JOUINAL OF VIROLOGY, Sept. 1977, p. 637-644Copyright 1977
American Society for Microbiology
Vol. 23, No. 3Printed in U.S.A.
Bacteriophage T4 Virion Baseplate Thymidylate Synthetaseand
Dihydrofolate ReductaseL. M. KOZLOFF,* M. LUTE, AND L. K.
Department ofMicrobiology and Immunology, University of Colorado
School ofMedicine, Denver,Colorado 80262
Received for publication 18 April 1977
Additional evidence is presented that both the phage T4D-induced
thymidyl-ate synthetase (gp td) and the T4D-induced dihydrofolate
reductase (gp frd) arebaseplate structural components. With regard
to phage td it has been foundthat: (i) low levels of thymidylate
synthetase activity were present in highlypurified preparations of
T4D ghost particles produced after infection with td+,whereas
particles produced after infection with td- had no measurable
enzymaticactivity; (ii) a mutation of the T4D td gene from td I to
td+ simultaneously pro-duced a heat-stable thymidylate synthetase
enzyme and heat-stable phage par-ticles (it should be noted that
the phage baseplate structure determines heatlability); (iii) a
recombinant of two T4D mutants constructed containing bothtd t and
frdts genes produced particles whose physical properties indicate
thatthese two molecules physically interact in the baseplate. With
regard to phagefrd it has been found that two spontaneous
revertants each oftwo different T4Dfrd's mutants to frd+ not only
produced altered dihydrofolate reductases butalso formed phage
particles with heat sensitivities different from their
parents.Properties ofT4D particles produced after infection with
parental T4D mutantspresumed to have a deletion of the td gene
and/or the frd gene indicate thatthese particles still retain some
characteristics associated with the presence ofboth the td and the
frd molecules. Furthermore, the particles produced by thedeletion
mutants have been found to be physically different from the
Considerable genetic and biochemical evi-dence has been
presented from several labora-tories (4, 12, 15, 16, 19-21) that
two early phage-induced enzymes, dihydrofolate reductase,called gp
frd (25) (referred to earlier as gp dfr), and thymidylate
synthetase, called gp td(3, 11), were components of the T-even
phagebaseplate. However, since neither the knownmissense mutations
(5) nor the known non-sense mutations (17) in these genes
preventedphage formation, the gene products were classi-fied by
Wood and Revel (25) as nonessentialcomponents of the baseplate.
This problem wascomplicated both by the failure to detect pro-teins
corresponding to these enzymes uponpolyacrylamide gel analysis of
phage particles(10, 24) and by isolation by viable phage parti-cles
presumably containing deletions of the tdand frd genes (7). Earlier
work from Mathews'laboratory (20) and this laboratory (11, 16)
indi-cated that these two gene products were notrequired as intact
functioning enzymes, butthat substantial portions of these
polypeptidescould play the required structural role. Itshould also
be noted that phage baseplates con-
tain an unusual form of folic acid, dihydropter-oyl
hexaglutamate, as a structural component,which is closely linked to
the two enzyme mole-cules (15). This paper and the
accompanyingreport by Mosher et al. (22), which deals largelywith
gp frd in phage particles, present addi-tional chemical, genetic,
and enzymatic datathat these two proteins are present in all
phageparticles yet examined. The thymidylate syn-thetase and the
dihydrofolate reductase mole-cules have been shown to be partially
buriedwithin the baseplate structure. Furthermore,these two reports
point out several anomalies inthe properties of the presumed T4D
deletionmutants of the frd and td genes that suggestthat the
physical and genetic mapping of thedeletion mutants may need
MATERIALS AND METHODSPreparation and purification of
stocks and isolation of mutants. Escherichia colibacteriophage
stocks were grown, purified, and as-sayed by standard procedures
(11, 12, 15). Whenused to measure thymidylate synthetase
activity,the purified phage particles were osmoticallyshocked, and
the ghost particles were further puri-
638 KOZLOFF, LUTE, AND CROSBY
fled on D2O-sucrose gradients (15). The T2L and T4Dstrains have
been used in this laboratory for sometime. The two T4D amber
mutants, one in the frdgene (frdll) and one in gene 11 (N9s), were
thosedescribed earlier (15). T4D td6, a missense mutationin the td
gene, was obtained from D. H. Hall (9). Theisolation and properties
of a T4D tdcs mutant (strain408) were described earlier (11). The
two frdts mu-tants, originally obtained from D. H. Hall, P1 andC31,
and their parent T4Do strain have also beendescribed (5, 12). T4
phage bearing deletions pre-sumed to be between genes 63 and 32,
originallyisolated by Homyk and Weil (7) and crossed byHomyk and
Weil into a T4D strain called 1589, wereobtained from C. Mathews.
The T4D 1589 straincontained a deletion in the rII gene that was
used asa DNA marker (7). These deletion strains are la-beled by the
names originally assigned by Homykand Weil as dell, del7, and del9.
The bacterial hoststrains used included E. coli B and three mutants
ofE. coli B affecting host dihydrofolate reductase ac-tivity, E.
coli B TRIM 102 and TRIM 103 (12) and E.coli B RT 500, obtained
from D. Baccanari (1). E. coliB201, which was td- and grew
adequately when themedium was supplemented with low
concentrationsof thymine, was obtained from C. Mathews. Thestandard
E. coli su+ strain used was CR63. E. coliOK305 was used to assay
for the "white halo"plaques produced by frd- and td- phage strains
(6).The enzyme assays and other methods have all
been described previously (12, 15). Standard re-agents were
obtained from commercial sources.
Thymidylate synthetase activity in phageghost particles. Capco
and Mathews (3) pre-sented the original evidence that the
phage-induced thymidylate synthetase molecule wasa phage component.
Later work from this labo-ratory indicated that this enzyme was a
base-plate component (11). In both laboratories,early attempts to
demonstrate thymidylatesynthetase activity in phage particles were
un-successful. Further efforts were now made withhigher
concentrations of phage ghost particlesin the highly sensitive
radioactive assay (18).The final concentration of phage ghosts
usedwas about 1013/ml in the reaction mixture.Those particles
showing thymidylate synthe-tase activity released radioactivity
from[3H]dUMP linearly for 1 to 2 h, and the enzy-matic rate was
calculated per particle added(Table 1). Although the baseplate
thymidylatesynthetase is known to be partially covered bygene 11
and 12 proteins (11), assays of T4Dghost particles lacking the 11
and 12 proteinsconsistently showed less thymidylate synthe-tase
activity than did preparations of wholeT4D ghosts. Similar amounts
of enzyme activ-ity were found in preparations of T4D, T4Dstrain
1589, and T2L, and somewhat less was
TABLz 1. Thymidylate synthetase activity inpreparations ofphage
Ghost particle preparation Activity" (mol/minper 10"3
T2L ...................... 0.07 0.01T4D ......................
0.16 0.02T4D frdll .................... 0.03 + 0.01T4D td6
T4 VIRION BASEPLATE COMPONENTS 639
sensitive. A single plaque was picked of themost heat labile of
these two mutants (strain408), and, using this stock, a search was
madefor a wild-type td+ revertant. This parent strain408 mutant
gave normal plaques on E. coliOK305 at 30'C but gave plaques with
whitehalos at 430C. Plating with E. coli B201 con-firmed that the
parent 408 was td- at 430C butwas td+ at 300C. One plaque (out of
13,000 ofstrain 408 examined) was of the normal type atthe high
temperature and was also td+ at 430Con B201. This revertant,
presumably due to asingle spontaneous mutation, was isolated,
andits properties were examined. The revertantnot only produced a
heat-stable thymidylatesynthetase, as compared with parent strain
408,but also produced phage particles that weremore heat stable
than the 408 and even moreheat stable than the original parent T4D
parti-cles (Fig. 1).
Preparation and properties of a T4D frdstds mutant. Evidence
from earlier work (11)had shown that the baseplate thymidylate
syn-thetase could physically interact with the base-plate
dihydrofolate reductase. This earlier
60 - Revertantof T4D tdts
work involved preparing and characterizing theproperties of a
double T4D mutant, using strain408, a td I mutant, as one parent
and an frdemutant known as P1 (9) as the other parent. Asecond
double mutant was now prepared byusing strain 408 again as one
parent but adifferent frd's mutant, known as C31, as theother
parent. The procedure was as describedearlier. A cross of the two
mutants was madewith strain 408 as the majority parent and C31,a
T4D far mutant, which is also resistant topyrimethamine (9), as the
minority parent. Theprogeny of the cross were plated on bacteria
onmedia containing pyrimethamine, and plaqueswere picked and tested
until one recombinantwas obtained that both produced
pryimetham-ine-resistant plaques and gave the td white-halo plaque
on OK305 only at the high temper-ature. The recombinant was also
td- on B201 atthe high temperature. The heat sensitivity ofthis
double mutant was compared to the major-ity parent 408, and Fig. 2
shows the typicalresults obtained in several experiments. Asfound
previously, the heat sensitivity of boththe 408 mutant and the
recombinant was a
Revertont 0of TUD tdts
FIG. 1. Heat sensitivity of various T4D particles and the
T4D-induced thymidylate synthetases. (a) Thephage particles were
heat inactivated in the standard way at 600C (11). (b) Extracts
were made ofE. coli Binfected with the various types ofphage. These
extracts were heated at 40"C, and the activity ofthe
remainingthymidylate synthetase was determined. Abbreviations: P,
Phosphate; ME, mercaptoethanol.
VOL. 23, 1977
640 KOZLOFF, LUTE, AND CROSBY
40xcC 31 (420)
0.1 M P Buffer
1.01,,,,15 30 45 60
FIG. 2. Heat sensitivity ofT4D particles producedby
recombination of T4D tdt' (408) and frd' (C31)mutants. The
recombinant T4D, which is tdto frdto,was prepared as described,
with the td's as the ma-jority parent. Phage stocks were grown
either at 28 to30C or at 42C, and the heat sensitivity at 60C ofthe
phage particles was determined in the usual way.P,
Phosphate.function of the temperature of assembly(whether at 42 or
at 30C). Irrespective of thetemperature of assembly, the double
mutantT4D tdusfrdts was more heat resistant than thetdts majority
parent. These changes in heat sen-sitivity indicate again that the
heat labilities ofthese two baseplate components are not
directlyadditive. The results support the conclusionthat these two
baseplate components influenceeach other physically, either
directly or indi-rectly.
Physical properties of revertants of T4Dfrd Is to frd+. The
original frd Is phage particleswere isolated as spontaneous mutants
by John-son and Hall (9) and were first characterized bytheir
ability to form plaques on bacteria in thepresence of
anti-dihydrofolate reductase com-pounds, such as pyrimethamine.
Johnson andHall (9) showed that two ofthese drug-resistantmutants
produced altered enzymes that wereboth drug resistant and heat
labile. Later, Koz-loff et al. (12) additionally characterized
theseT4D frdts mutants and showed that they pro-duced heat-labile
phage particles. A search wasmade for revertants oftwo of these
and C31, from frd's to frd+. Starting with sin-gle-plaque
isolates of P1 and C31 progeny,phage particles were examined for
their abilityto form normal plaques, i.e., without whitehalos, at
the high temperature (370C). Two re-vertants each of P1 and C31
were found, andthe plating properties of the revertants aregiven in
Table 2. All the revertants isolatedwere still resistant to the
addition of pyrime-thamine to the plating media, and it can
beconcluded that the change in temperature sen-sitivity of the free
dihydrofolate reductase, asshown by the plaque assays, was due to a
sec-ond mutation independent of the site sensitiveto
pyrimethamine.The heat sensitivities of the particles pro-
duced by the two revertants of P1 are shown inFig. 3, and the
heat sensitivities of the two C31revertant particles are shown in
Fig. 4. One ofthe P1 ts revertants had the same sensitivity asthe
parent P1, whereas the other revertant wasmore heat resistant. Both
of the ts revertants ofC31 produced phage particles with heat
sensi-tivities different from the parent C31 but in thiscase the
revertants produced particles thatwere more heat sensitive than the
parent C31.These properties support the conclusion
thatdihydrofolate reductase is a phage baseplatecomponent and
indicate that any change in thepolypeptide (such as to greater heat
resistanceof free enzyme) may change the heat sensitivityof the
baseplate to either greater heat resist-ance, such as the Pla
revertant, or more heatsensitivity, such as the C31a or C31b
TABLE 2. Plating properties of various T4D frdtsrevertants to
Plaques on OK305 at: Platingeffi-
Phagestrain ~~~~~ciency onPhage strain 037C32C OK305 +3700 3200
T4Do No halo No halo 0.02
P1 Halo No halo 1.06Revertant a No halo No halo 0.70Revertant b
No halo No halo 1.03
C31 Halo No halo 0.98Revertant a No halo No halo 1.02Revertant b
No halo No halo 0.92
a Similar to the medium used by Johnson andHall (9) but
containing lower concentrations of pyri-methamine (P) and
sulfanilamide (S); final P = 70,ug/ml, and final S = 4.5 Ag/ml. The
plating ef-ficiency is the ratio of plaques on OK305 + (P+S)to the
plaques formed on OK305 without (P+S).
T4 VIRION BASEPLATE COMPONENTS 641
100.0 the frd gene, are even more sensitive to thisantiserum
than is dell or parent T4D. (ii) Fur-thermore, genetic experiments
by Mosher et al.
50.0 have shown that the frd gene is not deleted inPi del7 or
del9, but that the enzymatic activity isRevertont bprobably
suppressed by some other, additionalmutation. (iii) A replotting of
the deletions onthe T4 map (22) shows that these deletions
0 \ must lie 2,000 nucleotide pairs more clockwise(to the left)
and, therefore, would not delete the
o2 P 1 frd gene and probably not the td gene.>lo.oM\
Additional chemical and physical propertiesP1Revertan~ae \ of these
deletion mutants were characterizedPi Revertont 0 and are
summarized in Fig. 5. When the dele-
tion mutants, dell, del7, and del9, are treated50 , with
pyridine nucleotides, such as NADPH and
NADH, they are inactivated. The inactivationT. 600 reaction has
been attributed to an interactionpH = 7.0 with a pyridine
nucleotide binding site on the0.1 M P Buffer phage baseplate (16).
It should be noted that
NADPH inactivated these particles somewhatmore rapidly than
NADH, in accord with theproperties of the dihydrofolate reductase
1.0 20 cule. The phage-binding site was also shown toMINUTES be
specific for one of the two isomers of thepyridine nucleotides (16)
and to include a bind-
FIG. 3. Heat sensitivity of T4D P1 frd" and P1 1OO.efrd+
revertant particles. Two revertants of P1 fromfrdt to frd+ were
isolated as described in Table 2.Phage stocks were grown at 370C,
and the heat sensi-tivities at 600C were determined individually in
the 50.0usual way. P. Phosphate.Properties of phage bearing
tween genes 63 and 32. Homyk and Weil (7)have described the
isolation of three viable T4strains presumably bearing long
deletions ofthe region between genes 63 and 32 on the T4linkage
map, which would include the gene fortd (dell) and both genes td
and frd for both del7 ,0.0\and del9. Whereas the enzymological
activities aof infected extracts (3) support the view that _these
genes do not produce the appropriate C31functional enzymes, other
properties suggest 5.0 0that the heteroduplex physical mapping of
theDNA is sufficiently inaccurate so that the for- T =600 C31mation
of td and frd gene-related polypeptides pH = 7.0 Revertont bcannot
be excluded. The problems of interpret- 0.1 M P Buffering the
nature of these deletions are given indetail in the companion paper
by Mosher et al. C31 Revertant a(22) and are summarized only
(i) Immunological studies have shown not .0 20 30only that these
deletion mutants produce cross- MIN2TESreacting material to T4
phage thymidylate syn- MINUTESthetase and T4 phage dihydrofolate
reductase FIG. 4. Heat sensitivity ofT4D C31 frdt' and C31uthtalse
than t4ephage dartihydro ate redu ,' frd+ revertant particles. Two
revertants of C31 frombut also that the phage particles are even
more frd's to frd+ were isolated as described in Table
2.susceptible to inactivation by antisera to these Phage stocks
were grown at 37C, and the heat sensi-enzymes (22) than is T4D.
Whereas the sensi- tivities at 60C of the parent C31 and the two
revert-tivity of dell to anti-frd serum is not surpris- ants were
determined in the same experiment. P.ing, both del7 and del9, which
presumably lack Phosphate.
VOL. 23, 1977
J. VIROL.642 KOZLOFF, LUTE, AND CROSBY
FIG. 5. Effect of various inactivation treatments on different
T4D strains. Stocks of the parent T4D strain1589 and the three
deletion mutants ofHomyk and Weil were prepared and subjected to
various treatmentsthat primarily affect the baseplate structure.
Details of these procedures are given earlier for NADPH andNADH
(12), conjugase (14), Cd(CN)3- (13), and L-homoarginine (23), and
the heat treatment was thestandard procedure used in this work.
ing site for the adenosine diphosphoribose por-tion of the
nucleotide (19). Furthermore, it wasfound that hog kidney
conjugase, an enzymespecific for hydrolyzing the y-glutamyl bonds
offolate polyglutamates, also inactivated theseparticles (14). In
several experiments, it wasnoted that the strain T4D 1589 (into
whichthese deletions were crossed) was always inacti-vated more
rapidly and to a greater extent thanthe three deletion mutants.
These propertiesshow that the three deletion mutants have a
tail structure somewhat different than T4D1589 but that they
still contain the phage folateand enough of the dihydrofolate
reductase mol-ecule so that the phage particles are sensitive
topyridine nucleotides.Three other properties of the baseplates
these three deletion mutants as compared tothose of the T4D
strain 1589 are also shown inFig. 5. All three deletion mutants
were readilyinactivated by the reagent Cd(CN)3 (13) or byincubation
with L-homoarginine (23), whereas
T4 VIRION BASEPLATE COMPONENTS 643
T4D strain 1589 was highly resistant to thesetwo reagents.
Further, T4D strain 1589 wasmore readily inactivated by heating at
600Cthan were the three deletion mutants. SinceCd(CN)3-,
L-homoarginine, and heating at600C (12, 15) all primarily perturb
the base-plate, these results confirm the observationsthat the
physical properties of the baseplates ofthe deletion mutants are
different from those ofthe T4D 1589 strain. However, deletions of
thephage genomes between genes 63 and 32 havenot been thought to
include sequences for anyessential phage structural components
(25).These results indicate that these deletions doaffect
structural components. The identifica-tion of the structural
components in these mu-tants as polypeptides related to
thymidylatesynthetase and dihydrofolate reductase mole-cules rests
on the immunological, enzymologi-cal, and genetic (22) data, the
inactivation ofthe phages by pyridine nucleotides, the pres-ence of
phage folic acid, and the restriction ofthese strains in certain
hospital strains of E.coli (22).
Properties of deletion mutants grown onvarious strains of E.
coli. Various E. colistrains were available with different
amountsor types of host dihydrofolate reductases.RT500, an E. coli
B strain resistant to highlevels of trimethoprim, has been shown by
Bac-canari et al. (1) to overproduce the normal E.coli B frd. We
isolated earlier several E. coli Bmutants that were also resistant
to trimetho-prim (12) and showed that the dihydrofolatereductases
produced in these mutants were sig-nificantly altered, asjudged by
their heat sensi-tivity. These bacterial strains offered an
oppor-tunity to compare the deletion mutant particlesassembled in
different cells. Since del7 and del9are presumed to lack the T4D
frd gene,whereas dell does not lack this gene, any sub-stitution of
host enzymes during viral morpho-genesis would be expected to alter
the heatsensitivity of del7 and del9 phage particles, ascompared
with either dell or T4D 1589. Thethree deletion mutants and T4D
strain 1589were grown on E. coli CR63, E. coli B RT500,and the E.
coli B TRIM 102 and TRIM 103 (12)mutants. It was found that the T4D
strain 1589produced in all these host cells was somewhatmore heat
sensitive than the three mutants(see Fig. 5). But all three
mutants, dell, del7,and del9, gave very similar heat sensitivities
toeach other, irrespective of the host cells uponwhich they were
grown (data not shown). Inaddition, since these particles are also
sensitiveto antisera to T4D dihydrofolate reductase anddo react
with the co-factors for this enzyme,NADPH and NADH, it can be
assumed that all
three deletion mutants do not, in fact, lack theability to
synthesize a protein similar to theoriginal T4D dihydrofolate
DISCUSSIONThe data in this paper and the accompanying
paper by Mosher et al. (22) provide confirma-tion that both the
T4D frd and the T4D td genesproduce components that are normal
baseplatecomponents of the T-even bacteriophages. Fur-thermore,
these papers support the earlierviews that these enzymes are
partially buriedwithin the complex tail structure. It is not
sur-prising that, whereas phage ghost preparationsdo exhibit both
detectable dihydrofolate reduc-tase and detectable thymidylate
synthetase ac-tivities, which have been identified as beingdue to
phage gene products, the activities areextremely low. The low
activities are, mostlikely, due to the occlusion of both enzymes
inthe phage structure and the close affinity of thefolate, which
would prevent most of these en-zyme molecules from reacting
catalytically.The data also suggest that these proteins neednot be
complete polypeptides to fulfill theirstructural role, since
polypeptide fragmentsproduced by amber mutants in the frd or tdgene
can participate in phage assembly (12).The quantitation of these
two polypeptides in
the baseplate remains a difficult problem, andMosher et al. (22)
have put an upper limit ofabout six molecules of each per phage
particleas the sensitivity limit of their analytical proce-dure.
This value would be in accord with asimilar value for the six
folates per phage parti-cle (14, 15).The question of whether the
two enzymes are
essential or dispensable gene products for as-sembling the
baseplate is not completely re-solved by these experiments. The
evidence forconcluding that they are essential componentsof the
baseplate rests on the following data. (i)They have been detected
directly or, in somecases, indirectly by some significant
chemicalor genetic property in all T4 strains so far ana-lyzed,
with the only possible exception beingsome partially characterized
T4D deletionstrains. The properties of the deletion mutants,which
are analyzed in detail for the presence offrd by Mosher et al. in
the companion paper(22) and for the presence of td in this
paper,suggest that these deletion mutants do containfrd- and
td-like polypeptides. Earlier observa-tions with the missense and
nonsense muta-tions support the view that these enzymes neednot be
functional for them to play a structuralrole. (ii) The evidence on
the heat sensitivity ofvarious phage frd's and td's mutants and
theirrevertants, coupled with the confirmation by
VOL. 23, 1977
644 KOZLOFF, LUTE, AND CROSBY
Mosher et al. (22) that the baseplate is the mostheat-sensitive
component of the virus particle,support the idea that these two
proteins areintegral parts of the baseplates and not adven-titious
constituents attached to the outside ofthe baseplate. (iii) The
question remainswhether there could be phage particles thatwere
completely infectious but that lackedthese two components. One
analogy would be tothe hoc and soc proteins, which have been
re-cently shown by Ishii and Yanagida (8) to benormal but
dispensable components of the T4phage head. It should be noted
that, whereasIshii and Yanagida (8) readily isolated T4Dmutants
lacking the hoc and soc components,the situation, at least with
regard to phage frd,is different. A search was made for a T4D
mu-tant (19) resistant to inactivation by the frdcofactor NADPH,
and no mutants were ob-tained from 1012 T4D particles. This
meanseither that the frd polypeptide with a bindingsite for NADPH
is absolutely required or that aT4D mutation lacking frd is
extremely rare.There is no comparable method for searchingfor the
absence of the phage td in phage mu-tants. Nonetheless, since the
conservation of agene product for both structural and
metabolicroles is unusual, the possibility must still beconsidered
that one or both of these unexpectedphage components are
ACKNOWLEDGMENTSThis research was supported by Public Health
research grant A106336 from the National Institute of Al-lergy
and Infectious Diseases.
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