Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

0022-538X/81/120635-10$02.00/0

Dual Functions of Bacteriophage

T4D

Gene 28 Product:

Structural Component of the Viral Tail

Baseplate Central

Plug and

Cleavage Enzyme for Folyl

Polyglutamates

I.

Identification of T4D

Gene

28

Product in the Tail

Plug

LLOYD M.

KOZLOFFt*

AND JORGE ZORZOPULOSt

Departmentof Microbiology and Immunology, Universityof Colorado Health SciencesCenter,

Denver, Colorado 80262

Received 18

July

1980/Accepted14

July

1981

The T4Dbacteriophagegene 28product isacomponentof thecentralplug of the tailbaseplate,asshown by thefollowingtwoindependent lines of evidence. (i)Ahighlysensitive method for radioactive labeling ofonly tailbaseplate plug

components was developed. These labeled plugcomponents were incorporated

byacomplementation procedure intonewphageparticles andwereanalyzed by radioautography after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Threenewstructural proteinswerefound in additiontothe three known tailplug proteins(i.e., gP29, gP27, and gP5). One of the three newly identifiedcomponents

hadamolecular weight of24,000 to 25,000and appeared to be a product of T4D

gene28. (ii)Characterization ofmutantsofEscherichia coli bacteriophageT4D

whichproducedalteredgene 28products alsoindicated that thegene 28product

was aviral tailcomponent. T4D

28'

phageparticles producedatthepermissive

temperaturehadaltered heat labilities compared withparentT4Dparticles. We

isolated a single-step temperature revertant of T4D

28'

and found that it

produced phage particles which phenotypically resembled the original T4D

particles. Since the properties of the phage baseplatecomponents usually

deter-mine heatlability, these two changes inphysical stability after twosequential single mutations ingene 28supported the other evidence that thegene 28product

was aviralbaseplatecomponent.Also, compared withparent T4Dparticles,T4D

28'

andT4D 28amviralparticles adsorbedatdifferentrates tovarious typesof

host cells. In addition, T4D

28'

particles exhibited adifferent host range than

parentT4D particles. ThisT4D mutant formed plaques with anextremely low efficiency onall E. coliK-12 strains tested. We found that although T4D

28'

particles

adsorbed

rapidly

andirreversiblytothe E. coliK-12strains, asjudged

by gene rescue

experiments,

these particles were not ableto inject their DNA

into theE. coli K-12 strains.

On

the otherhand, the T4D

28'

revertant had a

platingefficiency onE. coli K-12 strains that was quite similar to the plating efficiency of the original parent,T4D. These properties ofphage particles

con-taininganalteredgene 28product supported the analyticalfinding that the gene 28product isastructuralcomponent of the central plug of the T4D tail baseplate.

They also indicated that thiscomponentplaysaroleinboth hostcellrecognition

and viralDNAinjection.

The first characterization ofthe role ofthe the long tail fibers) were observed. The gene

Escherichia coli Bbacteriophage T4Dgene 28 examinedmappedinan areaof severalgenes,all

productwas by Epstein etal. in 1963 (8). The of which appeared to be involved in tail mor-onephagemutantwhich these authors described phogenesis. Later, Edgar and Lielausis (7) and

was a

conditionally

lethal amber mutant; upon King et al. (13-15) showed clearly that the T4D

infection of nonpermissive host bacteria viral gene 28 product was involved in forming the

DNAsynthesisoccurred andempty heads were complex tail baseplate upon which the rest of

formed,

but novisibletailstructures(exceptfor the tail structure was assembled. Using an

in-tPresentaddress:DepartmentofMicrobiology,University genious indirect genetic method, Snustad con-of

California,

SanFrancisco, CA 94143. cluded in 1968 (29) that the gene 28 product

635

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636

fuilfiled

some unknown "catalytic" function in

tail morphogenesis rather than functioning as

"stoichiometric" componentof thetail.

In 1965Kozloff and Lute (17) found that the

phage baseplate contained a folic acid as an

untMsual low-molecular-weight structural

com-ponent. Thefolatewasidentified lateras

dihy-dropteroylhexaglutamate (16, 18, 20-22, 26, 28).

Since the formation of thiscompound in infected cells might have involved catalytic reactions

necessary for phage baseplate morphogenesis,

Kozloff and Lute investigated the possibility

thatthe gene 28 product or some other phage

catalytic gene product was involved in folate metabolism (18). Baugh and Krumdieck (2) had developed methods to synthesize pteroyl hex-aglutamate, and this compound andsome

ana-logs of itwere tested for activity inphage tail morphogenesis. Complementation experiments

wereperformed byusingmixtures of folate-de-pleted extracts from host cells infected with different T4D tail mutants, such as T4D

7-extract complemented with T4D 10- extract.

Considerable increases in

phage

formationwere

observed when

synthetic pteroyl hexaglutamate

wasadded butnotwhen

pteroyl

penta- or hep-taglutamatewasadded (21).If the T4Dgene 28

productwas anenzymewhich

functioned

solely

in theformation of the

pteroyl hexaglutamate,

then it seemed possible that the addition of

synthetic pteroyl hexaglutamate

to extracts of nonpermissive bacteria infected with the T4D

gene 28 amber mutant

might

lead to

phage

formation.

Subsequently,

itwasfound that small

amountsofphagewereformed whenthe

phage-specific folate was added to extracts of cells infected with the T4D gene 28 ambermutant.

Nootherextractof bacteria infected with

only

onephagemutant

responded

totheaddition of thisfolate compound, and it seemed clear that

thegene 28productwas involved infolate

me-tabolism; however, itwas notapparentwhy the

response to the added

pteroyl

hexaglutamate

was solow. Inview of the evidence(19)

concern-ing the dual role of thegene 28product,

only

a

minimalresponsetothe addedfolatecompound

ingene 28- extracts wouldbeexpected.

Later,

experiments

(18, 28) showedthat

wild-type T4D infections caused host cell folatesto

beconverted frompopulations of molecules

con-tainingmostlythreeglutamate residuesto

pop-ulations of molecules in which the number of

folates containing six

glutamate

residues was

increased

substantially.

However, in T4D

28--infectedcells,up to7% of thefolate compounds

were in forms containing 12 to 14 glutamate

residues. Suchlarge folyl polyglutamates could

notbe detected ineither uninfected cellsor

wild-type T4D-infected cells. Kozloff and Lute

sug-gested (18) that the catalytic function of the

gene 28 product involved the cleavage of the

high-molecular-weight folates to hexaglutamyl forms.

In1975, Kikuchi andKing (10-12) described

the sequential roles of the gene products

in-volved intail baseplate formation. They found that the gene 28 product acted early in the pathway which ledtotheformation of the

cen-tralplug of the tail plate. The role of thegene 28 product was not defined further, and since theseinvestigatorswere notabletofind thegene 28 product as a tail plug constituent, they thought that it acted catalytically in some

un-known fashion by altering the properties of the

gene 29product.

These observations caused us to continue

in-vestigationsontherole of thegene 28product. The results described in this paper and in the accompanyingpaper (19) ledtothe conclusion

thatthe gene 28 product has two functions in

forming phage

particles.

We present evidence that the gene 28 product is not only a folyl

polyglutamate cleavage

enzyme but also is a

structural

component of the

plug

of the tail

baseplate.

In this paper we present

analytical

evidence that thegene 28

product

isatail

plug

component. Inaddition, T4D gene 28 mutants

which producedaltered gene products showed

both in vivo and in vitro characteristics which indicated that the gene 28 product has these two

functions and also indicated that the gene 28

productis localized on the distalportionofthe

central plugofthebaseplate.Inthe

accompany-ingpaper(19),Kozloff and Lute describe folate

metabolism

in infected cells, present a

partial

characterization of the

enzymatic

natureofthe folate

polyglutamate cleavage reaction,

and

con-firn thelocalization of this

enzymatic

activity

onthe distal

portion

of the

baseplate.

MATERIALS AND METHODS

Bacteriophage and bacterial strains. We used

standard methods forphageassays(1).The bacterial

strains used includedE.coli B anda mutantof E. coli

B, strain OK305 (9). The E. coli K-12 strains used

included prototype strain AB259(furnished byA. L.

Taylor) and anumber of otherE. coli K-12 strains

from theColi Genetic StockCenter,includingCGSC

4617 containingan

Su2+

gene(glutamine)andCGSC

2597containinganSu3" gene(tyrosine);wealso used

E. coli K-12 strain CR63, which contains an Sul+

(serine) gene. The E. coli bacteriophage strains used

havebeendescribedpreviously; these included

wild-typeT4D, the ambermutantof T4D in gene28(A452)

(18), other T4D ambermutantsin gene7(B16),gene

26(N131), gene27(N69), gene29(B7), and gene51

(529), and themultipleT4 ambermutantX6,which

containsamber mutations in gene23(B17), gene34

(B25 and A455), gene36(El),gene37(N52), and gene

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38 (C290). In addition,we obtaineda new (31) T4D

temperature-sensitivemutantin gene 28(A61).Since

the gene 28phagemutants wereof critic lixnportance

inthiswork,wepickedasingle plaqueiromthe T4D

28amand T4D28'mutantstocks received from

Cali-fornia Institute ofTechnology collection. The T4D

28amstock has been characterizedpreviously(18, 20,

28).The T4D28'stockwasbackcrossed twiceto

wild-type T4D (10 T4D 28+/1 T4D 28k) to reduce the

possibilityofmultiplemutations. A total of88plaques

werepicked from the secondbackcross,andonly2of

the 88 phage stocks produced temperature-sensitive

plaques. Thesetwo stockswerecomparedwith each

otherand with theoriginal A61stock. Allproperties

testedwere identical for the original A61 stock and

thetwostocks obtainedby backcrossing. These

prop-ertiesincluded thefollowing: (i) complementationat

39°C,asdeterminedbyspottests onplates containing

E.coli B(negativefor T4D28ambutpositivefor T4D

26am,T4D 27am, T4D 29am, and all othermutants

tested); (ii) temperaturesensitivity (plating efficiency

was 1.0 at 250C,0.7 at 32°C, and <0.01 at 370C or

higher); (iii) all stocksplated with identical low

effi-cienciesonall of the E.coli K-12 strains listedabove,

butthey plated equally wellonE. coli B and strain

OK305,aderivative of E. coli B.

One of thetwostocks obtained afterbackcrossing

was then used in the experiments described below

with the assumption that it differed from our

wild-typestrain of T4Donlyin gene28.

Media. We used M9 mediumpreparedasdescribed

by Kikuchi and King (10-12) to prepare 14C-labeled

extracts. Tryptonebroth containing 8 gof tryptone

(DifcoLaboratories)perliter and5gofNaCl per liter

wasusedtoprepareunlabeledextractsand the other

phage stocks.

Preparation of

"4C-labeled

extracts. An

over-night culture of E. coli B in tryptone brothwasdiluted

30-fold in M9 medium and grownto adensity of4x

108cells per mlat370C.400

pi

ofasolutioncontaining

40,uCiof

14C-labeled

amino acidswasaddedto25ml

of theculture,and the incubationwascontinuedfor5

min; then the cellswere infected with a T4 amber

mutant at amultiplicityof4phageperbacterium. The

culturewassuperinfectedwith thesamemultiplicity

ofphage 8 minlater. At 45 min after the firstinfection,

thecellswerecollectedby centrifugationat40Cfor 10

minat1,500xg, and theresulting pelletwasfrozenat

-70°C. To prepareanextract, 1 mlofasolution of

M9 mediumlacking glucose (1),whichpreviouslyhad

been diluted 10-fold with water, was added to the

frozenpellet,andasmallamountof DNasewasalso

added. This mixturewasincubatedat300Cuntil the

pellet melted; then the mixturewasfrozenat-60°C

and then incubatedfor15minat300C.Then1ml of

M9 medium diluted 10-fold and containing 10 mM

EDTA andasmallamountof RNasewasadded. The

resulting mixture wasincubated for20min at 300C

and thencentrifugedat27,000xg for1htoeliminate

cell debris andlargephage structures, suchasheads

and sheaths. Thesupernatantsolutionwasusedasthe

sourceoflabeledphage protein.

Preparationof unlabeledextracts.E. coli Bwas

grown withvigorous aeration to adensity of4 x 108

cells per ml in2.5liters of tryptone broth mediumat

370C.

This culturewasinfected withanarrbermutant

at amultiplicity of infection of4andsuperinfected8

min lateratthesamemultiplicity.

The cellswerecollectedby centrifugation at 1,500

xg for 10min at 2 h after the first infection, and the

pelletwasfrozenat-70°C. Then2ml of M9 medium

lackingglucose, diluted 10-fold, and containing small

amountsof DNase wasadded, and the mixture was

incubatedat300C until the pelletwas nolonger frozen.

This mixturewasfrozen andmeltedtwice, incubated

for15minat300C,and usedasthe crudeextractin

thecomplementation reactions.

Sucrose gradient centrifugation of extracts

containingbaseplates. A 2-mlamountofalabeled

extractwasplaced inadialysistube and surrounded

with dry Sephadex G-200 to reduce the volume to

about 400

pl.

Then theextract waslayeredontop of

a 5-ml 15 to 30% sucrose gradient in M9 medium lacking glucose diluted 10-fold. After centrifugation

for3hat45,000 rpmand5°C in an SW50.1 rotor of a

Spinco ultracentrifuge, 0.25-ml fractions were col-lected from the top of the gradient with a Buchler

autodensiflow apparatus andaperistaltic pump.

Complementation reaction to identify

base-plate tail plugs. A

25-pl

amount of each fraction

obtained after sucrosegradient centrifugation was

in-cubated overnight at 300C with 50,A of unlabeled

extractfrom E. coli infected with T4D 29- (a tail

plug-negative extract). Themixtureswerethen diluted and

platedontoE.coli CR63tomeasurephage formation.

The fractions identifiedasthe fractions thatcontained

central plugs by both sedimentation properties and

complementation activity were pooled, mixed with a large excess of the unlabeled gene 29- extract, and

incubated overnight at300C.Before this incubation,

10

pl

ofasuspension containing unlabeled T4D phage

(5 x 1012 particles per ml) was added in order to

saturateanypossible host membrane pieces

contain-ing T4 receptors, to avoid inactivation of the newly

formedphageparticles, and to act as a carrier in the

purification of the labeled new phage. After incubation

the phagewere purified by four standard cyclds of

differential centrifugation. Four cycles were enough to

obtaina highly purified phage preparation, as

indi-catedby electron microscopy and by the absence of

anycontaminantlabeled E. coli outer membrane

pro-teins, whicharesynthesizedevenafter infection. The

phagewerethen concentrated once more by

centrifu-gation and suspended in 100 to 200,l of a buffer

containing 2% sodium dodecyl sulfate, 10 mM

'tris-hydrochloride (pH 6.8), 10% glycerol, 0.7% 2-mercap-toethanol, and bromophenol blue dye.

Acrylamide gel electrophoresis and

autora-diography. The slab gels, buffers, and autoradiogra-phy techniques used have been described previously byKikuchi andKing(10-12).

Phage heat lability. Heat lability experiments

wereperformedasdescribed previously (24,25).Stnall

temperaturefluctuationsfromday to day made precise

comparisonsdifficult,and only samples heated

simul-taneouslywerecompared.

Chemical and other methods. All procedures

used have beendescribed previously (23).All

chemi-calswereanalytical grade. The radioactive amino acids

werepurchased fromNewEngland NuclearCorp.

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KOZLOFF AND ZORZOPULOS

RESULTS

Preparation and isolation of"C-labeled

baseplate tail plugs. We obtainedlabeled

cen-traltail plugs by usinganambermutantof T4D

ingene7whichwasnotabletoform the "wedge"

outer structuresof the baseplate under

nonper-missive conditions.

14C-amino

acidswereadded

at5min before host E. coli B cellswereinfected

to obtain early and late viral labeled proteins.

The labeled central plugswereseparated from

other large viral substructures bycentrifugation

(see above), and the plugswerepurifiedby

su-crosegradient centrifugation (10-12). The

frac-tion containing the central tail plugswas

iden-tified by a complementation reaction with an

extractfromanambermutantdefective in

cen-tral plug formation (T4D 29-). Figure 1 shows

thatwefound two peaks ofcomplementing

ac-tivity. The peak containing the centralplug had

asedimentationconstantof22S,asreportedby

Kikuchiand King (10-12). However, we found

that this fraction also containedalarge number

of labeled proteins and could not be used to

identify central plug components. To identify

thespecific central plug proteins,weperformed

a complementation reaction between the 22S

gradient fraction andalargeexcessofunlabeled

gene29-extract.Since the 22Ssucrosegradient

fraction contained preformed labeled central

plugs, thenewviralparticles formedduring the

complementation reaction should havebeen

la-beled only in this substructure. Subsequent

steps involved the addition of unlabeled T4

phage as a carrier and purification of phage

particles by differential centrifugation. The

pu-rifiedphage particleswerethenheated at95°C

in the presence of sodium dodecyl sulfate, and

theproteinswereanalyzed by 1% sodium

dode-cyl sulfate-polyacrylamide gel electrophoresis.

Thesubsequent radioautography reflected only

theprotein compositionof thebaseplate central

plug. Figure 2 shows the densitometrictraceof

agel obtainedasdescribed above. We foundsix

labeled proteins; based on their sizes, three of

these were known components of the central

plug, namely, the product of gene 29 (80,000

daltons), the product ofgene27(49,000daltons),

and theproduct ofgene5 (44,000 daltons)

(10-12). The other threecomponents (designated A,

B, and C) havenotbeen reportedpreviously.

Toidentify these three unknown central plug

components,weusedasimplified version of the

method described above. Sucrosegradient

sep-aration of the central plugwas notperformed,

butan extremely large excessofthe unlabeled

complementing extract was added. We hoped

that this wouldnotallowsignificant

incorpora-tionofanylabeledprotein into the newly formed

0

0 0

10x c

0

E0 41

01

0E

c

.

20 40 60 80 100

% of Gradient top

.

rI0

0

0

x

0

E

._

o 0 -Q

u 0

1-u

tY

FIG. 1. Sedimentation of baseplate central tail

plugs in a 15 to 30% sucrose gradient. Radioactive

lysateswerepreparedfrom cells infected with aT4D

ambermutantdefective in gene7. Centrifugation was

performedfor3hat45,000 rpmwith anSW50.1 rotor

at5°C.A

25-gIl

amountof eachfraction was mixed

with50ulofaT4D 29- extract, and the mixture was

incubatedat37°C overnight. The viable phage were

assayed for plaque-forming activity after the

incu-bation. The centralplugwaslocalized by comparing

the complementation pattem with the

complementa-tion patternobtainedbyKikuchi and King(10-12).

viral

particles,

with the

major exception

ofthe viral components

missing

from the unlabeled

extract. To test this

method,

we

prepared

the

same labeled extract of bacteria infected with the T4ambermutantdefective in gene 7.

Any

possible intacttails dueto evensmallamounts

of leakiness in gene 7 and other

large phage

structures

(such

as

phage heads)

wereremoved

by

centrifugation,

and the supernatant extract was

complemented

with a

large

excess

(more

than100-fold)ofanunlabeledextractfrom

bac-teriainfectedwith T4 am29.Carrier

phage

was

added,andthenewviral

particles

were

purified

andanalyzed. Figure3ashows that thelabeled proteins

incorporated

into thenewviral

particles

werethe same

proteins

obtained

previously (Fig.

2).Thismodified

technique

permitted

theuseof

various T4D ambermutantsdefectiveinone or

more of the central

plug

components in the

prepar4tion of both labeled and unlabeled

ex-tracts. The unlabeled extract contained the

othercentral

plug

components,which could

in-corporate the labeled missing gene

product

to

form atail

plug

and thena

complete

newviral

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[image:4.500.263.450.62.289.2]

(a) (b)

I

fI

(c) (d) (e) (f)

gP29 gP27

7-+29- 4 gP5

A B

FIG. 2. Densitometric traceshowing the

distribu-tion of radioactive proteins incorporated into new

viral particles thatwereobtained by

complementa-tion of labeled tailplugs withanunlabeledT4D

29-extract. The fractions obtained from sucrose

gra-dients thatcontained the central plugsweremixed

with largeexcessesof unlabeled T4D29-extract,and

unlabeledphagewasaddedas acarrier. After

over-night incubation, the newparticles werepurified,

mixed, and boiled with buffer containing 2% sodium

dodecyl sulfate, 10mM Tris (pH 6.8), 10%glycerol,

0.7 Mmercaptoethanol, and bromophenolblue dye.

Theradioactive proteins wereanalyzed by 10%

so-diumdodecylsulfate-polyacrylamide gel

electropho-rests. The densitometrictraceofthe autoradiogram

obtainedfrom the dry gel is shown. The arrowsat

thetopindicate the positions of themolecular weight

standards,asfollows:arrow a,phosphorylaseb

(mo-lecularweight,94,000);arrowb, bovineserum

albu-min (67,000); arrow c, ovalbumin (43,000); arrowd,

carbonicanhydrase (30,000);arrow e, soybean

tryp-sininhibitor (20,100);arrowf,a-lactalbumin(14,400).

particle. Usingdifferent combinations ofmutant

extracts, we identified the unknown proteins.

Under these circumstances more than one

la-beled protein could be incorporated in

signif-icantamountsinto thevirusparticleifasecond

labeledprotein forned a complexor

substruc-turewith the labeledproteinwhichwasabsent

in the unlabeled extract. Figure 3b shows the

results obtained after complementation of

la-beled extracts from bacteria infected with the

T4D 51- amber mutant with an unlabeled

ex-tract of bacteria infected with the T4D 28-

am-bermutant.Peak C and thepeak corresponding

togene product27 werethe onlytwo proteins

incorporated insignificant amounts intophage

particles. The simplest explanation of these

re-sults was to assume that protein C was the

product of gene 28 and that gP27 formed a

strongcomplex with gP28,sothat this complex

was incorporated into the new viral particles

without undergoing dilution by the unlabeled

gP27 in the gene 28- extract. All ofthe other

viral proteins appeared to have been diluted successfully by the unlabeled products. (It should be pointed out that Kikuchi and

King

[12]

showed that in a gene 28- extract only a

smallamountofgP27 formedatailplug precur-sor and thatmostof thegP27 remained at the

top of the

gradient.

This observation supports

thehypothesis that gP27canformastable

com-plex with gP28.) Figure 3c shows the results obtained by using a

complementation

mixture containingaT4D51-labeledextractandaT4D

26-

unlabeled

extract. Inthiscase,

only protein

AandgP5were

incorporated

into thenewviral particles. By

using

similar

reasoning,

protein

A

should

correspond

tothe

product

ofgene26, anid

(a) 7 * +29- gP29 gP27

(b)Sl- +28- gP

llC

9P5

A

(c)51*+265

gP27gPS

[image:5.500.52.239.58.223.2]

(d)28-*+51 S

;A

FIG. 3. Densitometric traces showing the

distri-butionsofradioactiveproteins incorporatedintonew

viralparticlesthat wereobtained by

complementa-tionbetweenlabeled andunlabeledextractsofE.coli

B infected with different T4 amber mutants. The

analyticalprocedures usedweretheprocedures

de-scribedin thelegendtoFig.2. (a) 7- labeled+

29-unlabeled. (b) 51- labeled+ 28- unlabeled. (c)

51-labeled+26-unlabeled.(d)28-labeled+51-

unla-beled.

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[image:5.500.254.441.245.577.2]

it appears that gP26 formed a complex with gP5.

Figure 3d shows the results forcomplementation

of a gene 28- labeled extract with a gene

51-unlabeled extract. In this case, the three labeled

proteins incorporatedweregP27, gP5, and

(ten-tatively) gP26, andnoother labeledproteinwas

incorporated. With regard to peak C, this

com-ponent waslabeledwhen the 14C-labeled extract

containedintact gP28 (Fig. 3b); however, in the

reciprocal experiment, when intact gP28 was not

presentin the labeledextract (Fig. 3d), peak C

was not present in the new phage particles.

Theseresultsstrongly supportedthehypothesis

thatpeakC wasthe gene 28product. Fromthe

mobilityofgP28duringsodiumdodecyl

sulfate-polyacrylamide gel electrophoresis, the

molecu-lar weightof this compound (peak C)appeared

tobe24,000to25,000.

Peak A had an apparent molecular weight of

41,000; based on the results shown in Fig. 3c,

this peak appeared to be gP26. However, this

identification needs confirmation. The

molecu-lar weight ofpeak B was29,000 to 30,000, and

peak B appeared to be the monomer of

thymi-dylate synthetase (3, 4, 26), which has been

reported to be abaseplate structural component

based on a variety of evidence (4, 14). The

assembly pathway for the central plug of the

baseplate involves not only more components, butalsoasomewhat differentsequence of

mor-phogenetic steps than originally proposed by

Kikuchi andKing (10-12). This scheme has been

discussed elsewhere inapaperdealingwith the

assembly of thymidylate synthetase into the

centralplugandtheuseof thefolatecompound

to link the central plug to the outer wedge

components (Proceedings ofthe Seventh

Bac-teriophageAssemblyMeeting, in press).

Heatlabilities ofT4D, T4D 28t8, and T4D

28tsrevertants. Kozloffetal. have shown that

the complex multicomponent tail baseplate of

T4D is the most labile portion of the phage

particleand that thestructure of thisbaseplate determines the heat lability ofa particle (23).

This isnotsurprisingsincethis structure

under-goesaradical conformational change during

in-fection. When temperature-sensitive

bacterio-phage gene products are incorporated into the

baseplates ofphageparticles, theheatlabilities

of these phage particles are changed and are

usually,butnotnecessarily,increased(23). The

temperature lability ofT4D

28'

particles was

compared with that of parent wild-type T4D

particles at 62°C (Fig. 4). Under these

condi-tions,T4D28' particleswereconsiderablymore

heatlabile thanT4Dparticles.

To be certain that itwas achange in gene28

protein that was the cause of the altered heat

stability of the T4D 28ts particles, we isolated

MINUTES

FIG. 4. Heat labilities of T4D, T4D2818,and aT4D

28'S revertant. Purifiedphage particles were treated

with DNase and diluted to a concentration of106

cells per ml in 0.1Mphosphate buffer(pH 7.0).After

equilibration at room temperature for 30 min, the

phage were diluted 1:10 into preheated buffer at 62°C

and assayed at differenttimes.

T4D

28"

revertants. Theserevertants were

iso-lated by plating independent plaque-derived

stocks at 44°C; 1 heat-resistant phagewas

ob-tained per 106phage plated.The phenotype of

one isolate (designated

a,)

was consistent with thehypothesisthatit wasa true revertantofa

point mutation; Fig.4shows that a, phage

par-ticleswere moreheatresistantthan their parent T4D

28"

particles andwere

essentially

similar

towild-type T4Dparticles.

HostrangesandadsorptionratesofT4D

mutantsin gene 28. Since the gene28

product

was located on the phage

baseplate,

we

exam-ined the effects ofchangesinthe gene28product

onhost range,adsorptionrates,and DNA

injec-tion. We found that the change in the gene 28

product which made this protein heat labile

affected theabilityof thisphagemutant toform

plaquesonall strains of E. coli K-12 examined. For example, onE. coli K-12strain AB259 the

efficiency

ofplaqueformationat30°Cwas<0.3%

of the efficiencyonE. coliB, and itwas1% on

E. coliK-12 strainCR63

(Su"+)

and 0.1% onE.

coli K-12

(Su2+)

(Table1).OnE.coliK-12strain

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[image:6.500.262.454.72.346.2]

CGSC

2597

(Su3+),

the

efficiency

of

plaque

for-mationwasless than 0.1%compared with E. coli B. These plating properties suggested that the

gene 28productwasinvolved sinceafteraT4D

28tsspontaneous revertantlost itstemperature

sensitivity, it

regained

the

ability

to

form

plaquesonE. coli K-12 strains

(Table 1).

Pugsly and Schnaitman (28) have shown that thecell walls ofE. coliBand E. coli K-12are

different with respect to protein composition andfunction.

Recently,

work inour

laboratory

(32) hasfocusedonthe role ofcell wall

protein

b instabilizingphagereceptors.Since therewas

no reason to suspectthat thechange inthe gene

28protein which made the phagetemperature

sensitive when it was grown on E. coli B in-hibited the biosyntheticprocessesin E. coli

K-12 hosts even at permissive temperatures, we

examined the initialstepsofthe interaction

be-tween T4D mutants and E. coli K-12 strains.

Cultures of the E. coli K-12

Su2+

strain and E. coli Bwere grown,washed in saline, and then heatedat

650C

for1h. Heat-killed bacterial cell preparationsarequite stable and havebeen used

widely to measure phage adsorption rates (1).

After treatment with DNase, the heated

cells

werewashed in saline and storedin thecoldat a concentration of 4 x

108

cells per ml. For adsorption rate

determinations,

the heat-killed bacteriawereusedat afinal concentrationof2.5 x

10"

cells perml, and the phage preparation wasaddedsothat therewere twophageparticles

perkilledcell. This bacterial suspension contain-ing the added phagewasincubatedat

270C,

and

sampleswereremoved andassayed for live (i.e., unadsorbed) phage.

Theratesofadsorption of three phage stocks

differing only in thegene 28protein are shown

fora

typical

experiment in Fig.5.Allthreetypes

ofphageparticles readily attachedtotheE. coli K-12

Su2`

strain, but thereweresignificant

dif-ferences in the initial adsorptionrates as

mea-sured by 50%

adsorption.

T4D

28's

particles,

TABLE 1.

Platingproperties

of T4D,T4D28'8,and

aT4D28'8revertant onvarious bacterialstrainsa

Plating efficiencyon:

Phage

~~~~~~~E.

coli

Phage E.coi E.coliK- K-12 E.

col

K-B 12AB259 CR63 12(Su2+)

(Sul+)

T4D 1.0 0.9 1.0 1.0

T4D28ts 1.0 <0.003 0.01 <0.001

T4D28'sre- 1.0 0.9 0.9 1.0

vertant a,

5The

plating efficiency for each virus strain was

definedas1.0onE. coliB grownonOK305 medium

(6)andincubatedat300C.

z

U

uJ

100.11 100.0

80.0 0 50.0 TAD

TAD 28S '-. TAD 28am

1 2 3I

10.0 a

TAD

5.0 TA0D 28S

TAD 28am

i.e1%I

MINUTES

FIG. 5. Adsorption of T4D, T4D

28",

and T4D

28am to heat-killed E. coli K-12SU2+ cells. Phage

particles were added to heat-killed E. coli K-12SU2+

cellsataconcentration of 2.5 x 108 cells per ml in

broth. There weretwophage particles per cell, and

the suspension was incubated at 27°C. Samples

re-movedatdifferent times and assayed for live

unad-sorbedphage.

whichdidnotform

plaques

when

plated

onthis host, adsorbedmorerapidly than wild-typeT4D

particles. Furthermore,

T4D 28am which was

prepared

by growth

onthe E.coliK-12 permis-sive strain

CR63,

carrieda

different

mutation in

gene 28than T4D

28',

and

produced

adifferent

gene 28

product

than T4D 28X, adsorbed even

more

rapidly

thanT4D

28's.

Sixseparate

adsorp-tionexperimentswereperformed by using either this strain ofE. coli K-12or

heat-killed

E. coli

B. Inthedifferentexperimentsthetotalamount

ofphage adsorption varied from60 to97%, but therelative initial

adsorption

ratesfor thethree phage mutants were

always

similar and

inde-pendent

of thetype ofhostcell. Inthe

experi-mentshown in the insert inFig.5, theadsorption

rate (1) forT4D

28'

was 1.9 times faster than

the rate forwild-type T4D,whereas the

adsorp-tion ratefor T4D 28amwas2.6timesfaster than

the rate forwild-type T4D. Forthe six

experi-ments, theadsorptionratefor T4D

28'

was 1.8

±0.3times faster than therateforT4D,whereas

theadsorptionrate for T4D 28am was 2.2 + 0.4

timesfaster than the rateforwild-typeT4D.

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[image:7.500.248.446.56.344.2] [image:7.500.47.240.541.634.2]

DNA injection efficiency of T4D 28t'into

E. coli K-12. Since the reactions leading to

irreversible adsorption on E. coli K-12 strains

appeared to be functioning efficiently, we

ex-aminedthe DNAinjection efficiency of T4D28t"

particles by gene rescue experiments. Cultures of either E. coli K-12 strain AB259 (which

con-tains no tRNA suppressor and is infected by

T4D

28'

at anefficiency of only<0.3%) and E.

coli B were grown to densities of 2 x 108 cells

per ml and were simultaneously multiply

in-fected with T4D 28ts and various T4D amber

mutants. Both typesof phage quickly adsorbed

to the E. coli K-12 strain and to the E. coli B

strain. After incubation at30°C for 1 h, sodium

azide was added to lyse the cultures, and the

lysates were assayedonE. coli B at permissive

andnonpermissive temperatures. Based on the

knowndistances between T4D gene 7, T4D gene

10, orthe multiple genes in T4D amX6 and gene

28 (8, 32), recombination would be extremely

likely in all three cases, leading to the production

of allpossiblerecombinants, including the

wild-type parent, T4D. In all cases, the amount of

wild-type T4D formed afteramixedinfection of

E.coliK-12strain AB259wasonlyafew percent

of theamountformed afteramixed infection of

E. coli B (Table 2). We concluded that the

efficiency of DNA injection of the T4D

28'

phageparticlesinto this E.coliK-12strain was

very low, so that verylittle rescue of the T4D

28ts genes, such as gene 7+ and gene 10+,

oc-curred.

TABLE 2. Rescueof variousT4D mutant amber genesby simultaneousinfection withT4D28ts in

varioustypesofhostcellsa

Titerofwild-type T4Dphage produced

in: Ratio of

Expt Phagecross titersb

E. coli E.coli B K-12

1 T4Dam7 x 1.0x106 1.2 x108 0.008 T4D28tS

2 T4D amlOx 4.0x 106 1.0x108 0.04 T4D28LS

3 T4DamX6 9.0X 106 1.6x108 0.05 xT4D28L

In these experiments E. coli K-12 strainAB259

andE. coli Batfinal concentrations of2 x 108cells perml in separate tubes of brothweresimultaneously

multiply infectedwithaT4D ambermutantand T4D

28'.Each type ofphage was presentat afinal concen-tration of5 x 108 phage per ml. The cultures were incubatedat30°Cfor1h,and thensodium azide was addedto a final concentration of 0.01%. The titer of

wild-typeT4Dproducedineachcrosswasdetermined

by measuring the plaque titer under nonpermissive

conditionsfor both parents(i.e.,onE.coli Bat44°C).

bRatio oftiter ofwild-typeT4Dproducedin E.coli K-12totiter ofwild-typeT4Dproducedin E. coli B.

DISCUSSION

Thedata in this paper and in the

accompany-ing paper (19) support the hypothesis that the

T4D gene28productactscatalyticallyand also

forms a

bU

uctural component of the tail plate.

Thiscatalyticfunction is in agreement with the

proposals ofSnustad(29) and Kikuchi andKing

(10-12), whohavesuggestedacatalytic function

for the gene 28 product, and with previous

re-sults from this laboratory on the absence of a

folyl polyglutamate cleavage activity in T4D

28--infected cells.

The conclusion that the gene 28 product is a

structuralcomponentof theplug portionof the

baseplate and plays a role in phageadsorption

and DNAinjection dependsonanalyticalresults

and the properties of phages with chemically

different gene28products. The analytical

tech-niques described hereseem to be useful in the

identification of viral structural components

even if these componentsare synthesized early

duringinfectionoriftheyareminor components and therefore difficult to detect with

conven-tionaltechniques.Wefound threenewproteins

which arestructural componentsof the central

plug ofthe T4baseplate. Basedontheir

migra-tion distances during sodium dodecyl

sulfate-polyacrylamide gel electrophoresis,the apparent

molecular weights of theseproteins are 41,000,

30,000, and 24,000 to 25,000. The smallest

pro-tein (24,000 to 25,000daltons) wasidentifiedas

theproduct ofgene28by

reciprocal

complemen-tationexperiments, usingachase

technique.

The

othertwocomponentswere identifiedonly

ten-tativelyasproductsof gene26(the

41,000-dalton

component) and thymidylate synthetase (the 30,000-dalton component). Detailson the

iden-tity of these two componentswill be

published

elsewhere. It isinterestingthat thesize of gene

28 product on the genetic map of Wood and

Revel(31) isrelatively small,in agreementwith our finding that it has a molecular weight of

24,000to25,000.

Thepropertiesof T4Dparticles havingaltered gene 28 products support the analytical

evi-dence. It may be that T4D 28'

particles

pro-duced even atpermissive temperatures do not

contain the normalamountsofstructural folate

in theirtailplatesand that thisdeficiencyleads

toanincreasedheatlabilitycomparedwiththe

parent, T4D. Measurements of the folate

con-tents ofphageparticles are notprecise

enough

(18, 22) to answer this question since values

varying from three to six folates per

particle

havebeen obtained. However, thepropertiesof

therevertantin gene 28argue againstthis

pos-sibility. The mutant obtained by selecting for

particles which plated at 44°C was more heat

stable than the parentT4D

28'

and hadaheat

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sensitivity

similar to that

of

the

original

wild

type, T4D. The data in Table 1 and in the

accompanyingpaper(19) showthatrevertant

a,

simultaneously regainednotonly its heat

stabil-itybut alsoto asignificant extent its resistance to sulfadiazine and pyrimethamine; it also

re-gained its

ability

toinfect E.coli K-12 strains. Theinfluence of changes in the gene 28

pro-tein on the adsorption rate indicated that the

gene 28 product itselfparticipates in host cell

adsorptionorinteracts directlyor indirectly with

the

baseplate

componentsthat affect adsorption. Furthermore, there is the direct demonstration that the failure of T4D

28'

toinfectatypical E. coli K-12 strain, such as AB259, is not in the adsorptionstepbutin the DNAinjection

mech-anism of the phage particle. We believe that

componentsof the central tail plugareinvolved

intheinjectionprocess, asthisprocessinvolves release of viral DNA.

There remain the related questions of how

many molecules of the gene 28

product

arein the tail plug and why previous polyacrylamide

gel

analyses of tail structures (6, 7, 10-15, 31)

havenotrevealed apolyacrylamide gel

electro-phoresisprotein band correspondingtothisgene

product. Basedonthetracings shown in Fig. 2

and3 for the

plug

componentsandthe

quanti-tationgiven by Crowtheretal.

(5)

for other tail components,itappearsthat thereare

only

about sixmolecules of

gP28

percentral

plug.

Astothe failuretodetect this

plug

component

previously,

itshould be noted thatnot

only

is

gP28

present

in small amounts, but it would have been ob-scured in

previous

conventional

analyses by pY,

another small

baseplate wedge

component

re-ported by Kikuchi and King, which is quite similar in size

(10-12).

Itshould also be noted that a

complementation analysis

of

baseplate

plug

precursors

(10-12)

failedtodetect thegene

28productas astructuralcomponent.This

neg-ative result now is understandable because of thedual role of thegene 28

product.

Inextracts

of E.coliBinfected withT4D28am,not

only

is there very little of the required

hexaglutamyl

form offolicacid, but significant concentrations of

dihydropteroyl glu12

14 arealso present (28). This

compound

shouldcompetewithanyof the phage folate compounds and block

baseplate

morphogenesis. Based on these considerations

and others discussed elsewhere(16), we propose

that thephage dihydropteroylhexaglutamateis

necessaryto join or link the

baseplate

central

tailplugtothe sixouterwedgelikestructuresof

the

baseplate.

ACKNOWLEDGMENT

This research wassupported by Public Health Service researchgrant AI06336from the NationalInstitute ofAllergy andInfectious Diseases.

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