compositioni of tail fibers p.6
TABLE 3. Amino acid composition of T-evenz phagetail tubes

TABLE 3.

Amino acid composition of T-evenz phagetail tubes p.7
FIG. 4.lysozyme,The Isoelectric points for tail fibers and tubes. values were calculated by using known values for trypsini, and bovine serum albumin.
FIG. 4.lysozyme,The Isoelectric points for tail fibers and tubes. values were calculated by using known values for trypsini, and bovine serum albumin. p.7
FIG. 5.materialasmicroscopic(A) ellipsoidal Tail fiber model. The dimensions given for intact fibers were determined from a micrograph such as A
FIG. 5.materialasmicroscopic(A) ellipsoidal Tail fiber model. The dimensions given for intact fibers were determined from a micrograph such as A p.8
FIG. 6.proteins.arrowseitherthein A Tail tube model. The tail tube is depicted as a helical substructure composed of the 57-, 38-, and 25-K In this model, the diameters of the tube were obtained from measuring at least 20 separate tubes, and length was obt
FIG. 6.proteins.arrowseitherthein A Tail tube model. The tail tube is depicted as a helical substructure composed of the 57-, 38-, and 25-K In this model, the diameters of the tube were obtained from measuring at least 20 separate tubes, and length was obt p.9

References