Bacteriophage T4 baseplate components. III. Location and

JouRNAL OF ViROLoGy, Dec. 1975, p. 1409-1419 Vol. 16, No. 6Copyright © 1975 American Society for Microbiology Printed in U.SA.

Bacteriophage T4 Baseplate ComponentsIII. Location and Properties of the Bacteriophage Structural

Thymidylate SynthetaseLLOYD M. KOZLOFF,* LOUISE K. CROSBY, AND MURL LUTE

Department of Microbiology, University of Colorado Medical School, Denver, Colorado 80220

Received for publication 17 April 1975

Two T4D thymidylate synthetase (td) temperature-sensitive mutants havebeen isolated and characterized. Both mutants produce heat-labile phage parti-cles. This observation supports the view that this viral-induced protein is aphage structural component. Further, antiserum to td has been shown to blocka specific step in tail plate morphogenesis. The results indicated that the tdprotein is largely covered by the T4D tail plate gene 11 protein. Since the phage-induced dihydrofolate reductase (dfr) also is partially covered by the gene 11protein, it appears that td was adjacent to the tail plate dfr. This location hasbeen confirmed by constructing a T4D mutant which is dfrlstdls and showing thatthese two tail plate constituents interact and give altered physical properties tothe phage particles produced. A structural relationship for the tail plate folate,dfr, and td has been reported.

In 1973 Capco and Mathews (2) presentedevidence that the T4 phage-induced thymidyl-ate synthetase (td) was a phage capsid compo-nent. They found that antiserum preparedagainst purified T4D td enzyme inactivated T4phage. Further, when the T6 td gene was genet-ically transferred to T4 phage particles the phys-ical properties of the T4D tdT6, especially itssensitivity to heat, were quite different fromeither parent. However, there was also the re-port that two td amber mutants, originally iso-lated by D. H. Hall and studied extensively byKrauss et al. (17), could produce viable phageparticles in the apparent absence of the com-plete td polypeptide after infection of the non-permissive host. It should be noted that the tdgene is a neighbor of the dihydrofolate reduc-tase (dfr) gene in the phage genome and thatboth gene products previously were thought tobe solely early enzymes and to be "non-essen-tial." Mutations that destroyed enzymatic activ-ity, whether missense or nonsense, decreasedphage yield by about 50%, but they did notprevent phage production (8).

While searching for temperature-sensitivemutants of T4D, we isolated two strains ofT4Dtdts (L. M. Kozloff and L. K. Crosby, Abstr.Annu. Meet. Am. Soc. Microbiol., 1974, V69, p.212). It was shown that these two strains pro-duced heat-labile phage particles and that thetd protein appeared to be a tail plate constitu-ent. This paper gives the detailed properties ofthese two mutants and presents additional evi-

dence that the td gene product is a tail platecomponent adjacent to the dfr in the tail plate.A tentative scheme for the structural relation-ships of the tail plate folate (14), dfr (12), andtd is proposed.

MATERIALS AND METHODSPreparation and purification of bacteriophage

stocks and substructures. The procedures de-scribed in the accompanying papers (12, 14) werealso used in this work. In addition, T4D td6 (T4Dwh6), a mutation in the td gene (7), was furnished byD. H. Hall. OnEscherichia coli B3, a strain ofE. coliB lacking td activity, also furnished by D. H. Hall,the td6 mutant gives very small or no plaques in thepresence of limiting thymidine (7). Two other tdmutants, td N43 and td N54, both amber mutants inthe td gene, were also furnished by D. H. Hall.Enzyme assays. The assay procedure for dfr

was that described earlier (16). td activity wasmeasured by a modification of the procedure of Lo-mac and Greenberg (19). The reaction mixture con-tained 0.04 M Tris, pH 7.4, 0.1 M 2-mercaptoethanol,0.001 M EDTA, 0.025 M MgCl2, 0.0002 M tetrahydro-folic acid, 100 1.l of enzyme extract, and 0.015 Mformaldehyde and was initiated by the addition of[3H]dUMP, which had a final molarity in the reac-tion of 5 x 10-5 M. The reaction was carried out atroom temperature in a total volume of 200 1.l. Priorto use in the assay 3H-labeled dUMP (10-4 M) wasdiluted with 20 volumes ofdistilled water and lyophi-lized and resuspended in 20 volumes of unlabeleddUMP (5 x 10-4 M). The reaction was stopped byremoving 20-,ul aliquots to a 200-Id suspension ofNorite (50 mg/ml) in 0.4 M perchloric acid. Thesamples were centrifuged at room temperature, and

1409

1410 KOZLOFF, CROSBY, AND LUTE

100 ul of the supernatant was dissolved in Aquasoland counted in a liquid scintillation counter. Thesupernatant fluid contains the 3H released fromdUMP when the methyl group is added.The tetrahydrofolic acid and unlabeled dUMP

were obtained from Sigma, and both the labeleddUMP and Aquasol were from Amersham/Searle.The FdUMP was a gift from S. S. Cohen, and theshowdomycin, (3-f8-n-ribofuranosylmaleimide) was

purchased from Calbiochem.Isolation of whus mutants. T4D at 1.1 x 1012/ml

was mutagenized with 0.4 M NH2OH in the pres-

ence of 0.1 M sodium phosphate buffer, pH 6.0,and 0.001 M EDTA, pH 6.0 (5). The reaction was

stopped after 36 h by a 1:100 dilution into coldbroth containing 1.0 M NaCl and 0.001 M EDTA.A total of 8,800 plaques were examined for theformation of "white halos" (wh) on the platingbacteria OK305 when incubated at 42 C. The whcharacteristic reflects a mutation in either the td ordfr gene. Two hundred and fifty-seven wh plaqueswere selected and replated for the wh characteristicat 30 C. Forty-one stocks were obtained which were

wh- at 42 C and wh+ at 30 C. Of these 41, two stocksformed heat-sensitive virus particles. These twostocks were backcrossed against excess wild-typeparental T4D (10 parental T4D/1 wh) a total of fourtimes. This resulted in the preparation of twostocks, mutant 408 and mutant 604, each of whichpossessed only the whus mutation. The characteriza-tion of these two mutants as T4D tdus is given later.A globulin fraction from antiserum against homo-

geneous T2L td was generously provided by FrankMaley (6). This serum is known to cross-react withboth the T4 and T6 td's. The serum preparationcontained 30 mg of globulin per ml.

RESULTS

Identification of T4D whts mutants. Thetwo temperature-sensitive mutants, 408 and604, which gave "white halo" plaques at 42 Cand normal plaques at 30 C, were characterizedafter they were genetically purified by back-crossing four times against parental T4D. Sinceplaques with a white halo can be produced byeither a mutation in the dfr or the td gene (8),both plating tests and the heat sensitivity of theenzyme activities were compared to parentalT4D and to nonsense (or missense) mutations inboth genes. When plated on E. coli B3, a thy-host, in the presence of 4 ag of thymidine perml which is limiting, both 408 and 604 formedno plaques and behaved like the known td mu-tant at the nonpermissive temperature, butboth formed normal plaques like wild type (td+or dfr mutant) at the permissive temperature(Table 1). When plated on E. coli B in the pres-ence of trimethoprim (0.55 ,ug/ml), an inhibitorofE. coli dfr activity, these two mutants formedplaques like wild type and thus were differentfrom the known dfr mutant. These plating prop-

TABLE 1. Plating properties of T4Dwh'8 mutantsa

Plaque size (mm) Plaque sizeon E. coli B-3 (mm) on E. coli(broth and B (broth and

Phage type thymidine) trimethoprim)

30C 43C 32C 42C

T4D dfr+td+ 1.2-2.0 0.3 1.0 2.5T4D dfr-td + 1.2-2.5 0.2 0.1 0.1T4D dfr+td- 0 0 1.0 1.5T4D wh'5-408 1.0-2.0 0 1.0 1.5T4D whls-604 1.8-2.2 0 1.0 1.5

a E. coli B-3 was grown with 4 ,ug ofthymidine perml in Burroughs-Wellcome broth. E. coli B wasgrown in tryptone broth (Difco), and the plates con-tained 0.55 ,tg of trimethoprim per ml. T4D dfr-td+was the whl mutant of Hall (7), and T4D dfr+td-was the td6 mutant of Hall (7).

erties strongly suggested that both mutant 408and mutant 604 were tdts mutants.

Extracts were prepared in the usual mannerto measure the heat sensitivity of the inducedtd and dfr activities. The heat sensitivity ofthe td activities induced in E. coli B3 by wild-type T4D and the two mutants is shown in Fig.1. Both mutants induced a td activity whichwas considerably more heat labile than thatinduced by the parental T4D. On the otherhand, the heat sensitivities of dfr activitiesinduced by 408 and 604 were identical to thatinduced by the parental phage (Fig. 2). Theseenzymological properties confirm the conclu-sion from the plating properties that 408 and604 are both tdts mutants.

Properties of T4D tdts mutant particles.The heat lability of preparations of tdls mutants408 and 604 as compared to parental T4D areshown in Fig. 3 and 4. Both stocks were grownat low and high temperatures and then heatedat 60 C in the standard way. Eight separatepreparations of mutant 408 and four separatepreparations of mutant 604 were grown. Allphage stocks prepared at either temperaturewere always more heat labile then parentalT4D. Further, when assembled at the high tem-perature the phage particles have significantlydifferent heat labilities than when assembledat a lower temperature.These results, which areboth qualitatively and quantitatively quite sim-ilar to those with dfsts far mutants, indicatethat the temperature-sensitive td gene productmust be a viral structural component. In viewof the fact that the tail plate is the most heat-labile phage structural component (16), theseobservations suggest that the td protein is atail plate constituent.

J. VIROL.

PHAGE BASEPLATE THYMIDYLATE SYNTHETASE

> 40

z

uJ

604

20

408

5 10 15 20

M I N U T E S AT 40

FIG. 1. Heat inactivation oftd induced by T4D mutants 408 and 604 and wild-type T4D. The extracts wereprepared after infection at 30 C as described earlier for the measurement of dfr activity (12).

The different heat labilities of 408 (and 604)when assembled at the high temperature ascompared to phage formed at a lower tempera-ture suggest that td can be used as a struc-tural component with some variations in itstertiary or quaternary structure. Presumablyat one temperature a segment of the td poly-peptide assumes a heat-labile configuration,and later, upon heating at 60 C, it is denaturedmore rapidly than when synthesized and usedfor phage assembly at the different tempera-ture.

Preparation and properties of a T4ddfrtstdls mutant. A phage double mutant was

obtained by infecting E. coli B simultaneouslywith the T4D tdts 408 mutant as the majorityparent (multiplicity of infection of 10) and withthe T4D far mutant, P1, which is dfrus (9), as theminority parent (multiplicity of infection of 1).The progeny of this cross were plated on E. coliB on plates containing pyrimethamine, andplaques were picked at random. The picked

plaques were then tested for their plating prop-erties, and one recombinant was found whichhad the properties shown in Table 2. Thisphage stock was both resistant to pyrimetha-mine and did not give plaques at the nonper-missive temperature on E. coli B3. These plat-ing tests and the enzymological properties de-scribed in Table 3 indicate that this stock isT4D tdtddfrtsThe heat sensitivity of this double mutant

was compared to the majority parent 408. Fig-ure 5 shows typical results obtained in severalexperiments. When assembled at 42 C the dou-ble mutant, as expected, was more heat labilethan 408 asembled at 42 C, but when assembledat 30 C the double mutant was less heat labilethan the 408 mutant assembled at this tempera-ture. These properties indicate that heat labili-ties ofthese two viral components are not neces-sarily additive. The heat labilities of these twocomponents appear to be dependent upon thequaternary or tertiary structure of each other.

1411VOL. 16, 1975


O_ I I I ' I

10-0

60

zLUU 40 0

20~~~

0~~~~~~

10 20 30 40 50

MINUTES AT 370FIG. 2. Heat inactivation ofdfr induced by T4D mutants 408 and 604 and by wild-type T4D. The extracts

were prepared after infection at 30 C as described earlier (12).

The properties of this double mutant, which istdIsdfrus, suggest that these two components in-teract physically and are probably adjacent toeach other in the baseplate.

Effect of antiserum to TD on tail plateassembly. Capco and Mathews reported thathigh concentrations of antiserum to purifiedbut not homogeneous T4D td inactivated T4D(2). Recently Galivan et al. (6) prepared antise-rum to homogeneous td isolated from bacteriainfected with T2L. This antiserum was reportedto cross-react with td induced by T4 or T6phages. It was found that the antiserum to theT2-induced td did not inactivate either T2, T4,or T6 phage particles even at a dilution of 1:3when incubated for as long as 18 h.Using procedures identical to those in the

accompanying papers (12, 14), this serum wasincubated with either incomplete T4D particleslacking both the 11 and 12 proteins, T4D 11-, or

with T4D particles lacking just 12P, T4D 12-.Later, the extract containing liP and 12P wasadded to treated particles. It can be seen in thetypical experiment shown in Fig. 6a and b thatantiserum to T2 td inhibits the step in morpho-genesis involving addition of the 11 protein tothe baseplate, but that this antiserum had noeffect on the addition of the 12 protein to 12-particles. These observations confirm that tdis a T4D baseplate component lying largelyunder the 11 protein in close proximity to thebaseplate dfi (12) and folate (14).

Effect of cofactors or inhibitors of td ac-tivity on addition of 11 protein to 11- phageparticles. Since cofactors or inhibitors of dfrreact with both whole phage particles and evenmore readily with incomplete particles lackingthe 11 (and 12) protein (12, 20), the effect ofanalogous compounds known to bind to td wasexamined. Intact T4D, T2L, and T2H were incu-

J. VIROL.


bated with either dUMP, FdUMP, showdomy-cin, or tetrahydrofolate plus formaldehyde.dUMP and the mixture of tetrahydrofolate plusformaldehyde are normal substrates for td,whereas FdUMP (18, 21) and showdomycin (10)are known to react stoichiometrically and possi-bly covalently with the enzyme and to causeirreversible inhibition. None of the four com-pounds in any combination affected phage via-bility or the rate of morphogenesis involving 11(and 12) protein addition to 11- (and 12-) parti-cles. It seems likely that the folate-binding siteon the tail plate td is occupied by the phagedihydropteroyl hexaglutamate. Further, it ap-pears that the binding site for dUMP (and show-domycin) is buried within the complex tail platestructure and is not free to react with these low-molecular-weight compounds.

Properties of T4D having an amber muta-tion in its td gene. Two T4D mutants, N43 andN54, derived from T4D Cl, having an ambermutation in their td gene were originally iso-

LUuiI-

I-.

0

Ia-I-

zLSu

LUJa-

lated by D. H. Hall and partially characterizedby Krauss et al. (17). In view of the evidence byCapco and Mathews (2) that td is a phagecomponent and that tds mutants form heat-labile particles and other evidence presentedearlier in this report, the properties of particlesproduced by these two td amber mutants wereexamined. Phage stocks were grown at 28 or42 C on both the nonpermissive bacterial host(E. coli B) and the permissive bacterial host(CR63).

The heat sensitivities of these four phagepreparations are shown in Fig. 7. It is apparentthat the heat sensitivity of the phage particlesis dependent on the td mutation, since thesensitivity reflects both the temperature of as-sembly and the completion or noncompletion ofthe td polypeptide. When grown at 28 C all thestocks were considerably more heat sensitivethan when grown at 42 C. Further, N54 whengrown at 42 C on E. coli B is much more sensi-tive than when it was grown at 42 C on CR63

MINUTES

FIG. 3. Heat inactivation at 60 C of T4D and T4D mutant 408 assembled either at 30 or 42 C.

1413VOL. 16, 1975


10(

I

U

t.

M NUT ES

FIG. 4. Heat inactivation at 60 C of T4D and T4D mutant 604 assembled either at 27 or 42 C.

TABLE 2. Plating properties of various T4D mutants

Plaque size(mm) on E. coli Pyrimeth-

T4D phage typea B-3 aminesensitivity

30C 43C

td+far+ 1.6 0.5 Sensitive408-(tds) 1.5 0 SensitiveP1-far-(dfrIs) 1.4 0.4 Resistant408 x Pl-dfrstdts 1.4 0 Resistant

a far = folate analogue resistant = dfr resistant.The plating conditions are those described for Table1.

and was more sensitive than N43 grown at 42 Con either host.A similar small, but reproducible, influence

of the host cell and temperature of assembly inphage particles produced is seen in Fig. 8, de-scribing the interaction of these four T4D tdamber mutant stocks with anti-dfr serum.The ability of the serum globulin to react with

TABLE 3. Properties ofenzymes induced by infectionwith various T4D mutantsa

dfr activ-td activity in pres-

Phage ity after 10 enceof4 xPhage min at 40 C 10-6 pyri-(%) metham-

ine (%)

T4D 62 7T4D tdts (408) 18 6T4D dfrls (P1) 82 47Recombinant T4D 30 64

tdtsdfrtsa The activities were measured as described in

Materials and Methods.

the phage tail plate dfr was influenced by thenature of the td formed. For three of thestocks, N43 grown on CR63 and N54 grown oneither host, the phage assembled at 42 C has itsdfr more exposed to the antiserum than phageproduced at 28 C.

J. VIROL.

PHAGE BASEPLATE THYYMIDYLATE SYNTHETASE

It should be emphasized again that parentalT4D particles have identical properties withregard to both heat sensitivity and inactivationrates with anti-dfr serum irrespective of theirhost or temperature of assmbly. It can be con-cluded that the td gene product is a constitu-ent of the virion, in agreement with the data ontd" mutants, and is most likely a tail plate com-ponent adjacent to the dfr. These propertiessupport the view that the partial peptide prod-ucts of the td gene in N43- and N54-infectedcells are large enough to serve this structuralrole.

DISCUSSIONA proposal for the structural relationship be-

tween baseplate folate (14), dfr (12), and td isshown in Fig. 9. All three components areshown partially or completely buried within thetail plate. They are tentatively placed close tothe center of the tail plate near the center

100.0

\o0

o UI\ 0

X 10.0

z

lx ~~~~408 xa.

F \

"plug" and the tail core. One reason for thislocation is that the structure consisting of thetail core plus plate contains folic acid but freetail plates do not contain the folate. Duringmorphogenesis the addition of the tail core tothe plate must stabilize the structure and helpbury the folate within the tail plate.

This complex structure illustrates the ex-tremely tight binding of the folate to the phagetail protein, as shown by the fact that folatecannot be removed from whole phage particlesor even 11- particles by stirring with a suspen-sion of activated charcoal for 18 h. The ability oftwo pteridine-binding proteins (the antifolateserum and milk protein [14]) to greatly inhibitthe rate of completion of morphogenesis of 11-particles, but to only decrease somewhat theamount of final phage formed, suggests thatthe pteridine is buried and that, whereas thepteridine binds reversibly with these two pro-teins, the addition of the 11 protein to this

M I N U T E S

FIG. 5. Heat sensitivity of recombinant T4D phage stock prepared from a cross of408 x Pl compared toparental T4D 408 mutant. Pl is dfru and 408 is td", so the recombinant is T4D dfrt' td'5 (see Tables 2 and 3).The procedure for preparing this stock is given in the text.

1415VOL. 16, 1975

60 0o

o Anti td /Anti td SerumI- ~~~~~~Serum

Z 40LUU~~~~

20~

T4D 11| T4D 12

1 2 3 4 1 2 3HOURS

FIG. 6. (a) Effect ofantiserum to td on the addition ofPll (and P12) to T4D 11- particles. The antiserumpreparation was incubated with T4D 11 particles overnight in the cold at a final globulin concentration of12mg/mil. The extract containing the Pll (and P12) was added later, as described earlier (14). The control titerwas4 x 106, and after3 h at30 C the titer was 4.4 x 109 ( 100%). (b) Effect ofantiserum to td on the additionofP12 to T4D 12- particles. The antiserum was incubated as in (a), and the reaction was started by additionofthe same extract as in (a). The initial titer was 12 x 108, and after3 h the T4D titer was 9.8 x 109 (= 100%).

100.0

280 420

,_ N54 (CR63)I.-

10.0

FIG.7.HeatN43 (B)0

42Cwiegoninetrteprsiehs(R3orhnoprsiehsN43(CR63)

z

U- 1.0uiL N43 (B) N54 (B)

N54 (B)

N43(CR63)

N54 (CR63)

0.1 I

30 60 30 60MINUTES

FIG. 7. Heat sensitivities of two T4D td amber mutants, N43 and N54. The stocks were assembled at28 or42 C while growing in either the permissive host (CR63) or the nonpermissive host (B). Phage were diluted toan initial titer of105 in 0.1 M potassium phosphate buffer, pH 7.0, and heated in a water bath at 60 C.

1416


280 420

0U2420

r 100 . . . I

N43 CR63-Grown N54 CR63-GrownzLuu

LU0..

10

280280

420 420

1 l20 40 60 20 40 60

MINUTES

FIG. 8. The inactivation of two T4D td amber mutants, N43 and N54, by antiserum to dfr. The stockswere assembled at 42 or 28 C while growing in either the permissive (CR63) or the nonpermissive host (B).Phage stocks and antiserum were diluted in tryptone broth adjusted to pH 5.0. The reaction mixture was

incubated at 37 C. The initial phage concentration was 5 x 106/ml, and the serum was used at a final dilutionof1:51.

structure is irreversible. The tip of the folatepolyglutamate portion is shown extending

T6ilCOr',O somewhat from the bottom of the baseplate,since it is known that hog kidney conjugate canreact with this portion of the folate molecule(3). The claim by D~awes and Goldberg (3 that,

ProteingIu6 for some phage strains, the inactivation by con-21tProtei Baseplatjugase is due to conjugase binding to the poly-

Base plate glutamate rather than to enzymatic cleavage ofthe terminal glutamate residues suggestseither that only a small portion extends fromthe tail plate or that it lies in some protected

FIG. 9. Suggested location for T4D baseplate dihy- fold or crevice of the proteins on the bottom ofdropteroyl hexaglutamate (pte-glud, dfr, and td. the baseplate.

1417VOL. 16, 1975


The dfr is shown in Fig. 9 as interactingwith the pteridine polyglutamate and the tdand to be only partially covered by the 11 pro-tein. It is known that the dfr must be buriedin the tail, since it required urea treatment ofplates to demonstrate dfr activity (16). How-ever, anti-dfr serum (22) does react with, andkill, whole phage particles. This serum killed11- particles at least fourfold more rapidly sothe dfr can only be partially covered by the 11protein. A portion of the dfr is shown exposedat the bottom of the tail, not only to react withantiserum but also to indicate a site for theinteraction with reduced nicotinamide adeninedinucleotide (NADPH). NADPH does bind toand inactivate phage and, as reported earlier(16), over 1010 phage can be inactivated byNADPH treatment with first-order kinetics.Further, no NADPH-resistant particles havebeen detected. It seems that this feature of thedfr is essential for phage viability. It shouldbe emphasized again that, in in vitro comple-mentation experiments, the interaction of var-ious nucleotides with this NADPH site beforethe phage is completely formed also affects therate of morphogenesis. For the formation of thephage structure then, the dfr polypeptidemust contain a NADPH binding site, a dihydro-folate binding site, and a td interacting site.The rest of the dfr molecule so far appears tobe unnecessary, and these experiments indicatethat a partial polypeptide can serve as a struc-tural element.The occlusion of the td molecule within the

tail plate is suggested by the data that thepresence of the 11 protein prevents inactivationby the anti-T2 td serum of Galivan et al. (6).However, Capco and Mathews (2) did get inacti-vation of T4 by high concentrations of antise-rum to T4 td. We have been unable to demon-strate any td activity in phage particles, evenusing gentle methods to disrupt incomplete 11-particles. This is perhaps not surprising in viewof the great lability of td reported by Krauss etal. (17) and Capco et al. (1). Finally, with re-gard to the location of the td, the td bindingsites for dUMP (or FdUMP or showdomycin)appear to be buried within the tail structure,possibly near the tail core since 11- particles donot react with these reagents. The physicalproperties of the two T4D amber td mutantsagain indicate that, like dfr, partial peptidesof td can still play a structural role. This viewis supported by the recent observation byMosher and Mathews (personal communica-tion) that their antiserum to T4D td does inac-tivate both N43 and N54 td amber stocks atrates slightly higher than for T4D.

The interaction of td and dfr shown in Fig.9 rests (i) on their common location under the11 protein in the baseplate and (ii) on the proper-ties of the T4D tdlsdfrls double mutant, whichsuggest a physical interaction of the two pro-teins. In addition, both enzymes have a sitewhich binds dihydrofolic acid. Whereas a physi-cal interaction between these two enzymes haslong been postulated, only recently has enzymo-logical evidence been presented, by Kawai andHillcoat (11), indicating that dfr and td fromL1210 cells do form physical complexes in solu-tion. It is of some interest that Tomich et al.(23) have recently reported that in T4-infectedcells td forms a complex with other early en-zymes and has less enzymatic activity thanexpected.The observations in these three papers, espe-

cially the properties of the tdls and dfrls mu-tants, reflect the inherent unstable nature ofthe tail plate. This phage structure must beflexible enough to undergo morphologicalchanges during infection, and this would be inaccord with its heat lability.A difficult final problem is still the determina-

tion of the number of folates and dfr and tdmolecules in each baseplate. Perhaps the bestestimate is still two or possibly six of each pertail plate. This low concentration, especially ofearly proteins, is perhaps the reason the latelabeling of T4 capsid components, followed bydisruption and polyacrylamide gel analysis ofeither phage or of infected cells, has not re-vealed these two components (4, 24). However,the failure of the current analytical proceduresto detect these components does not argueagainst the conclusion that these phage-in-duced components, dfr, td, and folate, notonly aid DNA synthesis but also play a criticalstructural role in forming the tail plate.

ACKNOWLEDGMENTSWe are grateful to Frank Maley for the generous gift of

antibodies to T2 thymidylate synthetase.This work was supported by Public Health Service grant

AI-06336 from the National Institute of Allergy and Infec-tious Diseases.

LITERATURE CITED1. Capco, G. R., J. R. Krupp, and C. K. Mathews. 1973.

Bacteriophage-coded thymidylate synthetase: charac-teristics of the T4 and T5 enzymes. Arch. Biochem.Biophys. 158:726-735.

2. Capco, G. R., and C. K. Mathews. 1973. Bacteriophage-coded thymidylate synthetase. Evidence that the T4enzyme is a capsid protein. Arch. Biochem. Biophys.158:736-743.

3. Dawes, J., and E. B. Goldberg. 1973. Functions of base-plate components in bacteriophage T4 infection. I.Dihydrofolate reductae and dihydropteroyl gluta-mate. Virology 55:380-390.

J. VIROL.


4. Dickson, R. C., and S. L. Barnes. 1970. Structural pro-teins ofbacteriophage T4. J. Mol. Biol. 53:461-473.

5. Freese, E., E. Bautz-Freese, and E. Bautz. 1961. Hy-droxylamine as a mutagenic and inactivating agent.J. Mol. Biol. 3:133-143.

6. Galivan, J., G. F. Maley, and F. Maley. 1974. Purifica-tion and properties of T2 bacteriophage-induced thy-midylate synthetase. Biochemistry 13:2282-2289.

7. Hall, D. H. 1967. Mutants of bacteriophage T4 unable toinduce dihydrofolate reductase activity. Proc. Natl.Acad. Sci. U.S.A. 58:584-591.

8. Hall, D. H., I. Tessman, and 0. Karlstrom. 1967. Link-age ofT4 genes controlling a series of steps in pyrimi-dine biosynthesis. Virology 31:442-448.

9. Johnson, J. R., and D. H. Hall. 1973. Isolation andcharacterization of mutants of bacteriophage T4 re-sistant to folate analogues. Virology 53:413-426.

10. Kalman, T. I. 1972. Inhibition of thymidylate synthe-tase by showdomycin and its 5'-phosphate. Biochem.Biophys. Res. Commun. 49:1007-1013.

11. Kawai, M., and B. L. Hillcoat. 1974. Interaction ofthymidylate synthetase and dihydrofolate reductaseenzymes in vitro and in vivo. Cancer Res. 34:1619-1626.

12. Kozloff, L. M., L. K. Crosby, M. Lute, and D. H. Hall.1975. Bacteriophage baseplate components. II. Bind-ing and location ofthe bacteriophage-induced dihydro-folate reductase. J. Virol. 16:1401-1408.

13. Kozloff, L. M., and M. Lute. 1965. Folic acid, a struc-tural component of T4 bacteriophage. J. Mol. Biol.12:780-792.

14. Kozloff, L. M., M. Lute, and L. K. Crosby. 1975. Bacte-riophage baseplate components. I. Binding and loca-tion of the folic acid. J. Virol. 16:1391-1400.

15. Kozloff, L. M., M. Lute, L. K. Crosby, N. Rao, V. A.Chapman, and S. S. DeLong. 1970. Bacteriophage tail

components. I. Pteroyl polyglutamates in T-even bac-teriophages. J. Virol. 5:726-739.

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1419VOL. 16, 1975

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Bacteriophage T4 baseplate components. III. Location and